The present invention aims to provide a novel process for producing isomaltose and isomaltitol, and uses thereof, and it solves the object by establishing a process for producing isomaltose comprising a step of contacting a saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with an .alpha.-isomaltosyl-transferring enzyme and an .alpha.-isomaltosylglucosaccharide-forming enzyme derived from a specific microorganism; a process for producing isomaltitol using the isomaltose produced by the above process; saccharide compositions comprising the isomaltose and/or the isomaltitol produced by the above processes; and uses thereof.

Inventors:  Kubota; Michio (Okayama, JP), Nishimoto; Tomoyuki (Okayama, JP), Sonoda; Tomohiko (Okayama, JP), Fukuda; Shigeharu (Okayama, JP), Miyake; Toshio (Okayama, JP) 
Assignee: Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo (Okayama, JP)
 
Appl. No.:  10/492,932
Filed:  October 18, 2002
PCT Filed:  October 18, 2002 
PCT No.:  PCT/JP02/10846 
371(c)(1),(2),(4) Date:  April 19, 2004 
PCT Pub. No.:  WO03/033717 
PCT Pub. Date:  April 24, 2003 

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Foreign Application Priority Data

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Oct 18, 2001 [JP]   2001-321182
Aug 30, 2002 [JP]   2002-252609
 

Current U.S. Class: 435/74 ; 435/101
Current International Class:  C12P 19/44 (20060101)

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References Cited [Referenced By]

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U.S. Patent Documents
  
4521252 June 1985 Miyake et al.
5679781 October 1997 Goldscher
7192746 March 2007 Kubota et al.
 

Foreign Patent Documents
     
 0 138 687  Apr., 1985  EP
 0 608 636  Aug., 1994  EP
 0 875 585  Nov., 1998  EP
 1229112  Aug., 2002  EP
 1284286  Feb., 2003  EP
 1335020  Aug., 2003  EP
 1361274  Nov., 2003  EP
 1 382 687  Jan., 2004  EP
 1382687  Jan., 2004  EP
 2 106 912  Apr., 1983  GB
 72598/83  Apr., 1983  JP
 145020/87  Jun., 1987  JP
 216493/88  Sep., 1988  JP
 101862/89  Apr., 1989  JP
 255988/02  Sep., 2002  JP
 WO 99/27124  Jun., 1999  WO
 WO 01/90338  Nov., 2001  WO
 WO 02/10361  Feb., 2002  WO
 WO 02/40659  May., 2002  WO
 WO 02/055708  Jul., 2002  WO
 WO 02/088374  Nov., 2002  WO
 

Other References
Cote, Gregory L. et al "Enzymically produced cyclic .alpha.-1,3-linked and .alpha.-1,6-linked oligosaccharides of D-glucose," European Journal of Biochemistry, (1994), vol. 226, pp. 641-648. cited by other .
Iwai, Atsushi et al "Molecular Cloning and Expression of an Isomalto-Dextranase Gene from Arthrobacter globiformis T6," Journal of Bacteriology, Dec. 1994, vol. 176, pp. 7730-7734. cited by other .
Sawai, Teruo et al, "A Bacterial Dextranase Releasing only Isomaltose from Dextrans," Journal of Biochemistry, (1974), vol. 75, pp. 105-112. cited by other .
Sawai, Teruo et al "Purification and Some Properties of the Isomaltodextranase of Actinomadura Strain R10 and Comparison with that of Arthrobacter globiformis T6," Carbohydrate Research, (1981), vol. 89, pp. 289-299. cited by other .
Yamamoto, Kazuya et al "Purification and Some Properties of Dextrin Dectranase from Acetobacter capsulatus ATCC 11894," Bioscience Biotechnology and Biochemistry, (1992), vol. 56(2), pp. 169-173. cited by other. 

Primary Examiner: Desai; Anand U
Attorney, Agent or Firm: Browdy and Neimark, P.L.L.C.

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Claims

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The invention claimed is:

1. A process for producing isomaltitol, comprising the steps of: (a) allowing an .alpha.-isomaltosylglucosaccharide-forming enzyme, which forms an .alpha.-isomaltosylglucosaccharide with a glucose polymerization degree of at least three and having both the .alpha.-1,6 glucosidic linkage as the linkage at the non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, via the .alpha.-glucosyl-transfer from a material saccharide having a glucose polymerization degree of at least two and having the .alpha.-1,4 glucosidic linkage as the linkage at the non-reducing end, without substantially increasing the reducing power of the material saccharide, to act on a saccharide with a glucose polymerization degree of at least two and having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end to form said .alpha.-isomaltosylglucosaccharide wherein said .alpha.-isomaltosylglucosaccharide-forming enzyme has the following physicochemical properties: (1) Molecular weight Having a molecular weight of about 117,000 to about 160,000 daltons when determined on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); (2) Isoelectric point Having an isoelectic point of about 4.7 to about 5.7 when determined on isoelectrophoresis using ampholine; (3) Optimum temperature Having an optimum temperature of about 40.degree. C. to about 45.degree. C. when incubated at a pH of 6.0 for 60 min; Having an optimum temperature of about 45.degree. C. to about 50.degree. C. when incubated at a pH of 6.0 for 60 min in the presence of 1 mM Ca.sup.2+; (4) Optimum pH Having optimum pH of about 6.0 to about 6.5 when incubated at 35.degree. C. or 60 min; (5) Thermal stability Being stable up to a temperature of about 35.degree. C. to 40.degree. C. when incubated at a pH of 6.0 for 60 min, Being stable up to a temperature of about 40.degree. C. to 45.degree. C. when incubated at a pH of 6.0 for 60 min in the presence of 1 mM Ca.sup.2+, (6) pH Stability Having a stable pH range at about 4.5 to about 10.0 when incubated at 4.degree. C. for 24 hours; (b) allowing an isomaltodextranase to act on the resulting mixture in the step (a) to form isomaltose; (c) hydrogenating either the resulting mixture in the step (b) directly or the isomaltose, which has been separated from the mixture to form isomaltitol; and (d) collecting the formed isomaltitol.

2. The process of claim 1, wherein one or more enzymes selected from the group consisting of .alpha.-isomaltosyl-transferring enzyme, which forms a cyclotetrasaccharide having the structure of cyclo{.fwdarw.6) -.alpha.-D-glucopyranosyl- (1.fwdarw.3) -.alpha.-D-glucopyranosyl- (1.fwdarw.6) -.alpha.-D-glucopyranosyl- (1.fwdarw.3) -.alpha.-D-glucopyranosyl- (1.fwdarw.}from said .alpha.-isomaltosylglucosaccharide and has the following physicochemical properties: (1) Molecular weight Having a molecular weight of about 82,000 to about 136,000 daltons when determined on SDS-PAGE; (2) Isoelectic point (pI) Having a pI about 5.0 to about 6.1 when determined on isoelectrophoresis using ampholine; (3) Optimum temperature Having an optimum temperature of about 45.degree. C. to about 50.degree. C. when incubated at a pH of 6.0 for 30 min; (4) Optimum pH Having an optimum pH of about 5.5 to about 6.0 when incubated at 35.degree. C. for 30 min; (5) Thermal stability Being stable up to a temperature of about 40.degree. C. when incubated at a pH of 6.0 for 60 min; and (6) pH Stability Having a stable pH range at about 4.0 to about 9.0 when incubated at 4.degree. C. for 24 hours; cyclomaltodextrin glucanotransferase and starch debranching enzyme are further allowed to act on said saccharide with a glucose polymerization degree of at least two and having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end in the step (a).

3. The process of claim 1, wherein glucoamylases is further allowed to act on the reaction mixture after the enzymatic reaction of said isomaltodextranase in the step (b).

4. A process of claim 1, wherein said saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, is one or more saccharides selected from the group consisting of maltooligosaccharides, maltodextrins, amylodextrins, amyloses, amylopectins, soluble starches, liquefied starches, gelatinized starches, and glycogens.

5. The process of claim 1, characterized in that it employs a column chromatography using an alkaline metal- and/or alkaline earth metal-strong-acid-cation-exchange-resin and optionally employs a step of pulverization or crystallization in the step (d).

6. The process of claim 1, wherein said isomaltitol is collected in the form of a syrup, powder, or crystal in the step (d).

7. The process of claim 1, wherein the collected isomaltitol in the step (d) is a high isomaltitol content syrup comprising isomaltitol in an amount of at least 40% (w/w), on a dry solid basis.
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Description

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CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 application of PCT/JP02/10846, filed Oct. 18, 2002, which claims benefit of JP 2001-321182, filed Oct. 18, 2001 and JP 2002-252609, filed Aug. 30, 2002, all of which are herein incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel process for producing isomaltose and isomaltitol, and uses thereof, more particularly, to a process for producing isomaltose and/or isomaltitol in a relatively high yield from a saccharide which has the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, and uses thereof.

BACKGROUND ART

Isomaltose is a rare saccharide that merely exists in nature in fermented foods in a slight amount, and it is known to be produced by conventional methods such as partial hydrolysis reactions using acid catalysts, enzymatic reactions using dextranase or isomaltodextranase, reverse-synthetic reactions for forming isomaltose from glucose using glucoamylase or acid catalysts, and glucose-transferring reactions for forming isomaltose from maltose or maltodextrins using .alpha.-glucosidase. However, the above conventional methods are far from a satisfactory industrial-scale production of isomaltose, because the isomaltose contents in the reaction mixtures, obtained by the above conventional processes, are only about 10 to about 25% (w/w) (the symbol "% (w/w)" is abbreviated as "%" throughout the specification, unless specified otherwise), on a dry solid basis (d.s.b.) and their purities are relatively low. To improve this drawback, for example, a column chromatography, disclosed in Japanese Patent Kokai No. 72,598/83, can be mentioned. According to the method, a relatively high purity isomaltose can be produced from material saccharide solutions with an isomaltose content of about 10 to about 25%, d.s.b. However, even if the method is employed, there still remains a problem of that the purity and the yield of the produced isomaltose inevitably depend on the isomaltose content in the material saccharide solutions used.

Under these circumstances, there has been required a novel process for producing isomaltose on an industrially scale and in a lesser cost and a higher yield.

While isomaltitol is a sugar alcohol having satisfactory non-reducibility, low sweetness, and moisture-retaining ability, and it is a useful sugar alcohol which has been extensively used in food products, cosmetics, pharmaceuticals, etc., in the form of a saccharide mixture with sorbitol, maltitol, and glucosyl-1,6-mannitol.

Isomaltitol can be theoretically prepared by hydrogenating, i.e., reducing the reducing group of paratinose or isomaltose, as a reducing oligosaccharide, into an alcohol group. In particular, although isomaltitol has been prepared from isomaltose in a relatively high yield, the desired industrial supply of material isomaltose has not been satisfactory. Isomaltose is known to be prepared by the methods such as partial hydrolytic reactions of dextrans using acid catalysts, enzymatic reactions using dextranase or isomaltodextranase, reverse-synthetic reaction for forming isomaltose from glucose using glucoamylase or acid catalysts, glucose-transferring reactions for forming isomaltose from maltose or maltodextrins using .alpha.-glucosidase. However, the above conventional methods are far from a satisfactory industrial-scale production of isomaltitol, because the isomaltose contents in the reaction mixtures, obtained by the above conventional processes, are only about 10 to about 25%, d.s.b., and the purity of isomaltitol, obtained by hydrogenating the above-identified isomaltose, is relatively low. To improve the drawback, for example, by applying column chromatography disclosed in Japanese Patent Kokai No. 72,598/83, a relatively high purity isomaltose can be obtained from material saccharide solutions with a relatively low isomaltose content of about 10% to about 25%, d.s.b., and then hydrogenated to obtain isomaltitol. Even in the process for producing isomaltitol, as a drawback, the yield and the cost of isomaltitol inevitably depend on the isomaltose content of the material saccharide solutions used, and this lowers the yield and increases the production cost of isomaltitol.

While in the case of producing isomaltitol from paratinose, the material paratinose is known to be prepared, for example, from sucrose through glucose-transferring reaction using .alpha.-glucosyl transferase. However, since the resulting reaction mixture comprises, as by products, trehalulose as an isomer of paratinose and others such as glucose and fructose as hydrolyzates of paratinose, the paratinose content in the reaction mixture could not be over about 85%, d.s.b. In producing isomaltitol from paratinose, glucosyl-1,6-mannitol is formed along with isomaltitol in a production ratio of, usually, 1:1 by weight, and this lowers the purity and the yield of isomaltitol as a drawback.

Under these circumstances, a novel process for producing isomaltitol on an industrial scale and in a lesser cost and a higher yield has been strongly required.

DISCLOSURE OF INVENTION

Considering the above prior arts, the object of the present invention is to establish a process for producing isomaltose and isomaltitol on an industrial scale and in a lesser cost and a higher yield, and uses thereof. Namely, the object of the present invention is to establish a process for producing isomaltose and isomaltitol on an industrial scale and in a lesser cost and a higher yield, saccharide mixtures comprising isomaltose and/or isomaltitol, and uses thereof.

During the present inventors had been eagerly studying on solving the above objects, it was reported in European Journal of Biochemistry, Vol. 226, pp. 641-648 (1994) a cyclic tetrasaccharide, having the structure of cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.} (may be called "cyclotetrasaccharide" throughout the specification), having the structure of isomaltose intramolecularly, formed by contacting a hydrolyzing enzyme, i.e., alternanase, with alternan composed of four glucose molecules linked together via the alternating .alpha.-1,3 and .alpha.-1,6 bonds.

As previously disclosed in Japanese Patent Application No. 2000-229557 (International Publication No. WO 01/90338), the present inventors established a process for producing cyclotetrasaccharide using an .alpha.-isomaltosyl-transferring enzyme which forms cyclotetrasaccharide from amylaceous saccharides such as panose, and in Japanese Patent Application No. 2000-234937 (International Publication No. WO 02/10361), they established another process for producing cyclotetrasaccharide in a higher yield by allowing an .alpha.-isomaltosyl-transferring enzyme and an .alpha.-isomaltosylglucosaccharide-forming enzyme which forms .alpha.-isomaltosylglucosaccharide from maltooligosaccharides. Further, as disclosed in Japanese Patent Application No. 2001-130922 (International Publication No. WO 02/04166), the present inventors established another process for producing isomaltose in a higher yield by allowing an .alpha.-isomaltosylglucosaccharide-forming enzyme and an isomaltose-releasing enzyme to act on material starches.

Thereafter, the present inventors discovered .alpha.-isomaltosylglucosaccharides and an .alpha.-isomaltosylglucosaccharide-forming enzyme, which can be used in the above process for producing isomaltose, and also found that isomaltose is produced on an industrial scale and in a lesser cost and a higher yield by using these enzymes. The present inventors further studied the method for producing isomaltitol from isomaltose; they studied the enzymatic reaction mechanisms of such .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme and found that the production yield of isomaltose is dramatically increased by allowing an .alpha.-isomaltosylglucosaccharide-forming enzyme and an .alpha.-isomaltose-releasing enzyme capable of releasing isomaltose to act on a saccharide having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme, and that isomaltitol is easily produced on an industrial scale and in an increased yield by hydrogenating the isomaltose thus obtained. The present inventors also established the uses of the isomaltitol thus obtained and accomplished this invention; they solved the above object by establishing a process for producing isomaltose comprising a step of contacting a saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with one or more .alpha.-isomaltosylglucosaccharide-forming enzymes derived from Bacillus globisporus N75 strain (FERM BP-7591) (hereinafter may be called "N75 strain"), Arthrobacter globiformis A19 strain (FERM BP-7590) (hereinafter may be called "A19 strain"), and Arthrobacter ramosus S1 strain (FERM BP-7592) (hereinafter may be called "S1 strain"), which are disclosed in PCT/JP01/06412 (International Publication No. WO 02/10361) in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme derived from Bacillus globisporus N75 strain (FERM BP-7591) and/or Arthrobacter globiformis A19 strain (FERM BP-7590) to form .alpha.-isomaltosylglucosaccharides having the .alpha.-1,6 glucosidic linkage as the linkage of non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, and/or to form a saccharide with the structure of cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.}, contacting the products thus obtained with isomaltose-releasing enzyme to form isomaltose, and collecting the produced isomaltose; saccharide mixtures with such isomaltose; and uses thereof. As regards the above-identified Bacillus globisporus N75 strain (FERM BP-7591), the microorganism was deposited on May 16, 2001, and has been maintained in International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan. Arthrobacter ramosus S1 strain (FERM BP-7592) was deposited on May 16, 2001, and has been maintained in the above institute.

The present inventors further solved the object of the present invention by contacting a saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with .alpha.-isomaltosylglucosaccharide-forming enzyme in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme to form .alpha.-isomaltosylglucosaccharides, having the .alpha.-1,6 glucosidic linkage as the linkage of non-reducing end and .alpha.-1,4 glucosidic linkage other than the above linkage and having a glucose polymerization degree of at least three, and/or cyclotetrasaccharide; contacting the resulting saccharides with isomaltose-releasing enzyme to form isomaltose; hydrogenating the resulting mixtures containing isomaltose directly or after collecting isomaltose to form isomaltitol; and collecting the formed isomaltitol; saccharide mixtures containing isomaltitol; and uses thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an elution pattern of a saccharide, obtained by the enzymatic reaction using .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain, when determined on high-performance liquid chromatography.

FIG. 2 is a spectrum of nuclear magnetic resonance (.sup.1H-NMR) of cyclotetrasaccharide, obtained by the enzymatic reaction using .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 3 is a spectrum of nuclear magnetic resonance (.sup.13C-NMR) of cyclotetrasaccharide, obtained by the enzymatic reaction using .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 4 represents the structure of cyclotetrasaccharide, i.e., cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.}.

FIG. 5 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide forming enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 6 shows the pH influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 7 shows the thermal stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 8 shows the pH stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 9 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 10 shows the pH influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 11 shows the thermal stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 12 shows the pH stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C9 strain.

FIG. 13 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 14 shows the pH influence on .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 15 shows the thermal stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 16 shows the pH stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 17 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 18 shows the pH influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 19 shows the thermal stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 20 shows the pH stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus C11 strain.

FIG. 21 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 22 shows the pH influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 23 shows the thermal stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 24 shows the pH stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 25 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 26 shows the pH influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 27 shows the thermal stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 28 shows the pH stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Bacillus globisporus N75 strain.

FIG. 29 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 30 shows the pH influence on the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 31 shows the thermal stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 32 shows the pH stability of .alpha.-isomaltosylglucosaccharide-forming enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 33 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 34 shows the pH influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 35 shows the thermal stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 36 shows the pH stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 37 is a figure for a restriction map of a recombinant DNA "pAGA4", where the part with a bold line is a DNA encoding a polypeptide having an .alpha.-isomaltosyl-transferring enzyme activity, derived from a microorganism of the species Arthrobacter globiformis A19 strain.

FIG. 38 shows the thermal influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 39 shows the pH influence on the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 40 shows the thermal stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 41 shows the pH stability of .alpha.-isomaltosyl-transferring enzyme from a microorganism of the species Arthrobacter ramosus S1 strain.

FIG. 42 is a spectrum of nuclear magnetic resonance (.sup.1H-NMR) of .alpha.-isomaltosylmaltotriose, obtained by the enzymatic reaction using .alpha.-isomaltosylglucosaccharide-forming enzyme.

FIG. 43 is a spectrum of nuclear magnetic resonance (.sup.1H-NMR) of .alpha.-isomaltosylmaltotetraose, obtained by the enzymatic reaction using .alpha.-isomaltosylglucosaccharide-forming enzyme.

FIG. 44 is a spectrum of nuclear magnetic resonance (.sup.13C-NMR) of .alpha.-isomaltosylmaltotriose, obtained by the enzymatic reaction using .alpha.-isomaltosylglucosaccharide-forming enzyme.

FIG. 45 is a spectrum of nuclear magnetic resonance (.sup.13C-NMR) of .alpha.-isomaltosylmaltotetraose, obtained by the enzymatic reaction using .alpha.-isomaltosylglucosaccharide-forming enzyme.

FIG. 46 is a spectrum of nuclear magnetic resonance (.sup.1H-NMR) of product A.

FIG. 47 is a spectrum of nuclear magnetic resonance (.sup.13C-NMR) of product A.

FIG. 48 is an x-ray powder diffraction pattern of isomaltitol crystal obtained by the method of the present invention.

FIG. 49 is a spectrum of nuclear magnetic resonance (.sup.1H-NMR) of isomaltitol crystal obtained by the method of the present invention.

FIG. 50 is a spectrum of nuclear magnetic resonance (.sup.13C-NMR) of isomaltitol crystal obtained by the method of the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The .alpha.-isomaltosylglucosaccharide-forming enzyme as referred to as in the present invention means those which forms .alpha.-isomaltosylglucosaccharides such as .alpha.-isomaltosylglucose (or panose), .alpha.-isomaltosylmaltose, .alpha.-isomaltosylmaltotriose, and .alpha.-isomaltosyltetraose; .alpha.-isomaltosylglucosaccharide-forming enzymes derived from microorganisms of the species Bacillus globisporus C9 strain (FERM BP-7143) (hereinafter may be called "C9 strain"), Bacillus globisporus C11 strain (FERM BP-7144) (hereinafter may be called "C11 strain"), Bacillus globisporus N75 strain (FERM BP-7591), and Arthrobacter globiformis A19 strain (FERM BP-7590), which are disclosed in PCT/JP01/06412 (International Publication No. WO 02/10361); and recombinant polypeptides having an activity of .alpha.-isomaltosylglucosaccharide-forming enzyme, which is disclosed in Japanese Patent Application No. 2001-5441 (International Publication No. WO 02/055708). Among these enzymes, those from Bacillus globisporus N75 strain (FERM BP-7591) and Arthrobacter globiformis A19 strain (FERM BP-7590) are most preferably used in the present invention. As regards the above-identified Bacillus globisporus C9 strain (FERM BP-7143), and Bacillus globisporus C11 strain (FERM BP-7144) were deposited on Apr. 25, 2000, and have been maintained in National Institute of Bioscience and Human-Technology Agency of Industrial Science and Technology, now changed into International Patent Organism Depositary National Institute of Advanced Industrial Science and Technology, AIST Tsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, 305-8566, Japan.

The .alpha.-isomaltosylglucosaccharide-forming enzyme as referred to as in the present invention is a generic term for enzymes and polypeptides which have an activity of .alpha.-isomaltosylglucosaccharide-forming enzyme, and it is an enzyme which forms, via the .alpha.-glucosyl-transfer, a saccharide, having a glucose polymerization degree of at least three and having both the .alpha.-1,6 glucosidic linkage as the linkage of non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, from a material saccharide having a glucose polymerization degree of at least two and having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end, without substantially increasing the reducing power of the material saccharide used; has no dextran-forming ability; and which is inhibited by EDTA (ethylenediaminetetraacetic acid). More particularly, the above material saccharide, having both a glucose polymerization degree of at least two and the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end, includes, for example, one or more saccharides selected from maltooligosaccharides, maltodextrins, amylodextrins, amyloses, amylopectins, soluble starches, gelatinized starches, and glycogens. The above .alpha.-isomaltosylglucosaccharide-forming enzyme has the following physicochemical properties:

(1) Action Forming a saccharide having a glucose polymerization degree of at least three and having both the .alpha.-1,6 glucosidic linkage as the linkage at the non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, via the .alpha.-glucosyl-transfer from a material saccharide having a glucose polymerization degree of at least two and having the .alpha.-1,4 glucosidic linkage as the linkage at the non-reducing end, without substantially increasing the reducing power of the material saccharide;

(2) Molecular weight Having a molecular weight of about 74,000 to about 160,000 daltons when determined on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis);

(3) Isoelectric point Having an isoelectric point of about 3.8 to about 7.8 when determined on isoelectrophoresis using ampholine;

(4) Optimum temperature Having an optimum temperature of about 40.degree. C. to about 50.degree. C. when incubated at a pH of 6.0 for 60 min; Having an optimum temperature of about 45.degree. C. to about 55.degree. C. when incubated at a pH of 6.0 for 60 min in the presence of 1 mM Ca.sup.2+; Having an optimum temperature of 60.degree. C. when incubated at a pH of 8.4 for 60 min; or Having an optimum temperature of 65.degree. C. when incubated at a pH of 8.4 for 60 min in the presence of 1 mM Ca.sup.2+;

(5) Optimum pH Having an optimum pH of about 6.0 to about 8.4 when incubated at 35.degree. C. for 60 min;

(6) Thermal stability Having a thermostable region at temperatures of about 45.degree. C. or lower when incubated at a pH of 6.0 for 60 min, Having a thermostable region at temperatures of about 50.degree. C. or lower when incubated at a pH of 6.0 for 60 min in the presence of 1 mM Ca.sup.2+, Having a thermostable region at temperatures of about 55.degree. C. or lower when incubated at a pH of 8.0 for 60 min, and Having a thermostable region at temperatures of about 60.degree. C. or lower when incubated at a pH of 8.0 for 60 min in the presence of 1 mM Ca.sup.2+;

(7) pH Stability Having a stable pH region at about 4.5 to about 10.0 when incubated at 4.degree. C. for 24 hours; and

(8) N-Terminal amino acid sequence tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleuci- ne, histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leu- cine, alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-gl- utamine-phenylalanine-glutamine-serine-glycine, or others.

The .alpha.-isomaltosyl-transferring enzyme used in the present invention means an enzyme, which forms cyclotetrasaccharide from .alpha.-isomaltosylglucosaccharides such as panose and isomaltosylmaltose, for example, .alpha.-isomaltosyl-transferring enzymes derived from Bacillus globisporus C9 strain (FERM BP-7143), Bacillus globisporus C11 strain (FERM BP-7144), Bacillus globisporus N75 strain (FERM BP-7591), Arthrobacter globiformis A19 strain (FERM BP-7590), and Arthrobacter ramosus S1 strain (FERM BP-7592), as well as recombinant polypeptides having an activity of .alpha.-isomaltosyl-transferring enzyme disclosed in PCT/JP01/10044 (International Publication No. WO 02/40659), which all have an .alpha.-isomaltosyl-transferring activity and are called as a general term of ".alpha.-isomaltosyl-transferring enzyme" in the present invention. Among these enzymes, those from Bacillus globisporus N75 strain (FERM BP-7591), Arthrobacter globiformis A19 strain (FERM BP-7590), and Arthrobacter ramosus S1 strain (FERM BP-7592) are most preferably used in the present invention. The .alpha.-isomaltosyl-transferring enzyme usable in the present invention has the following physicochemical properties:

(1) Action Forming a cyclotetrasaccharide having the structure of cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.} from a saccharide having a glucose polymerization degree of at least three and having both the .alpha.-1,6 glucosidic linkage as the linkage at the non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage;

(2) Molecular weight Having a molecular weight of about 82,000 to about 136,000 daltons when determined on SDS-PAGE;

(3) Isoelectric point (pI) Having a pI of about 3.7 to about 8.3 when determined on isoelectrophoresis using ampholine;

(4) Optimum temperature Having an optimum temperature of about 45.degree. C. to about 50.degree. C. when incubated at a pH of 6.0 for 30 min;

(5) Optimum pH Having an optimum pH of about 5.5 to about 6.5 when incubated at 35.degree. C. for 30 min;

(6) Thermal stability Having a thermostable range at temperatures of about 45.degree. C. or lower when incubated at a pH of 6.0 for 60 min;

(7) pH Stability Having a stable pH range at about 3.6 to about 10.0 when incubated at 4.degree. C. for 24 hours.

(8) N-Terminal amino acid sequence isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline, aspartic acid-threonine-leucine-serine-glycine-valine-phenylalanine-histidine-glyc- ine-proline, or others.

The isomaltose-releasing enzyme used in the present invention means an enzyme, which has an action of releasing isomaltose from .alpha.-isomaltosylglucosaccharides or cyclotetrasaccharide, such as isomaltodextranase (EC 3.2.1.94) derived from microorganisms of the species Arthrobacter globiformis T6 (NRRL B-4425) reported in Journal of Biochemistry, Vol. 75, pp. 105-112 (1974); Arthrobacter globiformis (IAM 12103) which is distributed and available from Institute of Molecular and Cellular Biosciences, the University of Tokyo, Tokyo, Japan; and Actinomadura R10 (NRRL B-11411) disclosed in Carbohydrate Research, Vol. 89, pp. 289-299 (1981).

The saccharide usable in the present invention, which has the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, means one or more saccharides selected from maltooligosaccharides, maltodextrins, amylodextrins, amyloses, amylopectins, soluble starches, liquefied starches, gelatinized starches, and glycogens. Examples of material starches for the above-identified soluble starches, liquefied starches, and gelatinized starches are, for example, terrestrial starches such as corns, rices, and wheats; subterranean starches such as potatoes, sweet potatoes, and tapioca; and partial hydrolyzates thereof, i.e., partial starch hydrolyzates. Preferably, such partial starch hydrolyzates can be generally prepared by suspending the above terrestrial or subterranean starches in water into starch suspensions with a concentration, usually, of at least 10%, preferably, 15 to 65%, and more preferably, 20 to 50%; and liquefying the starch suspensions with acids or enzyme preparations. The liquefaction degree of the above terrestrial and subterranean starches is preferably set to a relatively low level, usually, a DE (dextrose equivalent) of less than 15, preferably, a DE of less than 10, and more preferably, DE of 9 to 0.1. In the case of liquefying the above terrestrial or subterranean starches with acids, for example, employed are methods which comprise the steps of liquefying the starches with acids such as hydrochloric acid, phosphoric acid, and oxalic acid; and then usually neutralizing the resulting mixtures with one or more alkalis such as calcium carbonate, calcium oxide, and sodium carbonate to adjust the mixtures to a desired pH. In the case of liquefying the above terrestrial or subterranean starches with an enzyme such as .alpha.-amylase, particularly, thermostable liquefying .alpha.-amylase can be preferably used as such an enzyme in the present invention. Isomaltose can be obtained in a higher yield by contacting saccharides, having the .alpha.-1,4 glucosidic linkage as the linkage of their non-reducing ends and a glucose polymerization degree of at least two, with .alpha.-isomaltosylglucosaccharide-forming enzyme in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme to form cyclotetrasaccharide and/or .alpha.-isomaltosylglucosaccharides having the .alpha.-1,6 glucosidic linkage as the linkage of their non-reducing ends and the .alpha.-1,4 glucosidic linkage as a linkage other than that of their non-reducing ends; and contacting the formed saccharides with isomaltose-releasing enzyme to form isomaltose; and collecting the formed isomaltose. In the case of contacting the terrestrial or subterranean starches with .alpha.-isomaltosylglucosaccharide-forming enzyme in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme, one or more enzymes selected from .alpha.-isomaltosyl-transferring enzyme, cyclomaltodextrin glucanotransferase (abbreviated as "CGTase" hereinafter), .alpha.-glucosidase, glucoamylase, and starch debranching enzyme including isoamylase and pullulanase can be used in combination; or one or more enzymes selected from .alpha.-isomaltosyl-transferring enzyme, CGTase, .alpha.-glucosidase, glucoamylase, and isoamylase can be used after the action of .alpha.-isomaltosylglucosaccharide-forming enzyme in the presence or the absence of .alpha.-isomaltosyl-transferring enzyme, whereby isomaltose can be formed in a relatively high yield. In particular, the production yield of isomaltose from cyclotetrasaccharide can be increased to 100% as the highest possible level by allowing isomaltose-releasing enzyme to act on cyclotetrasaccharide, prepared by contacting .alpha.-isomaltosylglucosaccharide-forming enzyme with saccharides, having .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, in the presence of .alpha.-isomaltosyl-transferring enzyme. In practicing the present invention, the order of the enzymes used can be decided depending on the desired production yield of isomaltose, reaction time, reaction condition, etc., a plurality of enzymes can be used simultaneously; or a requisite amount of enzymes can be divided into portions and used at different timings. The pH for the enzymatic reactions of the enzymes used in the present invention is usually in the range of pH 4 to 10, preferably, pH 5 to 9. The temperature for the enzymatic reactions of the enzymes used in the present invention is usually in the range of 10 to 80.degree. C., preferably, 30 to 70.degree. C. The amount of enzymes used can be appropriately set depending on the reaction conditions and reaction times for each enzyme, and it is usually appropriately selected from 0.01 to 100 units/g substrate for .alpha.-isomaltosyl-transferring enzyme and .alpha.-isomaltosylglucosaccharide-forming enzyme, 1 to 10,000 units/g substrate for isomaltose-releasing enzyme and starch debranching enzyme, and 0.05 to 7,000 units/g substrate for CGTase, .alpha.-glucosidase, glucoamylase, and isoamylase. Varying depending on the amount of the enzymes used, the reaction time is appropriately set in view of the aimed production yield of isomaltose, usually, it is set to terminate the whole enzymatic reactions within 1 to 200 hours, preferably, 5 to 150 hours, and more preferably, 10 to 100 hours. The pH and temperature during each enzymatic reaction can be appropriately altered before completion of the enzymatic reactions of the present invention.

The content of isomaltose in the enzymatic reaction mixtures thus obtained usually reaches at least 30%, preferably, at least 40%, more preferably, at least 50%, and more preferably, 99% or more as the highest possible level. Particularly, enzymatic reaction mixtures having an isomaltose content of at least 50%, d.s.b., can be easily obtained by contacting .alpha.-isomaltosylglucosaccharide-forming enzyme, .alpha.-isomaltosyl-transferring enzyme, and isomaltose-releasing enzyme simultaneously or in this order with saccharides having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two. The above enzymatic reaction mixtures are usually subjected to conventional methods of filtration and centrifugation to remove insoluble impurities, followed by desalting to purify the resulting mixtures with ion exchangers in H- and OH-forms, and concentrating the resultants into syrups. The resulting syrups can be dried into solid or powdery products. If necessary, the above syrups and products can be purified into high isomaltose content products by using one or more fractionations using column chromatography using ion-exchangers, activated charcoals, and silica gels, etc.; separations using organic solvents such as alcohols and acetone; and separation methods using membranes, which can be used in an appropriate combination. In particular, as an industrial scale production method for high isomaltose content products, column chromatography using ion-exchange resins is advantageously used; column chromatography using one or more strong-acid cation exchange resins in an alkaline metal form of Na.sup.+, etc., or alkaline earth metal forms of Ca.sup.2+, Mg.sup.2+, etc., of styrene-divinylbenzene cross-linked copolymer resins with sulfonic group, as disclosed, for example, in Japanese Patent Kokai Nos. 23,799/83 and 72,598/83, facilitates the production of high isomaltose content products on an industrial scale and in a relatively high yield and low cost. Examples of commercialized products of the above-identified strong-acid cation exchange resins are "DOWEX 50W-X2.TM.", "DOWEX 50W-X4.TM.", and "DOWEX 50W-X8.TM.", commercialized by Dow Chemical Co., Midland, Mich., USA; "AMBERLITE CG-120.TM." commercialized by Rohm & Hass Company, PA, USA; "XT-1022E.TM.", commercialized by Tokyo Organic Chemical Industries, Ltd., Tokyo, Japan; "DIAION SK1B.TM.", "DIAION SK102.TM.", "DIAION SK104.TM.", etc., which are cation exchangers commercialized by Mitsubishi Chemical Corporation, Tokyo, Japan. In practicing such column chromatography using the above ion-exchange resins, any one of fixed-bed, moving bed, and semi-moving methods can be employed. With these methods, isomaltose can be increased its purity, d.s.b., usually, up to 60% or more, preferably, 80% or more, and more preferably, 99% or more, as the highest possible purity, in a relatively high yield. High isomaltose content products other than the isomaltose with the highest possible purity usually comprise isomaltose and one or more saccharides selected from glucose, maltose, maltotriose, maltotetraose, other partial starch hydrolyzates, .alpha.-isomaltosylglucosaccharide, cyclotetrasaccharide, and .alpha.-glucosyl-(1.fwdarw.6)-.alpha.-glucosyl-(1.fwdarw.3)-.alpha.-g- lucosyl-(1.fwdarw.6)-glucose (hereinafter may be abbreviated as "ring-opened tetrasaccharide") in a total amount, excluding that of isomaltose, usually, of 1 to 60%, d.s.b. To industrially produce isomaltitol by hydrogenating isomaltose, the above-identified desalting and purification steps using ion-exchangers in H- and OH-forms can be omitted, if necessary.

By hydrogenating the resulting isomaltose or isomaltose-containing products in the presence of reducing catalysts, isomaltitol and high isomaltitol content products can be produced in a relatively high yield. For example, the Raney Nickel catalyst is added to a 40-60% aqueous isomaltose solution. The mixture is placed in a high-pressure vessel, filled with hydrogen, increased its inner pressure, and stirred at temperatures of 100 to 120.degree. C. to hydrogenate the isomaltose until the hydrogen is no more consumed. In this case, isomaltose is reduced to isomaltitol, while reducing saccharides contained in isomaltose-containing products, such as glucose, maltose, maltotetraose, other partial starch hydrolyzates, reducing .alpha.-isomaltosylglucosaccharide, and ring-opened tetrasaccharide are simultaneously reduced to sugar alcohols. Cyclotetrasaccharide is a non-reducing saccharide which is not susceptible to hydrogenation. After removing the Raney nickel catalyst from the resulting isomaltitol solution, the resulting solution is decolored with activated charcoal, desalted for purification with ion-exchangers in H- and OH-forms, and concentrated into a syrupy product, and optionally further dried into a powdery product. In necessary, the syrupy product can be, for example, purified by one or more of the following methods alone or in an appropriate combination into a saccharide mixture with isomaltitol: Fractionation of column chromatography using ion-exchangers, activated charcoals, silica gels, etc.; crystallization; separation using organic solvents such as alcohols and acetone; and separation using membranes. The crystallization method for isomaltitol is usually effected by placing in a crystallizer a supersaturated solution of isomaltitol kept at 40 to 95.degree. C., gradually adding a seed to the solution in an amount, usually, of 0.1 to 20%, and gradually cooling the mixture under gently stirring conditions to crystallize the contents and to form a massecuite. Thereafter, the resulting massecuite is subjected to conventional methods such as separation, block pulverization, fluidized-bed granulation, and spray drying to obtain a powdery crystalline isomaltitol, which is usually an anhydrous crystalline isomaltitol. The above separation means usually a method for separating massecuite into isomaltitol crystal and syrup by using a basket-type centrifuge, where a small amount of cooled water is optionally sprayed over the formed crystal for washing to facilitate the production of non-hygroscopic crystalline isomaltitol with a higher purity. As regards the other three methods among the above-identified methods, they have a characteristic of a higher yield of crystalline isomaltitol, although the purity of isomaltitol in the resulting massecuite with crystalline isomaltitol is not substantially improved because they do not separate syrup. Therefore, such massecuite usually comprises crystalline isomaltitol and one or more saccharides from sorbitol, maltitol, maltotriitol, maltotetraitol, sugar alcohols derived from other partial starch hydrolyzates and .alpha.-isomaltosylglucosaccharides, cyclotetrasaccharide, and .alpha.-glucosyl-(1.fwdarw.6)-.alpha.-glucosyl-(1.fwdarw.3)-.alpha.-gluco- syl-(1.fwdarw.6)-sorbitol (hereinafter may be abbreviated as "reduced ring-opened tetrasaccharide"). In the case of spray drying, a massecuite with a concentration of 70 to 85% and a crystallization percentage of 25 to 60 is sprayed from a nozzle by a high-pressure pump; dried with air heated to a temperature, free of melting the formed powdery crystal, usually, a temperature of 60 to 100.degree. C.; and aged by blowing air heated to 30 to 60.degree. C. for about 1 to about 20 hours to facilitate the production of a non-hygroscopic or substantially-hygroscopic crystal with syrup. In the case of block pulverization, usually, a massecuite with a concentration of 85 to 95% and a crystallization percentage of about 10 to about 60% are allowed to stand for 0.5 to 5 days to crystallize and solidify the whole contents into a block, followed by pulverizing the block by the methods such as crushing and cutting, and drying the resultant to facilitate the production of a non-hygroscopic or substantially-hygroscopic crystal with syrup.

With these crystallization methods, isomaltitol with a purity, usually, of at least 40%, d.s.b., preferably, at least 60%, d.s.b., and more preferably, at least 99%, d.s.b., can be obtained in a higher yield. Also, saccharide mixtures with isomaltitol, which comprise maltitol and one or more saccharides from sorbitol, maltitol, maltotriitol, maltotetraitol, sugar alcohols prepared from other partial starch hydrolyzates and .alpha.-isomaltosylglucosaccharides, cyclotetrasaccharide, and reduced ring-opened tetrasaccharide in a total amount excluding that of isomaltitol, usually, of not higher than 70%, d.s.b., preferably, not higher than 60%, d.s.b., and more preferably, 1 to 50%, d.s.b., can be easily obtained. Among the aforementioned saccharide mixtures with isomaltitol, those, which comprise isomaltitol and one or more saccharides from cyclotetrasaccharide, reduced ring-opened tetrasaccharide, sorbitol, maltitol, maltotriitol, maltotetraitol, and sugar alcohols prepared from other partial starch hydrolyzates and .alpha.-isomaltosylglucosaccharide, are novel compositions. Examples of the form of the isomaltitol and saccharide mixtures with isomaltitol obtained by the present process include various forms of liquids, pastes, syrups, granules, powders, and solids.

The isomaltose, isomaltitol, saccharide mixtures of isomaltose and/or isomaltitol, and crystalline isomaltitol (which all may be generally called "the saccharides of the present invention" hereinafter) produced by the process of the present invention have a high quality and elegant sweetness, and have a feature of that they do not substantially form acids, as a causative of dental caries, by dental caries-inducing microorganisms. Thus, the saccharides of the present invention can be preferably used as sweeteners which do not substantially induce dental caries. Varying to some extent depending on the purity of isomaltose and isomaltitol, the saccharides of the present invention have substantially non- or insubstantial-hygroscopicity, satisfactory free-flowing ability, and desired shelf-life, do not substantially induce the Maillard reaction even in the presence of amino compounds such as amino acids and proteins, do not substantially affect the coexisting ingredients, and do not substantially change color in themselves. The saccharide mixtures and products with crystalline isomaltitol according to the present invention can be advantageously used as a sugar coating for tablet in combination with one or more conventional binders such as pullulan, hydroxyethyl starch, and polyvinylpyrrolidone. The saccharides of the present invention have also useful properties of osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, ability of saccharide-crystallization-preventing ability, substantial non-fermentability, and starch-retrogradation-preventing ability. Thus, the saccharides according to the present invention can be arbitrarily used as a sweetener, taste-improving agent, flavor-improving agent, flavor-retaining agent, quality-improving agent, stabilizer, filler-imparting agent in various compositions such as food products including health foods and health supplements, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

The saccharides according to the present invention can be also used as a seasoning for sweetening various products, and In necessary, they can be used in combination with one or more other sweeteners such as a corn syrup solid, glucose, fructose, lactosucrose, .alpha.,.alpha.-trehalose (alias trehalose), .alpha.,.beta.-trehalose (alias neotrehalose), .beta.,.beta.-trehalose, maltose, sucrose, isomerized sugar, honey, maple sugar. isomaltooligosaccharide, galactooligosaccharide, lactooligosaccharide, fructooligosaccharide, sorbitol, maltitol, lactitol, dihydrochalcone, stevioside, .alpha.-glycosyl stevioside, rebaudioside, glycyrrhizin, L-aspartyl L-phenylalanine methyl ester, sucralose, acesulfame K, saccharin, glycine, and alanine. If necessary, one or more fillers such as dextrins, starches, and lactose can be suitably used in combination.

The saccharides, particularly, those comprising the crystalline isomaltitol powder according to the present invention can be used alone, and optionally they can be used in combination with one or more of appropriate fillers, excipients, binders, sweeteners to make them into different shapes of granules, spheres, short rods, plates, cubes, tablets, films, or sheets.

The saccharides of the present invention have a sweetness that well harmonize with other tastable substances having sour-, acid-, salty-, astringent-, delicious-, and bitter-tastes; and have a satisfactorily high acid- and heat-tolerance. Thus, they can be favorably used to sweeten, improve the taste, or improve the quality of various foods and beverages, for example, amino acids, peptides, soy sauce, powdered soy sauce, miso, "funmatsu-miso" (a powdered miso), "moromi" (a refined sake), "hishio" (a refined soy sauce), "furikake" (a seasoned fish meal), mayonnaise, dressing, vinegar, "sanbai-zu" (a sauce of sugar, soy sauce and vinegar), "funmatsu-sushi-su" (powdered vinegar for sushi), "chuka-no-moto" (an instant mix for Chinese dish), "tentsuyu" (a sauce for Japanese deep-fat fried food), "mentsuyu" (a sauce for Japanese vermicelli), sauce, catsup, "yakiniku-no-tare" (a sauce for Japanese grilled meat), curry roux, instant stew mix, instant soup mix, "dashi-no-moto" (an instant stock mix), nucleotide seasonings, mixed seasoning, "mirin" (a sweet sake), "shin-mirin" (a synthetic mirin), table sugar, and coffee sugar. Also, the saccharide of the present invention can be arbitrarily used in "wagashi" (Japanese cakes) such as "senbei" (a rice cracker), "arare" (a rice cake cube), "okoshi" (a millet-and-rice cake), "mochi" (a rice paste) or the like, "manju" (a bun with a bean-jam), "uiro" (a sweet rice jelly), "an" (a bean jam) or the like, "yokan" (a sweet jelly of beans), "mizu-yokan" (a soft adzuki-bean jelly), "kingyoku" (a kind of yokan), jelly, pao de Castella, and "amedama" (a Japanese toffee); Western confectioneries such as a bun, biscuit, cracker, cookie, pie, pudding, butter cream, custard cream, cream puff, waffle, sponge cake, doughnut, Yorkshire pudding, chocolate, chewing gum, caramel, and candy; frozen desserts such as an ice cream and sherbet; syrups such as a "kajitsu-no-syrup-zuke" (a preserved fruit) and "korimitsu" (a sugar syrup for shaved ice); pastes such as a flour paste, peanut paste, fruit paste, and spread; processed fruits and vegetables such as a jam, marmalade, "syrup-zuke" (fruit pickles), and "toka" (conserves); pickles and pickled products such as a "fukujin-zuke" (red colored radish pickles), "bettara-zuke" (a kind of whole fresh radish pickles), "senmai-zuke" (a kind of sliced fresh radish pickles), and "rakkyo-zuke" (pickled shallots); premixes for pickles and pickled products such as a "takuan-zuke-no-moto" (a premix for pickled radish), and "hakusai-zuke-no-moto" (a premix for fresh white rape pickles); meat products such as a ham and sausage; products of fish meat such as a fish ham, fish sausage, "kamaboko" (a steamed fish paste), "chikuwa" (a kind of fish paste), and "tenpura" (a Japanese deep-fat fried fish paste); "chinmi" (relish) such as a "uni-no-shiokara" (a salted gut of sea urchin), "ika-no-shiokara" (a salted gut of squid), "su-konbu" (processed tangle), "saki-surume" (a dried squid strip), and "fugu-no-mirin-boshi" (a dried mirin-seasoned swellfish); "tsukudani" (foods boiled down in soy sauce) such as those of layer, edible wild plants, dried squid, small fish, and shellfish; daily dishes such as a "nimame" (cooked beans), potato salad, and "konbu-maki" (a tangle roll); milk products such as a yogurt and cheese; canned and bottled products such as those of meat, fish meat, fruit, and vegetables; alcoholic beverages such as sake, distilled spirit, shochu-based beverage, synthetic sake, liqueur, cocktail, and others; soft drinks such as a coffee, tea, cocoa, juice, isotonic beverage, carbonated beverage, sour milk beverage, and beverage containing lactic acid bacteria; instant food products such as an instant pudding mix, instant hot cake mix, "sokuseki-shiruko" (an instant mix of adzuki-bean soup with rice cake), and instant soup mix; and other foods and beverages such as solid foods for babies, foods for therapy, health/tonic drinks, peptide foods, frozen foods, and health foods. The saccharide of the present invention can be arbitrarily used to improve the taste preference of feeds and foods for animals and pets such as domestic animals, poultry, honey bees, silk warms, fishes, crustaceans including shrimps/prawns/lobsters, and crabs. In addition, the saccharides of the present invention can be used as a sweetener for solid products such as a tobacco, cigarette, tooth paste, lipstick/rouge, lip cream, internal liquid medicine, tablet, troche, cod liver oil in the form of a drop, cachou, oral refrigerant, or gargle. Also the saccharides can be used in the above products as a taste-improving agent, flavoring substance, quality-improving agent, stabilizer, or moisture-retaining agent.

The saccharides of the present invention are sugar alcohols which do not cause the Maillard reaction because of their non-reducibility. Therefore, the saccharides have no fear of deteriorating effective ingredients such as amino compounds and can be incorporated as a quality-improving agent and/or stabilizer into health foods and pharmaceuticals, which have effective ingredients, active components, or physiologically active substances, to obtain stabilized, high quality health foods or pharmaceuticals in the form of a liquid, paste, or solid. Examples of the above-identified effective ingredients and biologically active substances are lymphokines such as .alpha.-, .beta.- and .gamma.-interferons, tumor necrosis factor-.alpha. (TNF-.alpha.), tumor necrosis factor-.beta. (TNF-.beta.), macrophage migration inhibitory factor, colony-stimulating factor, transfer factor, and interleukins; hormones such as insulin, growth hormone, prolactin, erythropoietin, and follicle-stimulating hormone; biological preparations such as BCG vaccine, Japanese encephalitis vaccine, measles vaccine, live polio vaccine, smallpox vaccine, tetanus toxoid, Trimeresurus antitoxin, and human immunoglobulin; antibiotics such as penicillin, erythromycin, chloramphenicol, tetracycline, streptomycin, and kanamycin sulfate; vitamins such as thiamine, riboflavin, L-ascorbic acid, .alpha.-glycosyl ascorbic acid, cod liver oil, carotenoid, ergosterol, tocopherol, rutin, .alpha.-glycosyl rutin, naringin, .alpha.-glycosyl naringin, hesperidin, and .alpha.-glycosyl hesperidin; enzymes such as lipase, elastase, urokinase, protease, .beta.-amylase, isoamylase, glucanase, and lactase; extracts such as ginseng extract, bamboo leaf extract, Japanese plum extract, pine leaf extract, snapping turtle extract, chlorella extract, aloe extract, and propolis extract; viable microorganisms such as viruses, lactic acid bacteria, and yeasts; and royal jelly.

The methods for incorporating the saccharides of the present invention into the aforesaid compositions are those which can incorporate the saccharides into the compositions before completion of their processings, and which can be appropriately selected among the following conventional methods; mixing, dissolving, melting, soaking, penetrating, dispersing, applying, coating, spraying, injecting, crystallizing, and solidifying. The amount of the saccharides to be incorporated into each of the above compositions is usually in an amount of at least 0.1%, desirably, at least 1%, and more desirably, 2 to 99.9% by weight of each of the compositions.

The following experiments explain the process for producing isomaltose and isomaltitol according to the present invention:

EXPERIMENT 1

Preparation of Non-Reducing Cyclotetrasaccharide by Culturing

A liquid medium, consisting of 5% (w/v) of "PINE-DEX #1", a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan, 1.5% (w/v) of "ASAHIMEAST.TM.", a yeast extract commercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in a 500-ml Erlenmeyer flask in an amount of 100 ml, sterilized by autoclaving at 121.degree. C. for 20 min, cooled, and then seeded with a stock culture of Bacillus globisporus C9 strain (FERM BP-7143), followed by culturing under rotary-shaking conditions at 27.degree. C. and 230 rpm for 48 hours and centrifuging the resulting culture to remove cells and to obtain a supernatant. The supernatant was autoclaved at 120.degree. C. for 15 min and then cooled, and the resulting insoluble substances were removed by centrifugation to obtain a supernatant.

To examine the saccharides in the supernatant, they were separated by developing twice on silica gel thin-layer chromatography (abbreviated as "TLC" hereinafter) using, as a developer, a mixture solution of n-butanol, pyridine, and water (=6:4:1 by volume), and, as a thin-layer plate, "KIESELGEL.TM. 60", an aluminum plate (20.times.20 cm) for TLC commercialized by Merck & Co., Inc., Rahway, USA. The coloration of the separated total sugars by the sulfuric acid-methanol method and that of the reducing saccharides by the diphenylamine-aniline method detected a non-reducing saccharide positive at an Rf value of about 0.31 on the former detection method but negative on the latter detection method.

About 90 ml of the supernatant obtained in the above was adjusted to pH 5.0 and heated to 45.degree. C. and then incubated for 24 hours after admixed with 1,500 units per gram of solids of "TRANSGLUCOSIDASE L AMANO.TM.", an .alpha.-glucosidase specimen commercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan; and 75 units per gram of solids of a glucoamylase commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan. Thereafter, the resulting culture was adjusted to pH 12 by the addition of sodium hydroxide and boiled for two hours to decompose the remaining reducing sugars. After removing insoluble substances by filtration, the resulting solution was decolored and desalted with "DIAION PK218.TM." and "DIAION WA30.TM.", cation exchange resins commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan; and further desalted with "DIAION SK-1B.TM.", commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan, and "AMBERLITE IRA411.TM.", an anion exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan. The resulting solution was decolored with an activated charcoal, membrane filtered, concentrated by an evaporator, and lyophilized in vacuo to obtain about 0.6 g, d.s.b., of a saccharide powder. The analysis of the saccharide powder on high-performance liquid chromatography (abbreviated as "HPLC" hereinafter) detected a single peak at an elution time of 10.84 min as shown in FIG. 1, and revealed that it had a purity of as high as 99.9% or higher. The above HPLC was run using "SHOWDEX KS-801.TM. column", Showa Denko K.K., Tokyo, Japan, at a column temperature of 60.degree. C. and a flow rate of 0.5 ml/min of water, and "RI-8012", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. When measured for reducing power of the saccharide on the Somogyi-Nelson's method, the reducing power was below a detectable level, meaning that the saccharide was substantially a non-reducing saccharide.

EXPERIMENT 2

Structure Analysis of Non-reducing Saccharide

Fast atom bombardment mass spectrometry (called "FAB-MS") of a non-reducing saccharide, obtained by the method in Experiment 1, significantly detected a proton-addition-molecular ion with a mass number of 649, meaning that the saccharide had a mass number of 648. According to conventional manner, the saccharide was hydrolyzed with sulfuric acid and then analyzed for sugar composition on gas chromatography. As a result, D-glucose was detected only, revealing that the saccharide was composed of D-glucose molecules or a cyclotetrasaccharide composed of four D-glucose molecules based on the above mass number. Nuclear magnetic resonance analysis (called "NMR") of the saccharide gave a .sup.1H-NMR spectrum as shown in FIG. 2 and a .sup.13C-NMR spectrum as shown in FIG. 3, and these spectra were compared with those of conventional saccharides, revealing that the spectra were coincided with those of a non-reducing cyclic saccharide, cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.} disclosed in "European Journal of Biochemistry", pp. 641-648 (1994). The data confirmed that the saccharide obtained in this experiment is a cyclotetrasaccharide as shown in FIG. 4, i.e., cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.}.

EXPERIMENT 3

Production of .alpha.-isomaltosylglucosaccharide-forming Enzyme from Bacillus globisporus C9 Strain

A liquid culture medium, consisting of 4.0% (w/v) of "PINE-DEX #4.TM.", a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan, 1.8% (w/v) of "ASAHIMEAST.TM.", a yeast extract commercialized by Asahi Breweries, Ltd., Tokyo, Japan, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in an amount of 100 ml each, sterilized by autoclaving at 121.degree. C. for 20 min, cooled, and then seeded with a stock culture of Bacillus globisporus C9 strain (FERM BP-7143), followed by culturing under rotary-shaking conditions at 27.degree. C. and 230 rpm for 48 hours for a seed culture. About 20 L of a fresh preparation of the same liquid culture medium as used in the above seed culture were placed in a 30-L fermentor, sterilized by heating, and then cooled to 27.degree. C. and inoculated with 1% (v/v) of the seed culture, followed by culturing at 27.degree. C. and pH 6.0 to 8.0 for 48 hours under aeration-agitation conditions. After completion of the culture, the resulting culture, which had about 0.45 unit/ml of .alpha.-isomaltosylglucosaccharide-forming enzyme, about 1.5 units/ml of .alpha.-isomaltosyl-transferring enzyme, and about 0.95 unit/ml of a cyclotetrasaccharide-forming activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant. When measured for enzymatic activity, the supernatant contained about 0.45 unit/ml of .alpha.-isomaltosylglucosaccharide-forming enzyme, i.e., a total enzymatic activity of about 8,110 units; about 1.5 units/ml of .alpha.-isomaltosyl-transferring enzyme, i.e., a total enzymatic activity of about 26,900 units; and about 0.95 unit/ml of cyclotetrasaccharide-forming enzyme, i.e., a total enzymatic activity of about 17,100 units. These activities were assayed as follows: The activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was assayed by dissolving maltotriose in 100 mM acetate buffer (pH 6.0) to give a concentration of 2% (w/v) for a substrate solution, adding a 0.5 ml of an enzyme solution to a 0.5 ml of the substrate solution, enzymatically reacting the mixture solution at 35.degree. C. for 60 min, suspending the enzymatic reaction by boiling the solution for 10 min, and quantifying maltose, among the isomaltosyl maltose and maltose formed mainly in the reaction mixture, on HPLC disclosed in Experiment 1. One unit activity of .alpha.-isomaltosylglucosaccharide-forming enzyme is defined as the enzyme amount that forms one micromole of maltose per minute under the above enzymatic reaction conditions. Throughout the specification, the enzymatic activity of .alpha.-isomaltosylglucosaccharide-forming enzyme means the unit(s) assayed as above.

The activity of .alpha.-isomaltosyl-transferring enzyme was assayed by dissolving panose in 100 mM acetate buffer (pH 6.0) to give a concentration of 2% (w/v) for a substrate solution, adding a 0.5 ml of an enzyme solution to 0.5 ml of the substrate solution, enzymatically reacting the mixture solution at 35.degree. C. for 30 min, suspending the enzymatic reaction by boiling the solution for 10 min, and quantifying glucose, among the cyclotetrasaccharide and glucose formed mainly in the reaction mixture, by the glucose oxidase method. One unit activity of .alpha.-isomaltosyl-transferring enzyme is defined as the enzyme amount that forms one micromole of glucose per minute under the above enzymatic reaction conditions. Throughout the specification, the enzymatic activity of .alpha.-isomaltosyl-transferring enzyme means the unit(s) assayed as above.

The cyclotetrasaccharide-forming activity was assayed by dissolving "PINE-DEX #100.TM.", a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan, in 50 mM acetate buffer (pH 6.0) to give a concentration of 2% (w/v) for a substrate solution, adding 0.5 ml of an enzyme solution to 0.5 ml of the substrate solution, enzymatically reacting the mixture solution at 35.degree. C. for 60 min, suspending the enzymatic reaction by heating the solution at 100.degree. C. for 10 min, and then further adding to the resulting solution one milliliter of 50 mM acetate buffer (pH 5.0) with 70 units/ml of "TRANSGLUCOSIDASE L AMANO.TM.", an .alpha.-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and 27 units/ml of glucoamylase, commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, followed by incubating the mixture at 50.degree. C. for 60 min, inactivating the remaining enzymes by heating at 100.degree. C. for 10 min, and quantifying cyclotetrasaccharide on HPLC described in Experiment 1. One unit activity of cyclotetrasaccharide-forming enzyme is defined as the enzyme amount that forms one micromole of cyclotetrasaccharide per minute under the above enzymatic reaction conditions. Throughout the specification, the activity of cyclotetrasaccharide-forming enzyme means the unit(s) assayed as above.

EXPERIMENT 4

Preparation of Enzyme from Bacillus globisporus C9 Strain

EXPERIMENT 4-1

About 18 L of the supernatant in Experiment 3 were salted out in 80% saturated ammonium sulfate and allowed to stand at 4.degree. C. for 24 hours, and the formed sediments were collected by centrifugation at 10,000 rpm for 30 min, dissolved in 10 mM phosphate buffer (pH 7.5), and dialyzed against a fresh preparation of the same buffer to obtain about 400 ml of a crude enzyme solution with 8,110 units of an .alpha.-isomaltosylglucosaccharide-forming activity, 24,700 units of an .alpha.-isomaltosyl-transferring activity, and about 15,600 units of a cyclotetrasaccharide-forming activity. The crude enzyme solution was subjected to ion-exchange chromatography using 1,000 ml of "SEPABEADS FP-DA13 .TM." gel, an ion-exchange resin commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan. The .alpha.-isomaltosylglucosaccharide-forming enzyme and cyclotetrasaccharide were eluted as non-adsorbed fractions without adsorbing on the ion-exchange resin. The resulting enzyme solution was dialyzed against 10 mM phosphate buffer (pH 7.0) with 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities, and subjected to affinity chromatography using 500 ml of "SEPHACRYL HR S-200.TM.", a gel commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA. Enzymatically active components adsorbed on the gel and, when sequentially eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to 100 mM of maltotetraose, the .alpha.-isomaltosyl-transferring enzyme and the .alpha.-isomaltosylglucosaccharide-forming enzyme were separately eluted, i.e., the former was eluted with the linear gradient of ammonium sulfate at about 0 M and the latter was eluted with the linear gradient of maltotetraose at about 30 mM. Thus, fractions with an .alpha.-isomaltosyl-transferring activity and those with an .alpha.-isomaltosylglucosaccharide-forming activity were separatory collected. No cyclotetrasaccharide-forming activity was found in any of the above fractions but found in their mixture solution, and the fact revealed that the activity of forming cyclotetrasaccharide from partial starch hydrolyzates was exerted by the coaction of the activities of the above two types of enzymes.

Methods for separately purifying .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme are described below:

EXPERIMENT 4-2

Purification of .alpha.-isomaltosylglucosaccharide-forming Enzyme

Factions of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained in Experiment 4-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The dialyzed solution was centrifuged to remove insoluble impurities, and the resulting supernatant was fed to hydrophobic chromatography using 350 ml of "BUTYL-TOYOPEARL 650 M.TM.", a gel for hydrophobic chromatography commercialized by Tosoh Corporation, Tokyo, Japan. The enzyme was adsorbed on the gel and eluted at about 0.3 M ammonium sulfate when eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, followed by collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using "SEPHACRYL HR S-200.TM." gel to purify the enzyme. The amount of enzyme activity, specific activity, and yield of .alpha.-isomaltosylglucosaccharide-forming enzyme in each purification step are in Table 1.

TABLE-US-00001 TABLE 1 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 8,110 0.12 100 Dialyzed solution after 7,450 0.56 91.9 salting out with ammonium sulfate Eluate from ion-exchange 5,850 1.03 72.1 column chromatography Eluate from affinity 4,040 8.72 49.8 column chromatography Eluate from hydrophobic 3,070 10.6 37.8 column chromatography Eluate from affinity 1,870 13.6 23.1 column chromatography Note: The symbol "*" means .alpha.-isomaltosylglucosaccharide-forming enzyme.

The finally purified .alpha.-isomaltosylglucosaccharide-forming enzyme specimen was examined for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, i.e., a high purity enzyme specimen.

EXPERIMENT 4-3

Purification of .alpha.-isomaltosyl-transferring Enzyme

Fractions with .alpha.-isomaltosyl-transferring enzyme, which had been separated from the fractions with .alpha.-isomaltosylglucosaccharide-forming enzyme by affinity chromatography in Experiment 4-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzed solution was centrifuged to remove insoluble impurities and subjected to affinity chromatography using 350 ml of "BUTYL-TOYOPEARL 650M", a gel for hydrophobic chromatography commercialized by Tosoh Corporation, Tokyo, Japan, to purify the enzyme. The enzyme was adsorbed on the gel and eluted therefrom at a concentration of about 0.3 M ammonium sulfate when eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, followed by collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using "SEPHACRYL HR S-200" gel to purify the enzyme. The amount of enzyme activity, specific activity, and yield of .alpha.-isomaltosyl-transferring enzyme in each purification step are in Table 2.

TABLE-US-00002 TABLE 2 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 26,900 0.41 100 Dialyzed solution after 24,700 1.85 91.8 salting out with ammonium sulfate Eluate from ion-exchange 19,400 3.41 72.1 column chromatography Eluate from affinity 13,400 18.6 49.8 column chromatography Eluate from hydrophobic 10,000 21.3 37.2 column chromatography Eluate from affinity 6,460 26.9 24.0 column chromatography Note: The symbol "*" means the .alpha.-isomaltosyl-transferring enzyme.

EXPERIMENT 5

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 5-1

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 4-2, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 140,000.+-.20,000 daltons.

A fresh preparation of the above purified specimen was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 5.2.+-.0.5. The influence of temperature and pH on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in accordance with the assay for its enzyme activity, where the influence of temperature was examined in the presence or the absence of 1 mM Ca.sup.2+. These results are in FIG. 5 (influence of temperature) and FIG. 6 (influence of pH). The optimum temperature of the enzyme was about 40.degree. C. (in the absence of Ca.sup.2+) and about 45.degree. C. (in the presence of 1 mM Ca.sup.2+) when incubated at pH 6.0 for 60 min, and the optimum pH of the enzyme was about 6.0 to about 6.5 when incubated at 35.degree. C. for 60 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min in the presence or the absence of 1 mM Ca.sup.2+, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzymes was determined by keeping the testing enzyme solutions in the form of an appropriate 50 mM buffer having a prescribed pH at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 7 (thermal stability) and FIG. 8 (pH stability). As a result, the enzyme had thermal stability of up to about 35.degree. C. in the absence of Ca.sup.2+ and about 40.degree. C. in the presence of 1 mM Ca.sup.2+, and pH stability of about 4.5 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 3.

TABLE-US-00003 TABLE 3 Metal Relative activity Metal Relative activity ion (%) ion (%) None 100 Hg.sup.2+ 4 Zn.sup.2+ 92 Ba.sup.2+ 65 Mg.sup.2+ 100 Sr.sup.2+ 80 Ca.sup.2+ 115 Pb.sup.2+ 103 Co.sup.2+ 100 Fe.sup.2+ 98 Cu.sup.2+ 15 Fe.sup.3+ 97 Ni.sup.2+ 98 Mn.sup.2+ 111 Al.sup.3+ 99 EDTA 20

As evident form the results in Table 3, the enzyme activity was strongly inhibited by Hg.sup.2+, Cu.sup.2+, and EDTA, and it was also inhibited by Ba.sup.2+ and Sr.sup.2+. It was also found that the enzyme was activated by Ca.sup.2+ and Mn.sup.2+.

Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:1, i.e., tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleuci- ne in the N-terminal region.

EXPERIMENT 5-2

Property of .alpha.-isomaltosyl-transferring Enzyme

A purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 4-3, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 112,000.+-.20,000 daltons.

A fresh preparation of the above purified specimen was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 5.5.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in accordance with the assay for its enzyme activity. These results are in FIG. 9 (influence of temperature) and FIG. 10 (influence of pH). The optimum temperature of the enzyme was about 45.degree. C. when incubated at pH 6.0 for 30 min, and the optimum pH of the enzyme was about 6.0 when incubated at 35.degree. C. for 30 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the form of an appropriate 50 mM buffer having a prescribed pH at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 11 (thermal stability) and FIG. 12 (pH stability). As a result, the enzyme had thermal stability of up to about 40.degree. C. and pH stability of about 4.0 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 4.

TABLE-US-00004 TABLE 4 Relative activity Metal Relative activity Metal ion (%) ion (%) None 100 Hg.sup.2+ 1 Zn.sup.2+ 88 Ba.sup.2+ 102 Mg.sup.2+ 98 Sr.sup.2+ 101 Ca.sup.2+ 101 Pb.sup.2+ 89 Co.sup.2+ 103 Fe.sup.2+ 96 Cu.sup.2+ 57 Fe.sup.3+ 105 Ni.sup.2+ 102 Mn.sup.2+ 106 Al.sup.3+ 103 EDTA 104

As evident form the results in Table 4, the enzyme activity was strongly inhibited by Hg.sup.2+, and it was also inhibited by Cu.sup.2+. It was also found that the enzyme was not activated by Ca.sup.2+ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ. ID NO:2, i.e, isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline-asparagine-glycine in the N-terminal region.

EXPERIMENT 6

Production of .alpha.-isomaltosylglucosaccharide-forming Enzyme from Bacillus globisporus C11 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of "PINE-DEX #4", a partial starch hydrolyzate, 1.8% (w/v) of "ASAHIMEAST", a yeast extract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each, autoclaved at 121.degree. C. for 20 minutes to effect sterilization, cooled, inoculated with a stock culture of Bacillus globisporus C11 strain (FERM BP-7144), and incubated at 27.degree. C. for 48 hours under rotary shaking conditions of 230 rpm. The resulting cultures were pooled and used as a seed culture. About 20 L of a fresh preparation of the same nutrient culture medium as used in the above culture were placed in a 30-L fermentor, sterilized by heating, cooled to 27.degree. C., inoculated with 1% (v/v) of the seed culture, and incubated for about 48 hours while stirring under aeration-agitation conditions at 27.degree. C. and a pH of 6.0 to 8.0. The resultant culture, having about 0.55 unit/ml of an .alpha.-isomaltosylglucosaccharide-forming activity, about 1.8 units/ml of an .alpha.-isomaltosyl-transferring activity, and about 1.1 units/ml of a cyclotetrasaccharide-forming activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant. Measurement of the supernatant revealed that it had about 0.51 unit/ml of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, i.e., a total enzyme activity of about 9,180 units; about 1.7 units/ml of an .alpha.-isomaltosyl-transferring enzyme activity, i.e., a total enzyme activity of about 30,400 units; and about 1.1 units/ml of a cyclotetrasaccharide-forming enzyme activity, i.e., a total enzyme activity of about 19,400 units.

EXPERIMENT 7

Preparation of enzyme from Bacillus globisporus C11 Strain

EXPERIMENT 7-1

Purification of Enzyme from Bacillus globisporus C11 Strain

Eighteen litters of the supernatant, obtained in Experiment 6, were salted out in an 80% saturated ammonium sulfate solution and allowed to stand at 4.degree. C. for 24 hours. Then, the salted out sediments were collected by centrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer (pH 7.5), dialyzed against a fresh preparation of the same buffer as used in the above to obtain about 416 ml of a crude enzyme solution. The crude enzyme solution was revealed to have 8,440 units of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, about 28,000 units of an .alpha.-isomaltosyl-transferring enzyme activity, and about 17,700 units of a cyclotetrasaccharide-forming enzyme activity. When subjected to ion-exchange chromatography using "SEPABEADS FP-DA13" gel, disclosed in Experiment 4-1, the above three types of enzymes were eluted as non-adsorbed fractions without adsorbing on the gel. The non-adsorbed fractions with those enzymes were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to affinity chromatography using 500 ml of "SEPHACRYL HR S-200" gel to purify the enzyme. Active enzymes were adsorbed on the gel and sequentially eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate and a linear gradient increasing from 0 mM to 100 mM of maltotetraose, followed by collecting separate elutions of .alpha.-isomaltosyl-transferring enzyme and .alpha.-isomaltosylglucosaccharide-forming enzyme, respectively, where the former enzyme was eluted with the linear gradient of ammonium sulfate at a concentration of about 0.3 M and the latter enzyme was eluted with a linear gradient of maltotetraose at a concentration of about 30 mM. Therefore, fractions with the .alpha.-isomaltosylglucosaccharide-forming enzyme and those with the .alpha.-isomaltosyl-transferring enzyme were separately collected. Similarly as in the case of Bacillus globisporus C9 strain in Experiment 4, it was found that no cyclotetrasaccharide-forming activity was found in any fraction in this column chromatography, and that an enzyme mixture solution of both fractions of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme showed a cyclotetrasaccharide-forming enzyme activity, revealing that the activity of forming cyclotetrasaccharide from partial starch hydrolyzates was exerted in collaboration with the enzyme activities of the two types of enzymes.

Methods for separately purifying .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme are explained below:

EXPERIMENT 7-2

Purification of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A faction of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained in Experiment 7-1, was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The dialyzed solution was centrifuged to remove insoluble impurities, and the resulting supernatant was fed to hydrophobic chromatography using 350 ml of "BUTYL-TOYOPEARL 650 M", a gel commercialized by Tosoh Corporation, Tokyo, Japan. The enzyme adsorbed on the gel was eluted at about 0.3 M ammonium sulfate when eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, followed by collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using "SEPHACRYL HR S-200" gel to purify the enzyme. The amount of enzyme activity, specific activity, and yield of the .alpha.-isomaltosylglucosaccharide-forming enzyme in each purification step are in Table 5.

TABLE-US-00005 TABLE 5 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 9,180 0.14 100 Dialyzed solution after 8,440 0.60 91.9 salting out with ammonium sulfate Eluate from ion-exchange 6,620 1.08 72.1 column chromatography Eluate from affinity 4,130 8.83 45.0 column chromatography Eluate from hydrophobic 3,310 11.0 36.1 column chromatography Eluate from affinity 2,000 13.4 21.8 column chromatography Note: The symbol "*" means .alpha.-isomaltosylglucosaccharide-forming enzyme.

The finally purified .alpha.-isomaltosylglucosaccharide-forming enzyme specimen was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 7-3

Purification of .alpha.-isomaltosyl-transferring Enzyme

A faction of .alpha.-isomaltosyl-transferring enzyme, which had been separated from a fraction of .alpha.-isomaltosylglucosaccharide-forming enzyme by the affinity chromatography in Experiment 7-1, was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The dialyzed solution was centrifuged to remove insoluble impurities, and the resulting supernatant was fed to hydrophobic chromatography using 350 ml of "BUTYL-TOYOPEARL 650 M", a gel commercialized by Tosoh Corporation, Tokyo, Japan. The enzyme adsorbed on the gel and then it was eluted at about 0.3 M ammonium sulfate when eluted with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, followed by collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate. The resulting dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using "SEPHACRYL HR S-200" gel to purify the enzyme. The amount of enzyme activity, specific activity, and yield of the .alpha.-isomaltosyl-transferring enzyme in each purification step are in Table 6.

TABLE-US-00006 TABLE 6 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 30,400 0.45 100 Dialyzed solution after 28,000 1.98 92.1 salting out with ammonium sulfate Eluate from ion-exchange 21,800 3.56 71.7 column chromatography Eluate from affinity 13,700 21.9 45.1 column chromatography Eluate from hydrophobic 10,300 23.4 33.9 column chromatography Eluate from affinity 5,510 29.6 18.1 column chromatography Note: The symbol "*" means .alpha.-isomaltosyl-transferring enzyme.

EXPERIMENT 8

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 8-1

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 7-2, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 137,000.+-.20,000 daltons.

A fresh preparation of the same purified specimen as used in the above was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 5.2.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in accordance with the assay for its enzyme activity, where the influence of temperature was examined in the presence or the absence of 1 mM Ca.sup.2+. These results are in FIG. 13 (influence of temperature) and FIG. 14 (influence of pH). The optimum temperature of the enzyme was about 45.degree. C. in the absence of Ca.sup.2+ and about 50.degree. C. in the presence of 1 mM Ca.sup.2+ when incubated at pH 6.0 for 60 min. The optimum pH of the enzyme was about 6.0 when incubated at 35.degree. C. for 60 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) in the presence or the absence of 1 mM Ca.sup.2+ at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the from of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 15 (thermal stability) and FIG. 16 (pH stability). As a result, the enzyme had thermal stability of up to about 40.degree. C. in the absence of Ca.sup.2+ and up to about 45.degree. C. in the presence of 1 mM Ca.sup.2+. The pH stability of enzyme was about 5.0 to about 10.0.

The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 7.

TABLE-US-00007 TABLE 7 Relative activity Metal Relative activity Metal ion (%) ion (%) None 100 Hg.sup.2+ 4 Zn.sup.2+ 91 Ba.sup.2+ 65 Mg.sup.2+ 98 Sr.sup.2+ 83 Ca.sup.2+ 109 Pb.sup.2+ 101 Co.sup.2+ 96 Fe.sup.2+ 100 Cu.sup.2+ 23 Fe.sup.3+ 102 Ni.sup.2+ 93 Mn.sup.2+ 142 Al.sup.3+ 100 EDTA 24

As evident form the results in Table 7, the enzyme activity was strongly inhibited by Hg.sup.2+, Cu.sup.2+, and EDTA, and it was also inhibited by Ba.sup.2+ and Sr.sup.2+. It was also found that the enzyme was activated by Ca.sup.2+ and Mn.sup.2+. Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:1, i.e, tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleuci- ne in the N-terminal region. Comparison of the partial amino acid sequence in the N-terminal region with that derived from the .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9 strain in Experiment 5-1 revealed that they were the same and the consensus N-terminal amino acid sequence, commonly found in these .alpha.-isomaltosylglucosaccharide-forming enzymes, was an amino acid sequence of tyrosine-valine-serine-serine-leucine-glycine-asparagine-leucine-isoleuci- ne of SEQ ID NO:1 in the N-terminal region. Detailed method for assaying amino acid sequence is not shown in this specification because it is disclosed in detail in Japanese Patent Application No. 2001-519,441 (International Publication No. WO 02/055708), however, the .alpha.-isomaltosylglucosaccharide-forming enzyme has an amino acid sequence of 36-1284 amino acid residues shown in parallel in SEQ ID NO:21 similarly as that for the polypeptide, disclosed in the specification of the above-identified Japanese Patent Application No. 2001-5441.

EXPERIMENT 8-2

Property of .alpha.-isomaltosyl-transferring Enzyme

A purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 7-3, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 102,000.+-.20,000 daltons.

A fresh preparation of the same purified specimen as used in the above was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 5.6.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in accordance with the assay for its enzyme activity. These results are in FIG. 17 (influence of temperature) and FIG. 18 (influence of pH). The optimum temperature of the enzyme was about 50.degree. C. when incubated at pH 6.0 for 30 min. The optimum pH of the enzyme was about 5.5 to about 6.0 when incubated at 35.degree. C. for 30 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the form of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 19 (thermal stability) and FIG. 20 (pH stability). As a result, the enzyme had thermal stability of up to about 40.degree. C. and pH stability of about 4.5 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 8.

TABLE-US-00008 TABLE 8 Relative activity Metal Relative activity Metal ion (%) ion (%) None 100 Hg.sup.2+ 2 Zn.sup.2+ 83 Ba.sup.2+ 90 Mg.sup.2+ 91 Sr.sup.2+ 93 Ca.sup.2+ 91 Pb.sup.2+ 74 Co.sup.2+ 89 Fe.sup.2+ 104 Cu.sup.2+ 56 Fe.sup.3+ 88 Ni.sup.2+ 89 Mn.sup.2+ 93 Al.sup.3+ 89 EDTA 98

As evident form the results in Table 8, the enzyme activity was strongly inhibited by Hg.sup.2+, and it was also inhibited by Cu.sup.2+. It was also found that the enzyme was not activated by Ca.sup.2+ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:3, i.e., isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycine in the N-terminal region. Comparison of the partial amino acid sequence in the N-terminal region with that derived from the .alpha.-isomaltosyl-transferring enzyme from Bacillus globisporus C9 strain in Experiment 5-2 revealed that they had a consensus amino acid sequence of isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQ ID NO:4 in their N-terminal regions. Detailed method for assaying amino acid sequence is not shown in this specification because it is disclosed in detail in Japanese Patent Application No. 2000-350142 (International Publication No. WO 02/40659), however, the .alpha.-isomaltosyl-transforming enzyme has an amino acid sequence of amino acid residues 30-1093 shown in parallel in SEQ ID NO:22 similarly as that disclosed in the specification of the above-identified Japanese Patent Application No. 2000-350142.

EXPERIMENT 9

Amino Acid Sequence of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 9-1

Internal Partial Amino Acid Sequence of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A part of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 7-2, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with a fresh preparation of the same buffer as used in the above to give a concentration of about one milligram per milliliter. One milliliter of the dilute as a test sample was admixed with 10 .mu.g of trypsin commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and incubated at 30.degree. C. for 22 hours to hydrolyze the enzyme into peptides. To isolate the peptides, the resulting hydrolyzates were subjected to reverse-phase HPLC using ".mu.-Bondapak C18 column" with a diameter of 2.1 mm and a length of 150 mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and at ambient temperature, and using a liner gradient of acetonitrile increasing from 8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. The peptides eluted from the column were detected by monitoring the absorbance at a wavelength of 210 nm. Three peptide specimens named P64 with a retention time of about 64 min, P88 with a retention time of about 88 min, and P99 with a retention time of about 99 min, which had been well separated from other peptides, were separately collected and dried in vacuo and then dissolved in 200 .mu.l of a solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen was subjected to a protein sequencer for analyzing amino acid sequence up to eight amino acid residues to obtain amino acid sequences of SEQ ID NOs:5 to 7. The analyzed internal partial amino acid sequences are in Table 9.

TABLE-US-00009 TABLE 9 Peptide name Internal partial amino acid sequence P64 aspartic acid-alanine-serine-alanine- asparagine-valine-threonine-threonine P88 tryptophane-serine-leucine-glycine- phenylalanine-methionine-asparagine- phenylalanine P99 asparagine-tyrosine-threonine-aspartic acid- alanine-tryptophane-methionine-phenylalanine

EXPERIMENT 9-2

Internal Partial Amino Acid Sequence of .alpha.-isomaltosyl-transferring Enzyme

A part of a purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 7-3, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with a fresh preparation of the same buffer as used in the above to give a concentration of about one milligram per milliliter. One milliliter of the dilute as a test sample was admixed with 10 .mu.g of "Lysyl Endopeptidase" commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to react at 30.degree. C. for 22 hours to form peptides. The resultant mixture was subjected to reverse-phase HPLC to separate the peptides using ".mu.-Bondapak C18 column" having a diameter of 2.1 mm and a length of 150 mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and at ambient temperature, and using a liner gradient of acetonitrile increasing from 8% (v/v) to 40% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. The peptides eluted from the column were detected by monitoring the absorbance at a wavelength of 210 nm. Three peptide specimens named P22 with a retention time of about 22 min, P63 with a retention time of about 63 min, and P71 with a retention time of about 71 min, which had been well separated from other peptides, were separately collected and dried in vacuo and then dissolved in 200 .mu.l of a solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen was subjected to a protein sequencer for analyzing amino acid sequence up to eight amino acid residues to obtain amino acid sequences of SEQ ID NOs:8 to 10. The analyzed internal partial amino acid sequences are in Table 10.

TABLE-US-00010 TABLE 10 Peptide name Internal partial amino acid sequence P22 glycine-asparagine-glutamic acid-methionine- arginine-asparagine-glutamine-tyrosine P63 isoleucine-threonine-threonine-tryptophane- proline-isoleucine-glutamic acid-serine P71 tryptophane-alanine-phenylalanine-glycine- leucine-tryptophane-methionine-serine

EXPERIMENT 10

Production of .alpha.-isomaltosylglucosaccharide-forming Enzyme from Bacillus globisporus N75 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of "PINE-DEX #4", a partial starch hydrolyzate, 1.8% (w/v) of "ASAHIMEAST", a yeast extract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each, autoclaved at 121.degree. C. for 20 minutes to effect sterilization, cooled, inoculated with a stock culture of Bacillus globisporus N75 strain (FERM BP-7591), and incubated at 27.degree. C. for 48 hours under rotary shaking conditions of 230 rpm for use as a seed culture. About 20 L of a fresh preparation of the same nutrient culture medium as used in the above culture were placed in a 30-L fermentor, sterilized by heating, cooled to 27.degree. C., inoculated with 1% (v/v) of the seed culture, and incubated for about 48 hours while stirring under aeration-agitation conditions at 27.degree. C. and pH 6.0 to 8.0. The resultant culture, having about 0.34 unit/ml of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, about 1.1 units/ml of an .alpha.-isomaltosyl-transferring enzyme activity, and about 0.69 unit/ml of a cyclotetrasaccharide-forming enzyme activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant. Assay for enzyme activity of the supernatant revealed that it had about 0.33 unit/ml of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, i.e., a total enzyme activity of about 5,940 units; about 1.1 units/ml of an .alpha.-isomaltosyl-transferring enzyme activity, i.e., a total enzyme activity of about 19,800 units; and about 0.67 unit/ml of a cyclotetrasaccharide-forming enzyme activity, i.e., a total enzyme activity of about 12,100 units.

EXPERIMENT 11

Preparation of Enzyme from Bacillus globisporus N75 Strain

About 18 L of the supernatant obtained in Experiment 10 was salted out in a 60% saturated ammonium sulfate solution and allowed to stand at 4.degree. C. for 24 hours. Then, the salted out sediments were collected by centrifugation at 10,000 for 30 min, dissolved in 10 mM Tris-HCl buffer (pH 8.3), and dialyzed against a fresh preparation of the same buffer as used in the above to obtain about 450 ml of a crude enzyme solution, revealing to have 4,710 units of .alpha.-isomaltosylglucosaccharide-forming enzyme, about 15,700 units of .alpha.-isomaltosyl-transferring enzyme, and about 9,590 units of cyclotetrasaccharide-forming enzyme. The crude enzyme solution was subjected to ion-exchange chromatography using "SEPABEADS FP-DA13" gel, disclosed in Experiment 4-1. The enzyme was adsorbed on the gel, while .alpha.-isomaltosyl-transferring enzyme was eluted as a non-adsorbed fraction without adsorbing on the gel. When eluted with a linear gradient increasing from 0 M to 1 M NaCl, .alpha.-isomaltosylglucosaccharide-forming enzyme was eluted at a concentration of about 0.25 M NaCl. Under these conditions, fractions with an .alpha.-isomaltosylglucosaccharide-forming enzyme activity and those with an .alpha.-isomaltosyl-transferring enzyme were separately fractionated and collected. Similarly as in the case of Bacillus globisporus C9 strain in Experiment 4 and Bacillus globisporus C11 strain in Experiment 7, it was revealed that no cyclotetrasaccharide-forming activity was found in any of the above fractions collected separately in this column chromatography, and an enzyme solution, obtained by mixing both fractions of .alpha.-isomaltosylglucosaccharide-forming enzyme and of .alpha.-isomaltosyl-transferring enzyme, showed a cyclotetrasaccharide-forming activity, and these facts revealed that the activity of forming cyclotetrasaccharide from partial starch hydrolyzates is exerted by the coaction of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme.

The following experiments are methods for separately purifying .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme:

EXPERIMENT 11-2

Purification of .alpha.-isomaltosylglucosaccharide-forming Enzyme

Fractions with .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained in Experiment 11-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using 500 ml of "SEPHACRYL HR S-200" gel. The enzyme was adsorbed on the gel and then eluted therefrom sequentially with a linear gradient decreasing from 1 M to 0 M ammonium sulfate and with a linear gradient increasing from 0 mM to 100 mM maltotetraose. As a result, the .alpha.-isomaltosylglucosaccharide-forming enzyme adsorbed on the gel was eluted therefrom at a concentration of about 30 mM maltotetraose, followed by collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to hydrophobic chromatography using 350 ml of "BUTYL-TOYOPEARL 650M", a gel commercialized by Tosoh Corporation, Tokyo, Japan. The enzyme was adsorbed on the gel and then eluted with a linear gradient decreasing from 1 M to 0 M ammonium sulfate, resulting in an elution of the enzyme from the gel at a concentration of about 0.3 M ammonium sulfate and collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities and purified on affinity chromatography using 350 ml of "SEPHACRYL HR S-200" gel. The amount of enzyme activity, specific activity, and yield of the .alpha.-isomaltosylglucosaccharide-forming enzyme in each purification step are in Table 11.

TABLE-US-00011 TABLE 11 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 5,940 0.10 100 Dialyzed solution after 4,710 0.19 79.3 salting out with ammonium sulfate Eluate from ion-exchange 3,200 2.12 53.9 column chromatography Eluate from affinity 2,210 7.55 37.2 column chromatography Eluate from hydrophobic 1,720 10.1 29.0 column chromatography Eluate from affinity 1,320 12.5 22.2 column chromatography Note: The symbol "*" means .alpha.-isomaltosylglucosaccharide-forming enzyme.

The finally purified .alpha.-isomaltosylglucosaccharide-forming enzyme specimen was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 11-3

Purification of .alpha.-isomaltosyl-transferring Enzyme

Fractions of .alpha.-isomaltosyl-transferring enzyme, which had been separated from fractions of .alpha.-isomaltosylglucosaccharide-forming enzyme by ion-exchange chromatography in Experiment 11-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to affinity column chromatography using 500 ml of "SEPHACRYL HR S-200", a gel commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA. The enzyme was adsorbed on the gel and then eluted therefrom with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, resulting in an elution of the enzyme from the gel at a concentration of about 0.3 M ammonium sulfate and collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities and purified on hydrophobic chromatography using 380 ml of "BUTYL-TOYOPEARL 650M" gel. The enzyme was adsorbed on the gel and then eluted therefrom with a linear gradient decreasing from 1 M to 0 M ammonium sulfate, resulting in an elution of the enzyme at a concentration of about 0.3 M ammonium sulfate. The fractions with the enzyme activity were pooled and dialyzed against 10 mM Tris-HCl buffer (pH 8.0), and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to ion-exchange column chromatography using 380 ml of "SUPER Q-TOYOPEARL 650C" gel commercialized by Tosoh Corporation, Tokyo, Japan. The enzyme was not adsorbed on the gel and then eluted therefrom as non-adsorbed fractions which were then collected and pooled to obtain a finally purified enzyme preparation. The amount of enzyme activity, specific activity, and yield of the .alpha.-isomaltosylglucosaccharide-forming enzyme in each purification step are in Table 12.

TABLE-US-00012 TABLE 12 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 19,000 0.33 100 Dialyzed solution after 15,700 0.64 82.6 salting out with ammonium sulfate Eluate from ion-exchange 12,400 3.56 65.3 column chromatography Eluate from affinity 8,320 11.7 43.8 column chromatography Eluate from hydrophobic 4,830 15.2 25.4 column chromatography Eluate from ion-exchange 3,850 22.6 20.3 column chromatography Note: The symbol "*" means .alpha.-isomaltosyl-transferring enzyme.

The finally purified .alpha.-isomaltosyl-transferring enzyme specimen was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 12

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 12-1

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 11-2, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 136,000.+-.20,000 daltons.

A fresh preparation of the same purified specimen as used in the above was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 7.3.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in accordance with the assay for its enzyme activity, where the influence of temperature was examined in the presence or the absence of 1 mM Ca.sup.2+. These results are in FIG. 21 (influence of temperature) and FIG. 22 (influence of pH). The optimum temperature of the enzyme was about 50.degree. C. and about 55.degree. C. when incubated at pH 6.0 for 60 min in the absence of and in the presence of 1 mM Ca.sup.2+, respectively. The optimum pH of the enzyme was about 6.0 when incubated at 35.degree. C. for 60 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min in the absence of and in the presence of 1 mM Ca.sup.2+, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the form of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 23 (thermal stability) and FIG. 24 (pH stability). As a result, the enzyme had thermal stability of up to about 45.degree. C. and about 50.degree. C. in the absence of and in the presence of 1 mM Ca.sup.2+, respectively, and had pH stability of about 5.0 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 13.

TABLE-US-00013 TABLE 13 Relative Relative Metal ion activity (%) Metal ion activity (%) None 100 Hg.sup.2+ 1 Zn.sup.2+ 82 Ba.sup.2+ 84 Mg.sup.2+ 96 Sr.sup.2+ 85 Ca.sup.2+ 108 Pb.sup.2+ 86 Co.sup.2+ 93 Fe.sup.2+ 82 Cu.sup.2+ 7 Fe.sup.3+ 93 Ni.sup.2+ 93 Mn.sup.2+ 120 Al.sup.3+ 98 EDTA 35

As evident form the results in Table 13, the enzyme activity was strongly inhibited by Hg.sup.2+, Cu.sup.2+, and EDTA. It was also found that the enzyme was activated by Ca.sup.2+ and Mn.sup.2+. Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:11, i.e., histidine-valine-serine-alanine-leucine-glycine-asparagine-leucine-leucin- e in the N-terminal region. Comparison of the above partial amino acid sequence in the N-terminal region with that derived from the .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C11 strain in Experiment 8-1 revealed that they had a relatively high homology but differed in the amino acid residues 1, 4 and 9 in each of their partial amino acid sequences in their N-terminal regions. Detailed method for assaying amino acid sequence is not shown in this specification because it is disclosed in detail in Japanese Patent Application No. 2001-5441 (International Publication No. WO02/055708), however, the .alpha.-isomaltosylglucosaccharide-forming enzyme has an amino acid sequence of amino acid residues 36-1286 shown in parallel in SEQ ID NO:23 similarly as that disclosed in the specification of the above-identified Japanese Patent Application No. 2001-5441.

EXPERIMENT 12-2

Property of .alpha.-isomaltosyl-transferring Enzyme

A purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 11-3, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 112,000.+-.20,000 daltons.

A fresh preparation of the same purified specimen as used in the above was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 7.8.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in accordance with the assay for its enzyme activity. These results are in FIG. 25 (influence of temperature) and FIG. 26 (influence of pH). The optimum temperature of the enzyme was about 50.degree. C. when incubated at pH 6.0 for 30 min. The optimum pH of the enzyme was about 6.0 when incubated at 35.degree. C. for 30 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the from of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 27 (thermal stability) and FIG. 28 (pH stability). As a result, the enzyme had thermal stability of up to about 45.degree. C. and had pH stability of about 4.5 to about 10.0. The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 14.

TABLE-US-00014 TABLE 14 Relative activity Metal Relative activity Metal ion (%) ion (%) None 100 Hg.sup.2+ 0.5 Zn.sup.2+ 75 Ba.sup.2+ 102 Mg.sup.2+ 95 Sr.sup.2+ 91 Ca.sup.2+ 100 Pb.sup.2+ 69 Co.sup.2+ 92 Fe.sup.2+ 97 Cu.sup.2+ 15 Fe.sup.3+ 90 Ni.sup.2+ 91 Mn.sup.2+ 101 Al.sup.3+ 94 EDTA 92

As evident form the results in Table 14, the enzyme activity was strongly inhibited by Hg.sup.2+ and also inhibited by Cu.sup.2+. It was also found that the enzyme was not activated by Ca.sup.2+ and not inhibited by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:3, i.e., isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline-tyrosine-glycine at the N-terminal region. Comparison of the above partial amino acid sequence at the N-terminal region with that derived from the .alpha.-isomaltosyl-transferring enzymes from Bacillus globisporus C9 strain in Experiment 5-2 and from Bacillus globisporus C11 strain in Experiment 8-2 revealed that they had a consensus amino acid sequence of isoleucine-aspartic acid-glycine-valine-tyrosine-histidine-alanine-proline, as shown in SEQ ID NO:4 in their N-terminal regions. Detailed method for assaying amino acid sequence is not shown in this specification because it is disclosed in detail in PCT/JP01/04276 (International Publication No. WO 01/90338), however, the .alpha.-isomaltosyl-transferring enzyme obtained in Experiment 11-3 has an amino acid sequence of amino acid residues 30-1093 shown in parallel in SEQ ID NO:24 similarly as the polypeptide disclosed in the specification of PCT/JP01/04276.

EXPERIMENT 13

Internal Amino Acid Sequence of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 13-1

Internal Partial Amino Acid Sequence of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A part of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 11-2, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with a fresh preparation of the same buffer as used in the above to give a concentration of about one milligram per milliliter. One milliliter of the dilute as a test sample was admixed with 20 .mu.g of "Lysyl Endopeptidase" commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to react at 30.degree. C. for 24 hours to form peptides. The resultant mixture was subjected to reverse-phase HPLC to separate the peptides using ".mu.-Bondasphere C18 column" having a diameter of 3.9 mm and a length of 150 mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and at ambient temperature, and using a liner gradient of acetonitrile increasing from 8% (v/v) to 36% (v/v) in 0.1% (v/v) trifluoroacetate over 120 min. The peptides eluted from the column were detected by monitoring the absorbance at a wavelength of 210 nm. Three peptide specimens named PN59 with a retention time of about 59 min, PN67 with a retention time of about 67 min, and PN87 with a retention time of about 87 min, which had been well separated from other peptides, were separately collected and dried in vacuo and then dissolved in 200 .mu.l of a solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen was subjected to a protein sequencer for analyzing amino acid sequence up to eight amino acid residues to obtain amino acid sequences of SEQ ID NOs:12 to 14. The analyzed internal partial amino acid sequences are in Table 15.

TABLE-US-00015 TABLE 15 Peptide name Internal partial amino acid sequence PN59 aspartic acid-phenylalanine-serine- asparagine-asparagine-proline-threonine- valine PN67 tyrosine-threonine-valine-asparagine- alanine-proline-alanine-alanine PN87 tyrosine-glutamic acid-alanine-glutamic acid-serine-alanine-glutamic acid-leucine

EXPERIMENT 13-2

Internal Amino Acid Sequence of .alpha.-isomaltosyl-transferring Enzyme

A part of a purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 11-3, was dialyzed against 10 mM Tris-HCl buffer (pH 9.0), and the dialyzed solution was diluted with a fresh preparation of the same buffer as used in the above to give a concentration of about one milligram per milliliter. One milliliter of the dilute as a test sample was admixed with 20 .mu.g of "Lysyl Endopeptidase" commercialized by Wako Pure Chemical Industries, Ltd., Tokyo, Japan, and allowed to react at 30.degree. C. for 24 hours to form peptides. The resultant mixture was subjected to reverse-phase HPLC to separate the peptides using ".mu.-Bondasphere C18 column" having a diameter of 3.9 mm and a length of 150 mm, a product of Waters Chromatography Div., MILLIPORE Corp., Milford, USA, at a flow rate of 0.9 ml/min and at ambient temperature, and using a liner gradient of acetonitrile increasing from 4% (v/v) to 42.4% (v/v) in 0.1% (v/v) trifluoroacetate over 90 min. The peptides eluted from the column were detected by monitoring the absorbance at a wavelength of 210 nm. Three peptide specimens named PN21 with a retention time of about 21 min, PN38 with a retention time of about 38 min, and PN69 with a retention time of about 69 min which had been well separated from other peptides, were separately collected and dried in vacuo and then dissolved in 200 .mu.l of a solution of 0.1% (v/v) trifluoroacetate and 50% (v/v) acetonitrile. Each peptide specimen was subjected to a protein sequencer for analyzing amino acid sequence up to eight amino acid residues, but up to six amino acids residues for PN21, to obtain amino acid sequences of SEQ ID NOs: 15 to 17. The analyzed internal partial amino acid sequences are in Table 16.

TABLE-US-00016 TABLE 16 Peptide name Internal partial amino acid sequence PN21 asparagine-tryptophane-tryptophane- methionine-serine-lysine PN38 threonine-aspartic acid-glycine-glycine- glutamic acid-methionine-valine-tryptophane PN69 asparagine-isoleucine-tyrosine-leucine- proline-glutamine-glycine-aspartic acid

EXPERIMENT 14

Production of .alpha.-isomaltosylglucosaccharide-forming Enzyme from Arthrobacter globiformis A19 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of "PINE-DEX #4", a partial starch hydrolyzate, 1.8% (w/v) of "ASAHIMEAST", a yeast extract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each, autoclaved at 121.degree. C. for 20 minutes to effect sterilization, cooled, inoculated with a stock culture of Arthrobacter globiformis A19 strain (FERM BP-7590), and incubated at 27.degree. C. for 48 hours under rotary shaking conditions of 230 rpm for use as a seed culture. About 20 L of a fresh preparation of the same nutrient culture medium as used in the above culture were placed in a 30-L fermentor, sterilized by heating, cooled to 27.degree. C., inoculated with 1% (v/v) of the seed culture, and incubated for about 48 hours while stirring under aeration-agitation conditions at 27.degree. C. and pH 6.0 to 9.0. The resultant culture, having about 1.1 units/ml of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, about 1.7 units/ml of an .alpha.-isomaltosyl-transferring enzyme activity, and about 0.35 unit/ml of a cyclotetrasaccharide-forming enzyme activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant. Measurement of the supernatant revealed that it had about 1.06 units/ml of an .alpha.-isomaltosylglucosaccharide-forming enzyme activity, i.e., a total enzyme activity of about 19,100 units; about 1.6 units/ml of an .alpha.-isomaltosyl-transferring enzyme activity, i.e., a total enzyme activity of about 28,800 units; and about 0.27 unit/ml of a cyclotetrasaccharide-forming enzyme activity, i.e., a total enzyme activity of about 4,860 units. The activity of the .alpha.-isomaltosylglucosaccharide-forming enzyme from Arthrobacter globiformis A19 strain was similarly assayed as the method in Experiment 3 except for using 100 mM glycine-NaOH buffer (pH 8.4) as a buffer for substrate.

EXPERIMENT 15

Preparation of Enzyme from Arthrobacter globiformis A19 Strain

EXPERIMENT 15-1

Purification of Enzyme from Arthrobacter globiformis A19 Strain

About 18 L of the supernatant, obtained in Experiment 14, was salted out in a 60% saturated ammonium sulfate solution and allowed to stand at 4.degree. C. for 24 hours. Then, the salted out sediments were collected by centrifugation at 10,000 for 30 min, dissolved in 10 mM phosphate buffer (pH 7.0), dialyzed against a fresh preparation of the same buffer as used in the above to obtain about 850 ml of a crude enzyme solution. The crude enzyme solution was revealed to have 8,210 units of .alpha.-isomaltosylglucosaccharide-forming enzyme, about 15,700 units of .alpha.-isomaltosyl-transferring enzyme, and about 2,090 units of cyclotetrasaccharide-forming enzyme, followed by subjecting it to ion-exchange chromatography using 380 ml of "DEAE-TOYOPEARL 650S" gel. When eluted with a linear gradient increasing from 0 M to 0.5 M NaCl, .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme were separately eluted from the gel, the former was eluted at a concentration of about 0.2 M NaCl, while the latter was eluted at a concentration of about 0.3 M NaCl. Under these conditions, fractions with an .alpha.-isomaltosylglucosaccharide-forming enzyme activity and those with an .alpha.-isomaltosyl-transferring enzyme activity were separately fractionated and collected. Since the facts that no cyclotetrasaccharide-forming activity was found in any fraction obtained in this column chromatography, and an enzyme solution, obtained by mixing the fractions of .alpha.-isomaltosylglucosaccharide-forming enzyme and of .alpha.-isomaltosyl-transferring enzyme, showed a cyclotetrasaccharide-forming activity, it was revealed that the activity of forming cyclotetrasaccharide from partial starch hydrolyzates is exerted by the coaction of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme.

The following experiments describe a method for separately purifying .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme:

EXPERIMENT 15-2

Purification of .alpha.-isomaltosylglucosaccharide-forming Enzyme

Fractions with .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained in Experiment 15-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities and fed to affinity chromatography using 500 ml of "SEPHACRYL HR S-200" gel. The enzyme was adsorbed on the gel and then eluted therefrom with a linear gradient decreasing from 1 M to 0 M ammonium sulfate. As a result, the .alpha.-isomaltosylglucosaccharide-forming enzyme adsorbed on the gel was eluted therefrom at a concentration of about 0.2 M ammonium sulfate, followed by collecting fractions with the enzyme activity and pooling them for use as a finally purified specimen. The amount of enzyme activity, specific activity, and yield of .alpha.-isomaltosylglucosaccharide-forming enzyme in each purification step are in Table 17.

TABLE-US-00017 TABLE 17 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 19,100 0.11 100 Dialyzed solution after 8,210 0.48 43.0 salting out with ammonium sulfate Eluate from ion-exchange 6,890 4.18 36.1 column chromatography Eluate from affinity 5,220 35.1 27.3 column chromatography Note: The symbol "*" means .alpha.-isomaltosylglucosaccharide-forming enzyme.

The finally purified .alpha.-isomaltosylglucosaccharide-forming enzyme specimen was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 15-3

Purification of .alpha.-isomaltosyl-transferring Enzyme

Fractions of .alpha.-isomaltosyl-transferring enzyme, which had been separated from fractions of .alpha.-isomaltosylglucosaccharide-forming enzyme by ion-exchange chromatography in Experiment 15-1, were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to affinity column chromatography using 500 ml of "SEPHACRYL HR S-200" gel, a gel commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA. The enzyme was adsorbed on the gel and then eluted therefrom with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate, resulting in an elution of the enzyme from the gel at a concentration of about 0 M ammonium sulfate and collecting the resulting fractions with the enzyme activity for a partially purified specimen. The amount of enzyme activity, specific activity, and yield of .alpha.-isomaltosyl-transferring enzyme in each purification step are in Table 18.

TABLE-US-00018 TABLE 18 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 28,800 0.18 100 Dialyzed solution after 15,700 0.97 54.5 salting out with ammonium sulfate Eluate from ion-exchange 7,130 4.01 24.8 column chromatography Eluate from affinity 1,440 12.1 5.0 column chromatography Note: The symbol "*" means .alpha.-isomaltosyl-transferring enzyme.

The partially purified .alpha.-isomaltosyl-transferring enzyme specimen was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 16

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme and .alpha.-isomaltosyl-transferring Enzyme

EXPERIMENT 16-1

Property of .alpha.-isomaltosylglucosaccharide-forming Enzyme

A purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme, obtained by the method in Experiment 15-2, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 94,000.+-.20,000 daltons.

A portion of the above purified specimen was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 4.3.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosylglucosaccharide-forming enzyme was examined in accordance with the assay for its enzyme activity. The influence of temperature was determined in the presence of or the absence of 1 mM Ca.sup.2+. These results are in FIG. 29 (influence of temperature) and FIG. 30 (influence of pH). The optimum temperature of the enzyme was about 60.degree. C. and about 65.degree. C. when incubated at pH 8.4 for 60 min in the absence of and in the presence of 1 mM Ca.sup.2+, respectively. The optimum pH of the enzyme was about 8.4 when incubated at 35.degree. C. for 60 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM glycine-NaOH buffer (pH 8.0) at prescribed temperatures for 60 min in the absence of or the presence of 1 mM Ca.sup.2+, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme in 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 8.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 31 (thermal stability) and FIG. 32 (pH stability). As a result, the enzyme had thermal stability of up to about 55.degree. C. and about 60.degree. C. in the absence of and in the presence of 1 mM Ca.sup.2+, respectively, and had pH stability of about 5.0 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 19.

TABLE-US-00019 TABLE 19 Relative activity Metal Relative activity Metal ion (%) ion (%) None 100 Hg.sup.2+ 0 Zn.sup.2+ 56 Ba.sup.2+ 99 Mg.sup.2+ 97 Sr.sup.2+ 102 Ca.sup.2+ 106 Pb.sup.2+ 43 Co.sup.2+ 93 Fe.sup.2+ 36 Cu.sup.2+ 0 Fe.sup.3+ 35 Ni.sup.2+ 46 Mn.sup.2+ 98 Al.sup.3+ 37 EDTA 2

As evident form the results in Table 19, it was revealed that the enzyme activity was strongly inhibited by Hg.sup.2+, Cu.sup.2+, and EDTA. Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:18, i.e., alanine-proline-leucine-glycine-valine-glutamine-arginine-alanine-glutami- ne-phenylalanine-glutamine-serine-glycine in the N-terminal region. Detailed method for assaying amino acid sequence is not shown in this specification because it is disclosed in detail in Japanese Patent Application No. 2001-5441 (International Publication No. WO 02/055708), however, the .alpha.-isomaltosylglucosaccharide-forming enzyme has an amino acid sequence of amino acid residues 37-965 shown in parallel in SEQ ID NO:25 similarly as the polypeptide disclosed in the specification of the above Japanese Patent Application No. 2001-5441.

EXPERIMENT 16-2

Property of .alpha.-isomaltosyl-transferring Enzyme

A purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 15-3, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 113,000.+-.20,000 daltons.

A portion of the above purified specimen was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 4.2.+-.0.5.

The influence of temperature and pH on the above enzyme was examined in accordance with the assay for its enzyme activity. These results are in FIG. 33 (influence of temperature) and FIG. 34 (influence of pH). The optimum temperature of the enzyme was about 50.degree. C. when incubated at pH 6.0 for 30 min. The optimum pH of the enzyme was about 6.5 when incubated at 35.degree. C. for 30 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffer (pH 6.0) at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the form of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 35 (thermal stability) and FIG. 36 (pH stability). As a result, the enzyme had thermal stability of up to about 45.degree. C. and pH stability of about 4.5 to about 9.0. Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:19, i.e., asparagine-threonine-leucine-aspartic acid-glycine-valine-tryptophane-histidine-asparagine-proline-tyrosine-gly- cine-alanine-aspartic acid-glutamic acid-leucine-tyrosine-alanine-threonine-glutamine in the N-terminal region.

EXPERIMENT 16-3

Total Amino Acid Sequence of .alpha.-isomaltosyl-transferring Enzyme

According to the method in Japanese Patent Application No. 2001-5441 (International Publication No. WO 02/055708), chromosomal DNAs (cDNAs) were extracted from Arthrobacter globiformis A19 strain and purified. The purified cDNAs were hydrolyzed with a restriction enzyme, Not I, to obtain DNA fragments. While, "Bluescript II SK(+)", a plasmid vector commercialized by Stratagene Cloning Systems, California, USA, was completely cleaved with a restriction enzyme, Not I, and the resulting cleaved plasmid vector and the above DNA fragments using "DNA Ligation Kit" commercialized by Takara Shuzo Co., Ltd., Tokyo, Japan, to obtain a recombinant DNA. "Epicurian Coli XL2-Blue", commercialized by Stratagene Cloning Systems, California, USA, was transformed with the recombinant DNA to obtain a gene library. An oligonucleotide, represented by 5'-AAYACNCTNGAYGGNGTNTGGCAYAAYCCNTAYGGNGCNGAYGARCTNTGGAC-3', was chemically synthesized based on the amino acid sequence of amino acid residues 1-18 in SEQ ID NO:19, which had been revealed by the method in Experiment 16-2; and labeled with [.gamma.-.sup.32P]ATP and T4 polynucleotide kinase to obtain a probe. In accordance with the method in Japanese Patent Application No. 2001-5441 (International Publication No. WO 02/055708), the above gene library and the probe were subjected to the colony hybridization method, followed by selecting a transformant that strongly hybridized with the probe. The transformant was named "AGA4". According to conventional manner, a recombinant DNA was prepared from the transformant and analyzed for nucleotide sequence by conventional dideoxy method, revealing that the recombinant DNA thus obtained comprised the DNA of SEQ ID NO:26 consisting of 6153 base pairs, derived from Arthrobacter globiformis A19 strain. As shown in FIG. 37, in the recombinant DNA, the above DNA was linked to the downstream of the recognition site of Not I. When an amino acid sequence estimable from the above nucleotide sequence, which is shown in parallel in SEQ ID NO:26, was compared with the N-terminal amino acid sequence of the .alpha.-isomaltosyl-transferring enzyme that was confirmed by the method in Experiment 16-2, the amino acid sequence of SEQ ID NO:19 was completely coincided with the amino acid residues 50-69 shown in parallel in SEQ ID NO:26. Since the nucleotide sequence of nucleotide residues 4644-4646 in SEQ ID NO:26 encodes the termination codon (5'-TGA-3'), the C-terminus of .alpha.-isomaltosyl-transferring enzyme was revealed to be arginine, corresponding to amino acid residue 1121, shown in parallel in SEQ ID NO:26, which positions just before the termination codon. These results show that the .alpha.-isomaltosyl-transferring enzyme obtained in Experiment 15-3 comprises the amino acid residues 50-1121 shown in parallel in SEQ ID NO:26 and is encoded by a DNA comprising the nucleotide residues 1428-4643 shown in parallel in SEQ ID NO:26. A sequence of amino acid residues 1-49 shown in parallel in SEQ ID NO:26 was estimated to be an amino acid sequence of secretory signal for the polypeptide. These data revealed that the precursor peptide of the polypeptide before secretion comprises the amino acid sequence shown in parallel in SEQ ID NO:26 and is encoded by the nucleotide sequence shown in parallel in SEQ ID NO:26. Based on these, the recombinant DNA with its confirmed nucleotide sequence was named "pAGA4".

EXPERIMENT 17

Production of .alpha.-isomaltosyl-transferring Enzyme from Arthrobacter ramosus S1 Strain

A liquid nutrient culture medium, consisting of 4.0% (w/v) of "PINE-DEX #4", a partial starch hydrolyzate, 1.8% (w/v) of "ASAHIMEAST", a yeast extract, 0.1% (w/v) of dipotassium phosphate, 0.06% (w/v) of sodium phosphate dodecahydrate, 0.05% (w/v) magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each, autoclaved at 121.degree. C. for 20 min to effect sterilization, cooled, inoculated with a stock culture of Arthrobacter ramosus S1 strain (FERM BP-7592), and incubated at 27.degree. C. for 48 hours under rotary shaking conditions of 230 rpm for use as a seed culture. About 20 L of a fresh preparation of the same nutrient culture medium as used in the above culture were placed in a 30-L fermentor, sterilized by heating, cooled to 27.degree. C., inoculated with 1% (v/v) of the seed culture, and incubated for about 48 hours while stirring under aeration-agitation conditions at 27.degree. C. and pH 6.0 to 8.0. The resultant culture, having about 0.45 unit/ml of an .alpha.-isomaltosyl-transferring activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant having about 0.44 unit/ml of an .alpha.-isomaltosyl-transferring enzyme activity and a total enzyme activity of about 7,920 units.

EXPERIMENT 18

Purification of .alpha.-isomaltosyl-transferring Enzyme from Arthrobacter ramosus S1 Strain

About 18 L of a supernatant obtained in Experiment 17 were salted out in an 80% (w/v) ammonium sulfate solution at 4.degree. C. for 24 hours, and the resulting sediments were collected by centrifugation at 10,000 rpm for 30 min and dialyzed against 10 mM phosphate buffer (pH 7.0) to obtain about 380 ml of a crude enzyme solution having 6,000 units of .alpha.-isomaltosyl-transferring enzyme. The crude enzyme solution was dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The resulting supernatant was fed to affinity column chromatography using 500 ml of "SEPHACRYL HR S-200" gel. The enzyme was adsorbed on the gel and then eluted sequentially with a linear gradient decreasing from 1 M to 0 M of ammonium sulfate and with a linear gradient increasing from 0% (w/v) to 5% (w/v) maltotetraose, resulting in an elution of the enzyme from the gel at a concentration of about 2% (w/v) maltotetraose and collecting fractions with the enzyme activity. The fractions were pooled and dialyzed against 10 mM phosphate buffer (pH 7.0) containing 1 M ammonium sulfate, and the dialyzed solution was centrifuged to remove insoluble impurities. The supernatant thus obtained was fed to hydrophobic column chromatography using 380 ml of "BUTYL-TOYOPEARL 650M" gel. When eluted with a linear gradient decreasing from 1 M to 0 M ammonium sulfate, the .alpha.-isomaltosyl-transferring enzyme adsorbed on the gel was eluted therefrom at about 0.3 M ammonium sulfate, followed by collecting fractions with the enzyme activity for a purified enzyme specimen. The amount of enzyme activity, specific activity, and yield of the .alpha.-isomaltosyl-transferring enzyme in each purification step are in Table 20.

TABLE-US-00020 TABLE 20 Specific activity Enzyme* activity of enzyme* Yield Purification step (unit) (unit/mg protein) (%) Culture supernatant 7,920 0.47 100 Dialyzed solution after 6,000 3.36 75.8 salting out with ammonium sulfate Eluate from affinity 5,270 29.9 66.5 column chromatography Eluate from hydrophobic 4,430 31.1 55.9 column chromatography Note: The symbol "*" means .alpha.-isomaltosyl-transferring enzyme.

The purified .alpha.-isomaltosyl-transferring enzyme specimen obtained in this experiment was assayed for purity on gel electrophoresis using a 7.5% (w/v) polyacrylamide gel and detected on the gel as a single protein band, meaning a high purity enzyme specimen.

EXPERIMENT 19

Property of .alpha.-Isomaltosyl-transferring Enzyme

A purified specimen of .alpha.-isomaltosyl-transferring enzyme, obtained by the method in Experiment 18, was subjected to SDS-PAGE using a 7.5% (w/v) of polyacrylamide gel and then determined for molecular weight by comparing with the dynamics of standard molecular markers electrophoresed in parallel, commercialized by Japan Bio-Rad Laboratories Inc., Tokyo, Japan, revealing that the enzyme had a molecular weight of about 116,000.+-.20,000 daltons.

A portion of the above purified specimen was subjected to isoelectrophoresis using a gel containing 2% (w/v) ampholine commercialized by Amersham Corp., Div., Amersham International, Arlington Heights, Ill., USA, and then measured for pHs of protein bands and gel to determine the isoelectric point of the enzyme, revealing that the enzyme had an isoelectric point of about 4.2.+-.0.5.

The influence of temperature and pH on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in accordance with the assay for its enzyme activity. These results are in FIG. 38 (influence of temperature) and FIG. 39 (influence of pH). The optimum temperature of the enzyme was about 50.degree. C. when incubated at pH 6.0 for 30 min. The optimum pH of the enzyme was about 6.0 when incubated at 35.degree. C. for 30 min. The thermal stability of the enzyme was determined by incubating the testing enzyme solutions in the form of 20 mM acetate buffers (pH 6.0) at prescribed temperatures for 60 min, cooling the resulting enzyme solutions with water, and assaying the remaining enzyme activity of each solution. The pH stability of the enzyme was determined by keeping the testing enzyme solutions in the from of 50 mM buffers having prescribed pHs at 4.degree. C. for 24 hours, adjusting the pH of each solution to 6.0, and assaying the remaining enzyme activity of each solution. These results are respectively in FIG. 40 (thermal stability) and FIG. 41 (pH stability). As evident from these figures, the enzyme had thermal stability of up to about 45.degree. C. and had pH stability of about 3.6 to about 9.0.

The influence of metal ions on the activity of .alpha.-isomaltosyl-transferring enzyme was examined in the presence of 1 mM of each metal-ion according to the assay for its enzyme activity. The results are in Table 21.

TABLE-US-00021 TABLE 21 Metal Relative activity Metal Relative activity ion (%) ion (%) None 100 Hg.sup.2+ 0.1 Zn.sup.2+ 78 Ba.sup.2+ 97 Mg.sup.2+ 99 Sr.sup.2+ 101 Ca.sup.2+ 103 Pb.sup.2+ 85 Co.sup.2+ 91 Fe.sup.2+ 105 Cu.sup.2+ 2 Fe.sup.3+ 75 Ni.sup.2+ 87 Mn.sup.2+ 98 Al.sup.3+ 93 EDTA 91

As evident form the results in Table 21, it was revealed that the enzyme activity was strongly inhibited by Hg.sup.2+ and also inhibited by Cu.sup.2+. It was also revealed that the enzyme was neither activated by Ca.sup.2+ nor by EDTA.

Amino acid analysis on the N-terminal amino acid sequence of the enzyme by "PROTEIN SEQUENCER MODEL 473A", an apparatus of Applied Biosystems, Inc., Foster City, USA, revealed that the enzyme had a partial amino acid sequence of SEQ ID NO:20, i.e., aspartic acid-threonine-leucine-serine-glycine-valine-phenylalanine-histidine-glyc- ine-proline at the N-terminal region.

EXPERIMENT 20

Action on Saccharides

It was tested whether any saccharides can be used as substrates for .alpha.-isomaltosylglucosaccharide-forming enzyme. For the purpose, a solution of maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, isomaltose, isomaltotriose, panose, isopanose, .alpha.,.alpha.-trehalose, kojibiose, nigerose, neotrehalose, cellobiose, gentibiose, maltitol, maltotriitol, lactose, sucrose, erlose, selaginose, maltosyl glucoside, or isomaltosyl glucoside was prepared.

To each of the above solutions was added two units/g substrate of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from either Bacillus globisporus C9 strain obtained by the method in Experiment 4-2, Bacillus globisporus C11 strain obtained by the method in Experiment 7-2, Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, or Arthrobacter globiformis A19 strain obtained by the method in Experiment 15-2, and the resulting each solution was adjusted to give a substrate concentration of 2% (w/v) and incubated at 30.degree. C. and pH 6.0 for 24 hours, except for using pH 8.4 for the enzyme from Arthrobacter globiformis A19 strain. The enzyme solutions before and after the enzymatic reactions were respectively analyzed on TLC disclosed in Experiment 1 to confirm whether the enzymes acted on these substrates. The results are in Table 22.

TABLE-US-00022 TABLE 22 Enzymatic action Enzyme of Enzyme of Enzyme of Enzyme of Substrate C9 strain C11 strain N75 strain A19 strain Maltose + + + + Maltotriose ++ ++ ++ ++ Maltotetraose +++ +++ +++ +++ Maltopentaose +++ +++ +++ +++ Maltohexaose +++ +++ +++ +++ Maltoheptaose +++ +++ +++ +++ Isomaltose - - - - Isomaltotriose - - - - Panose - - - - Isopanose ++ ++ ++ ++ Trehalose - - - - Kojibiose + + + + Nigerose + + + + Neotrehalose + + + + Cellobiose - - - - Gentibiose - - - - Maltitol - - - - Maltotriitol + + + + Lactose - - - - Sucrose - - - - Erlose + + + + Selaginose - - - - Maltosyl glucoside ++ ++ ++ ++ Isomaltosyl glucoside - - - - Note: Before and after the enzymatic reaction, the symbols "-", "+", "++", and "+++", mean that it showed no change, it showed a slight reduction of the color spot of the substrate and the formation of other reaction product, it showed a high reduction of the color spot of the substrate and the formation of other reaction product, and it showed a substantial disappearance of the substrate spot and the formation of other reaction product, respectively.

As evident from the Table 22, it was revealed that the .alpha.-isomaltosylglucosaccharide-forming enzymes well acted on saccharides having a glucose polymerization degree of at least three and having a maltose structure at their non-reducing ends, among the saccharides tested. It was also found that the enzymes slightly acted on saccharides, having a glucose polymerization degree of two, such as maltose, kojibiose, nigerose, neotrehalose, maltotriitol, and erlose.

EXPERIMENT 21

Reaction product from Maltooligosaccharide

EXPERIMENT 21-1

Preparation of Reaction Product

To an aqueous solution containing one percent (w/v) of maltose, maltotriose, maltotetraose, or maltopentaose as a substrate was added a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme obtained by the method in Experiment 7-2 in an amount of two units/g solid, d.s.b., for the aqueous solutions of maltose and maltotriose; 0.2 unit/g solid, d.s.b., for the aqueous solution of maltotetraose; and 0.1 unit/g solid, d.s.b., for the aqueous solution of maltopentaose, followed by incubation at 35.degree. C. and pH 6.0 for eight hours. After a 10-min incubation at 100.degree. C., the enzymatic reaction was suspended. The resulting reaction solutions were respectively measured for saccharide composition on HPLC using "YMC PACK ODS-AQ303", a column commercialized by YMC Co., Ltd., Tokyo, Japan, at a column temperature of 40.degree. C. and a flow rate of 0.5 ml/min of water, and using as a detector "RI-8012", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. The results are in Table 23.

TABLE-US-00023 TABLE 23 Substrate Saccharide as Mal- reaction product tose Maltotriose Maltotetraose Maltopentaose Glucose 8.5 0.1 0.0 0.0 Maltose 78.0 17.9 0.3 0.0 Maltotriose 0.8 45.3 22.7 1.9 Maltotetraose 0.0 1.8 35.1 19.2 Maltopentaose 0.0 0.0 3.5 34.4 Maltohexaose 0.0 0.0 0.0 4.6 Isomaltose 0.5 0.0 0.0 0.0 Glucosylmaltose 8.2 1.2 0.0 0.0 Glucosyl- 2.4 31.5 6.8 0.0 maltotriose X 0.0 2.1 30.0 11.4 Y 0.0 0.0 1.4 26.8 Z 0.0 0.0 0.0 1.7 Others 0.6 0.1 0.2 0.0 Note: In the table, glucosylmaltose means .alpha.-isomaltosylglucose alias 6.sup.2-O-.alpha.-glucosylmaltose or panose; glucosylmaltotriose means .alpha.-isomaltosylglucose alias 6.sup.3-O-.alpha.-glucosylmaltotriose; X means the .alpha.-isomaltosylmaltotriose in Experiment 11-2, alias 6.sup.4-O-.alpha.-glucomaltotetraose; Y means the .alpha.-isomaltosylmaltotetraose in Experiment 11-2, alias 6.sup.5-O-.alpha.-glucosylmaltopentaose; and Z means an unidentified saccharide.

As evident from the results in Table 23, it was revealed that, after the enzymatic action, glucose and .alpha.-isomaltosylglucose alias 6.sup.2-O-.alpha.-glucosylmaltose or panose were mainly formed maltose as a substrate; and maltose and .alpha.-isomaltosylglucose alias 6.sup.3-O-.alpha.-glucosylmaltotriose were mainly formed along with small amounts of glucose, maltotetraose, .alpha.-isomaltosylglucose alias 6.sup.2-O-.alpha.-glucosylmaltose or panose, and a product X. Also, it was revealed that maltotriose and the product X were mainly formed from maltotetraose as a substrate along with small amounts of maltose, maltopentaose, .alpha.-isomaltosylglucose alias 6.sup.3-O-.alpha.-glucosylmaltotriose; and a product Y; and that maltotetraose and the product Y were mainly formed from maltopentaose as a substrate along with small amounts of maltotriose, maltohexaose, and the products X and Z. The product X as a main product from maltotetraose as a substrate and the product Y as a main product from maltopentaose as a substrate were respectively isolated and purified as follows: The products X and Y were respectively purified on HPLC using "YMC PACK ODS-A R355-15S-15 12A", a separatory HPLC column commercialized by YMC Co., Ltd., Tokyo, Japan, to isolate the product X having a purity of at least 99.9% from the reaction product from maltotetraose in a yield of about 8.3%, d.s.b., and the product Y having a purity of at least 99.9% from the reaction product from maltopentaose in a yield of about 11.5%, d.s.b.

EXPERIMENT 21-2

Structural Analysis on Reaction Product

The products X and Y, obtained by the method in Experiment 21-1, were subjected to methyl analysis and NMR analysis in a usual manner. The results on their methyl analyses are in Table 24. Regarding the results on their NMR analyses, FIG. 42 is a .sup.1H-NMR spectrum for the product X and FIG. 43 is for the product Y. The .sup.13C-NMR spectra for the products X and Y are respectively FIGS. 44 and 45. The assignment of the products X and Y are tabulated in Table 25.

TABLE-US-00024 TABLE 24 Analyzed Ratio methyl compound Product X Product Y 2,3,4-Trimethyl compound 1.00 1.00 2,3,6-Trimethyl compound 3.05 3.98 2,3,4,6-Tetramethyl compound 0.82 0.85

TABLE-US-00025 TABLE 25 Glucose Carbon Chemical shift on NMR (ppm) number number Product X Product Y a 1a 100.8 100.8 2a 74.2 74.2 3a 75.8 75.7 4a 72.2 72.2 5a 74.5 74.5 6a 63.2 63.1 b 1b 102.6 102.6 2b 74.2 74.2 3b 75.8 75.7 4b 72.1 72.1 5b 74.0 74.0 6b 68.6 68.6 c 1c 102.3 102.3 2c 74.2 74.2 3c 76.0 76.0 4c 79.6 79.5 5c 73.9 73.9 6c 63.2 63.1 d 1d 102.2 102.3 2d 74.0 (.alpha.), 74.4 (.beta.) 74.2 3d 76.0 76.0 4d 79.8 79.5 5d 73.9 73.9 6d 63.2 63.1 e 1e 94.6 (.alpha.), 98.5 (.beta.) 102.1 2e 74.2 (.alpha.), 76.7 (.beta.) 74.0 (.alpha.), 74.4 (.beta.) 3e 75.9 (.alpha.), 78.9 (.beta.) 76.0 4e 79.6 (.alpha.), 79.4 (.beta.) 79.8 5e 72.6 (.alpha.), 77.2 (.beta.) 73.9 6e 63.4 (.alpha.), 63.4 (.beta.) 63.1 f 1f 94.6 (.alpha.), 98.5 (.beta.) 2f 74.2 (.alpha.), 76.7 (.beta.) 3f 76.0 (.alpha.), 78.9 (.beta.) 4f 79.6 (.alpha.), 79.5 (.beta.) 5f 72.6 (.alpha.), 77.2 (.beta.) 6f 63.3 (.alpha.), 63.3 (.beta.)

Based on these results, the product X, formed from maltotetraose via the action of the .alpha.-isomaltosylglucosaccharide-forming enzyme, was revealed as a pentasaccharide, in which a glucose residue is linked via the .alpha.-linkage to OH-6 of the glucose positioning at the non-reducing end of maltotetraose, i.e., .alpha.-isomaltosylmaltotriose alias 6.sup.6-O-.alpha.-glucosylmaltotetraose, represented by Formula 1. .alpha.-D-Glcp-(1.fwdarw.6)-.alpha.-D-Glcp-(1.fwdarw.4)-.alpha.-D-Glcp-(1- .fwdarw.4)-.alpha.-D-Glcp-(1.fwdarw.4)-D-Glcp Formula 1:

The product Y formed from maltopentaose was revealed as a hexasaccharide, in which a glucosyl residue is linked via the .alpha.-linkage to OH-6 of the glucose at the non-reducing end of maltopentaose, i.e., .alpha.-isomaltosylmaltotetraose alias 6.sup.5-O-.alpha.-glucosylmaltopentaose, represented by Formula 2. .alpha.-D-Glcp-(1.fwdarw.6)-.alpha.-D-Glcp-(1.fwdarw.4)-.alpha.-D-Glcp-(1- .fwdarw.4)-.alpha.-D-Glcp-(1.fwdarw.4)-.alpha.-D-Glcp-(1.fwdarw.4)-D-Glcp Formula 2:

Based on these results, it was concluded that .alpha.-isomaltosylglucosaccharide-forming enzyme acts on maltooligosaccharides as indicated below: (1) The enzyme acts on as a substrate maltooligosaccharides having a glucose polymerization degree of at least two linked via the .alpha.-1,4 linkage, and catalyzes the intermolecular 6-glucosyl-transferring reaction in such a manner of transferring a glucosyl residue at the non-reducing end of a maltooligosaccharide molecule to C-6 of the non-reducing end of other maltooligosaccharide molecule to form both an .alpha.-isomaltosylglucosaccharide alias 6-O-.alpha.-glucosylmaltooligosaccharide, having a 6-O-.alpha.-glucosyl residue and an increased glucose polymerization degree by one as compared with the intact substrate, and a maltooligosaccharide with a reduced glucose polymerization degree by one as compared with the intact substrate molecule; and (2) The enzyme slightly catalyzes the 4-glucosyl-transferring reaction and forms both a maltooligosaccharide molecule, having an increased glucose polymerization degree by one as compared with the intact substrate, and a maltooligosaccharide having a reduced glucose polymerization degree by one as compared with the intact substrate molecule.

EXPERIMENT 22

Test on Reducing-power Formation

The following test was carried out to study whether .alpha.-isomaltosylglucosaccharide-formation enzyme had the ability of forming a reducing power. To a 1% (w/v) aqueous solution of maltotetraose as a substrate was added 0.25 unit/g substrate, d.s.b., of either of purified specimens of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9 strain obtained by the method in Experiment 4-2, Bacillus globisporus C11 strain obtained by the method in Experiment 7-2, Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, or Arthrobacter globiformis A19 strain obtained by the method in Experiment 15-2, and incubated at 35.degree. C. and pH 6.0, except that pH 8.4 was used for the enzyme from Arthrobacter globiformis A19 strain. During the enzymatic reaction, a portion of each reaction solution was sampled at prescribed time intervals and measured for reducing powder after keeping the sampled solutions at 100.degree. C. for 10 min to suspend the enzymatic reaction. Before and after the enzymatic reaction, the reducing saccharide content and the total sugar content were respectively quantified by the Somogyi-Nelson's method and the anthrone-sulfuric acid reaction method. The percentage of forming reducing power was calculated by the following equation:

Equation:

.times..times..times..times..times..times..times..times..times. ##EQU00001## AR: Reducing sugar content after enzymatic reaction. AT: Total sugar content after enzymatic reaction. BR: Reducing sugar content before enzymatic reaction. BT: Total sugar content before enzymatic reaction.

The results are in Table 26.

TABLE-US-00026 TABLE 26 Percentage of forming Reaction reducing power (%) time Enzyme of Enzyme of Enzyme of Enzyme of (hour) C9 strain C11 strain N75 strain A19 strain 0 0.0 0.0 0.0 0.0 1 0.0 0.1 0.1 0.0 2 0.1 0.0 0.0 0.1 4 0.1 0.1 0.0 0.0 8 0.0 0.0 0.1 0.1

As evident from the results in Table 26, it was revealed that .alpha.-isomaltosylglucosaccharide-forming enzyme did not substantially increase the reducing power of the reaction product when acted on maltotetraose as a substrate; the enzyme did not have any hydrolyzing activity or had only an undetectable level of such activity.

EXPERIMENT 23

Test on Dextran Formation

To examine whether .alpha.-isomaltosylglucosaccharide-formation enzyme has the ability of forming dextran, it was tested in accordance with the method in Bioscience Biotechnology and Biochemistry, Vol. 56, pp. 169-173 (1992). To a 1% (w/v) aqueous solution of maltotetraose as a substrate was added 0.25 unit/g substrate, d.s.b., of either of purified specimens of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9 strain obtained by the method in Experiment 4-2, Bacillus globisporus C11 strain obtained by the method in Experiment 7-2, Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, or Arthrobacter globiformis A19 strain obtained by the method in Experiment 15-2, and incubated at 35.degree. C. and pH 6.0, except that pH 8.4 was used for the enzyme from Arthrobacter globiformis A19 strain, for four or eight hours. After completion of the enzymatic reaction, the reaction was suspended by heating at 100.degree. C. for 15 min. Fifty microliters of each of the reaction mixtures were placed in a centrifugation tube and then admixed and sufficiently stirred with 3-fold volumes of ethanol, followed by standing at 4.degree. C. for 30 min. Thereafter, each mixture solution was centrifuged at 15,000 rpm for five minutes and, after removing supernatant, the resulting sediment was admixed with one milliliter of 75% ethanol solution and stirred for washing. The resulting each solution was centrifuged to remove supernatant, dried in vacuo, and then admixed and sufficiently stirred with one milliliter of deionized water. The total sugar content, in terms of glucose, of each of the resulting solutions was quantified by the phenol-sulfuric acid method. As a control, the total sugar content was determined similarly as in the above except for using either of purified specimens of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9 strain, Bacillus globisporus C11 strain, Bacillus globisporus N75 strain, and Arthrobacter globiformis A19 strain, which had been inactivated at 100.degree. C. for 10 min. The content of dextran formed was calculated by the following equation. Content of dextran formed (mg/ml)=[(Total sugar content for test sample)]-[(Total sugar content for control sample)].times.20 Equation

The results are in Table 27.

TABLE-US-00027 TABLE 27 Reaction Content of dextran formed (mg/ml) time Enzyme of Enzyme of Enzyme of Enzyme of (hour) C9 strain C11 strain N75 strain A19 strain 4 0.0 0.0 0.0 0.0 8 0.0 0.0 0.0 0.0

As evident from the results in Table 27, it was revealed that .alpha.-isomaltosylglucosaccharide-forming enzyme did not substantially have the action of forming dextran or had only an undetectable level of such activity because it did not form dextran when acted on maltotetraose.

EXPERIMENT 24

Transfer-acceptor Specificity

Using various saccharides, it was tested whether the saccharides were used as transferring-acceptors for .alpha.-isomaltosylglucosaccharide-forming enzyme. A solution of D-glucose, D-xylose, L-xylose, D-galactose, D-fructose, D-mannose, D-arabinose, D-fucose, D-psicose, L-sorbose, L-rhamnose, methyl-.alpha.-glucopyranoside, methyl-.beta.-glucopyranoside, N-acetyl-glucosamine, sorbitol, .alpha.,.alpha.-trehalose, isomaltose, isomaltotriose, cellobiose, gentibiose, glycerol, maltitol, lactose, sucrose, .alpha.-cyclodextrin, .beta.-cyclodextrin, .gamma.-cyclodextrin, or L-ascorbic acid was prepared. To each solution with a saccharide concentration of 1.6% was added "PINE-DEX #100", a partial starch hydrolyzate, as a saccharide donor, to give a concentration of 4%, and admixed with one unit/g saccharide donor, d.s.b., of either of purified specimens of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus C9 strain obtained by the method in Experiment 4-2, Bacillus globisporus C11 strain obtained by the method in Experiment 7-2, Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, or Arthrobacter globiformis A19 strain obtained by the method in Experiment 15-2, and incubated at 30.degree. C. and pH 6.0 for 24 hours, except that pH 8.4 was used for the enzyme from Arthrobacter globiformis A19 strain. The reaction mixtures of the post-enzymatic reactions were analyzed on gas chromatography (abbreviated as "GLC" hereinafter) for monosaccharides and disaccharides as acceptors, and on HPLC for trisaccharides as acceptors to confirm whether these saccharides could be used as the transfer acceptors of the above enzymes. In the case of performing GLC, the following apparatuses and conditions were used: GLC apparatus, "GC-16A" commercialized by Shimadzu Corporation, Tokyo, Japan; column, a stainless-steel column, 3 mm in diameter and 2 m in length, packed with 2% "SILICONE OV-17/CHROMOSOLV W", commercialized by GL Sciences Inc., Tokyo, Japan; carrier gas, nitrogen gas at a flow rate of 40 ml/min under temperature conditions of increasing from 160.degree. C. to 320.degree. C. at an increasing temperature rate of 7.5.degree. C./min; and detection, a hydrogen flame ionization detector. In the case of performing HPLC analysis, the following apparatuses and conditions were used: HPLC apparatus, "CCPD" commercialized by Tosoh Corporation, Tokyo, Japan; column, "ODS-AQ-303" commercialized by YMC Co., Ltd., Tokyo, Japan; eluent, water at a flow rate of 0.5 ml/min; and detection, a differential refractometer. The results are in Table 28.

TABLE-US-00028 TABLE 28 Product of transferring reaction Enzyme of Enzyme of Enzyme of Enzyme of Saccharide C9 strain C11 strain N75 strain A19 strain D-Glucose + + + + D-Xylose ++ ++ ++ + L-Xylose ++ ++ ++ + D-Galactose + + + .+-. D-Fructose + + + + D-Mannose - - - .+-. D-Arabinose .+-. .+-. .+-. .+-. D-Fucose + + + .+-. D-Psicose + + + + L-Sorbose + + + + L-Rhamnose - - - - Methyl-.alpha.- ++ ++ ++ ++ glucopyranoside Methyl-.beta.- ++ ++ ++ ++ glucopyranoside N-Acetylglucosamine + + + - Sorbitol - - - - Trehalose ++ ++ ++ ++ Isomaltose ++ ++ ++ + Isomaltotriose ++ ++ ++ .+-. Cellobiose ++ ++ ++ ++ Gentibiose ++ ++ ++ + Glycerol + + + + Maltitol ++ ++ ++ ++ Lactose ++ ++ ++ ++ Sucrose ++ ++ ++ ++ .alpha.-Cyclodextrin - - - - .beta.-Cyclodextrin - - - - .gamma.-Cyclodextrin - - - - L-Ascorbic acid + + + + Note: In the table, the symbols "-", ".+-.", "+", and "++" mean that no saccharide-transferred product was detected through transfer reaction to acceptor; a saccharide-transferred product was detected in an amount less than one percent through transfer reaction to acceptor; a saccharide-transferred product was detected in an amount over one percent but less than 10% through transfer reaction to acceptor; and a saccharide-transferred product was detected in an amount over ten percent through transfer reaction to acceptor.

As evident from the results in Table 28, it was revealed that .alpha.-isomaltosylglucosaccharide-forming enzymes utilizes different types of saccharides as transfer acceptors; the .alpha.-isomaltosylglucosaccharide-forming enzymes from C9, C11 and N75 strains advantageously transfer a saccharide(s), particularly, to D-/L-xylose, methyl-.alpha.-glucopyranoside, methyl-.beta.-glucopyranoside, .alpha.,.alpha.-trehalose, isomaltose, isomaltotriose, cellobiose, gentibiose, maltitol, lactose, and sucrose; then transfer to D-glucose, D-fructose, D-fucose, D-psicose, L-sorbose, N-acetylglucosamine, glycerol, and L-ascorbic acid; and further to D-arabinose. Particularly, the .alpha.-isomaltosylglucosaccharide-forming enzyme from A19 strain well transfers a saccharide(s), specifically, to methyl-.alpha.-glucopyranoside, methyl-.beta.-glucopyranoside, .alpha.,.alpha.-trehalose, cellobiose, maltitol, lactose, and sucrose; secondary transfers to D-glucose, D-/L-xylose, D-fructose, D-psicose, L-sorbose, isomaltose, gentibiose, glycerol, and L-ascorbic acid; and thirdly to D-galactose, D-mannose, D-arabinose, D-fucose, and isomaltotriose.

The properties of .alpha.-isomaltosylglucosaccharide-transferring enzyme as described above were compared with those of a previously reported enzyme having 6-glucosyl-transferring action; a dextrin dextranase disclosed in "Bioscience Biotechnology and Biochemistry", Vol. 56, pp. 169-173 (1992); and a transglucosidase disclosed in "Nippon Nogeikagaku Kaishi", Vol. 37, pp. 668-672 (1963). The results are in Table 29.

TABLE-US-00029 TABLE 29 .alpha.-Isomaltosyl-glucosaccharide- Dextrin forming enzyme of the present invention dextranase Transglucosidase Property C9 strain C11 strain N75 strain A19 strain Control Control Hydrolysis Negative Negative Negative Negative Negative Positive activity Optimum pH 6.0-6.5 6.0 6.0 8.4 4.0 to 4.2 3.5 Inhibition Positive Positive Positive Positive Negative Negative by EDTA

As evident from Table 29, .alpha.-isomaltosylglucosaccharide-forming enzyme had outstandingly novel physicochemical properties completely different from those of conventionally known dextrin dextranase and transglucosidase.

EXPERIMENT 25

Formation of Cyclotetrasaccharide

The test on the formation of cyclotetrasaccharide by .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme was conducted using saccharides. For the test, it was prepared a solution of maltose, maltotriose, maltotetraose, maltopentaose, amylose, soluble starch, "PINE-DEX #100" (a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan), or glycogen from oyster commercialized by Wako Pure Chemical Industries Ltd., Tokyo, Japan.

To each of these solutions with a concentration of 0.5%, one unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2 and 10 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from C11 strain obtained by the method in Experiment 7-3, and the resulting mixture was subjected to an enzymatic reaction at 30.degree. C. and pH 6.0. The enzymatic conditions were the following four systems: (1) After the .alpha.-isomaltosylglucosaccharide-forming enzyme was allowed to act on a saccharide solution for 24 hours, the enzyme was inactivated by heating, and then the .alpha.-isomaltosyl-transferring enzyme was allowed to act on the resulting mixture for 24 hours and then inactivated by heating; (2) After the .alpha.-isomaltosylglucosaccharide-forming enzyme and the .alpha.-isomaltosyl-transferring enzyme were simultaneously allowed to act on a saccharide solution for 24 hours, the enzymes were inactivated by heating; (3) After only the .alpha.-isomaltosylglucosaccharide-forming enzyme was allowed to act on a saccharide solution for 24 hours, the enzyme was inactivated by heating; and (4) After only the .alpha.-isomaltosyl-transferring enzyme was allowed to act on a saccharide solution for 24 hours, the enzyme was inactivated by heating.

To determine the formation level of cyclotetrasaccharide in each reaction mixture after the inactivation of enzyme(s) by heating, the reaction mixture was treated with .alpha.-glucosidase and glucoamylase similarly as in Experiment 1 to hydrolyze the remaining reducing oligosaccharides, followed by the quantitation of cyclotetrasaccharide on HPLC. The results are in Table 30.

TABLE-US-00030 TABLE 30 Yield of cyclotetrasaccharide (%) Substrate A B C D Maltose 4.0 4.2 0.0 0.0 Maltotriose 10.2 12.4 0.0 0.0 Maltotetraose 11.3 21.5 0.0 0.0 Maltopentaose 10.5 37.8 0.0 0.0 Amylose 3.5 31.6 0.0 0.0 Soluble starch 5.1 38.2 0.0 0.0 Partial starch 6.8 63.7 0.0 0.0 hydrolyzate Glycogen 10.2 86.9 0.0 0.0 Note: The symbols "A", "B", "C" and "D" mean that .alpha.-isomaltosylglucosaccharide-forming enzyme was first allowed to act on a substrate and then .alpha.-isomaltosyl-transferring enzyme was allowed acted on the substrate, the .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme were allowed to coact on a substrate, only .alpha.-isomaltosylglucosaccharide-forming enzyme was allowed to act on a substrate, and only .alpha.-isomaltosyl-transferring enzyme was allowed to act on a substrate.

As evident from the results in Table 30, no cyclotetrasaccharide was formed from any of the saccharides tested by the single action of either .alpha.-isomaltosylglucosaccharide-forming enzyme or .alpha.-isomaltosyl-transferring enzyme, but cyclotetrasaccharide was formed by the coaction of these enzymes. It was revealed that the formation level of cyclotetrasaccharide was relatively low, i.e., about 11% or lower, when .alpha.-isomaltosyl-transferring enzyme was allowed to act on the saccharides after the action of .alpha.-isomaltosylglucosaccharide-forming enzyme, while the formation level was increased when the enzymes were allowed to coact on any of the saccharides tested, particularly, it was increased to about 87% and about 64% when the enzymes were allowed to coact on glycogen and partial starch hydrolyzate, respectively.

Based on the reaction properties of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme, the formation mechanism of cyclotetrasaccharide by the coaction of these enzymes is estimated as follows: (1) .alpha.-Isomaltosylglucosaccharide-forming enzyme acts on a glucose residue at the non-reducing end of an .alpha.-1,4 glucan chain of glycogen and partial starch hydrolyzates, etc., and intermolecularly transfers the glucose residue to OH-6 of the glucose residue at the non-reducing end of other .alpha.-1,4 glucan chain of glycogen and partial starch hydrolyzates, etc., to form an .alpha.-1,4 glucan chain having an .alpha.-isomaltosyl residue at the non-reducing end; (2) .alpha.-Isomaltosyl-transferring enzyme acts on the .alpha.-1,4 glucan chain having an .alpha.-isomaltosyl residue at the non-reducing end and intermolecularly transfers the isomaltosyl residue to C-3 of a glucose residue at the non-reducing end of other .alpha.-1,4 glucan chain having an isomaltosyl residue at the non-reducing end to form an .alpha.-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl residue at the non-reducing end; (3) Then, .alpha.-isomaltosyl-transferring enzyme acts on the .alpha.-1,4 glucan chain having an isomaltosyl-1,3-isomaltosyl residue at the non-reducing end and releases the isomaltosyl-1,3-isomaltosyl residue from the .alpha.-1,4 glucan chain via the intramolecular transferring reaction to cyclize the released isomaltosyl-1,3-isomaltosyl residue into cyclotetrasaccharide; (4) From the released .alpha.-1,4 glucan chain, cyclotetrasaccharide is successively formed through the sequential steps (1) to (3). Thus, it is estimated that the coaction of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme increases the formation of cyclotetrasaccharide in such a cyclic manner as indicated above.

EXPERIMENT 26

Influence of Liquefaction Degree of Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calcium carbonate, adjusted to pH 6.0, and then mixed with 0.2 to 2.0% per gram starch of "TERMAMYL 60L.TM.", an .alpha.-amylase specimen commercialized by Novo Indutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95.degree. C. for 10 min. Thereafter, the reaction mixture was autoclaved at 120.degree. C. for 20 min, promptly cooled to about 35.degree. C. to obtain a liquefied starch solution with a DE (dextrose equivalent) of 3.2 to 20.5. To the liquefied starch solution were added two units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2, and 20 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from C11 strain obtained by the method in Experiment 7-3, followed by an incubation at 35.degree. C. for 24 hours. After completion of the reaction, the reaction mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes. Then, the reaction mixture thus obtained was treated with .alpha.-glucosidase and glucoamylase similarly as in Experiment 1 to hydrolyze the remaining reducing oligosaccharides, followed by quantifying the formed cyclotetrasaccharide on HPLC. The results are in Tale 31.

TABLE-US-00031 TABLE 31 Amount of .alpha.-amylase Yield of per starch (%) DE cyclotetrasaccharide (%) 0.2 3.2 54.5 0.4 4.8 50.5 0.6 7.8 44.1 1.0 12.5 39.8 1.5 17.3 34.4 2.0 20.5 30.8

As evident from the results in Table 31, it was revealed that the formation of cyclotetrasaccharide by the coaction of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme is influenced by the liquefaction degree of starch, i.e., the lower the liquefaction degree or the lower the DE, the more the yield of cyclotetrasaccharide from starch increases. On the contrary, the higher the liquefaction degree or the higher the DE, the lower the yield of cyclotetrasaccharide from starch decreases. It was revealed that a suitable liquefaction degree is a DE of about 20 or lower, preferably, a DE of about 12 or lower, more preferably, a DE of about five or lower.

EXPERIMENT 27

Influence of the Concentration of Partial Starch Hydrolyzate

Aqueous solutions of "PINE-DEX #100", a partial starch hydrolyzate with a DE of about two to about five, having a final concentration of 0.5 to 40%, were prepared and respectively admixed with one unit/g solid, d.s.b., of the purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2 and 10 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from C11 strain obtained by the method in Experiment 7-3, followed by the coaction of these enzymes at 30.degree. C. and pH 6.0 for 48 hours. After completion of the enzymatic reaction, the reaction mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes, and then treated with .alpha.-glucosidase and glucoamylase similarly as in Experiment 1 to hydrolyze the remaining reducing oligosaccharides, followed by quantifying the formed cyclotetrasaccharide on HPLC. The results are in Table 32.

TABLE-US-00032 TABLE 32 Concentration of Yield of PINE-DEX (%) cyclotetrasaccharide (%) 0.5 63.6 2.5 62.0 5 60.4 10 57.3 15 54.6 20 51.3 30 45.9 40 35.9

As evident from the results in Table 32, the yield of cyclotetrasaccharide was about 64% at a low concentration of 0.5%, while it was about 40% at a high concentration of 40%. The fact indicates that the yield of cyclotetrasaccharide increases depending on the concentration of partial starch hydrolyzate as a substrate. The result revealed that the yield of cyclotetrasaccharide increased as the decrease of concentration of partial starch hydrolyzate.

EXPERIMENT 28

Influence of the Addition of Cyclodextrin Glucanotransferase

A 15% aqueous solution of "PINE-DEX #100", a partial starch hydrolyzate, was prepared and admixed with one unit/g solid, d.s.b., of the purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2, 10 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from C11 strain obtained by the method in Experiment 7-3, and 0 to 0.5 unit/g solid, d.s.b., of CGTase from a microorganism of the species Bacillus stearothermophilus, followed by the coaction of these enzymes at 30.degree. C. and pH 6.0 for 48 hours. After completion of the reaction, the reaction mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes, and then treated with .alpha.-glucosidase and glucoamylase similarly as in Experiment 1 to hydrolyze the remaining reducing oligosaccharides, followed by quantifying the formed cyclotetrasaccharide on HPLC. The results are in Table 33.

TABLE-US-00033 TABLE 33 Amount of CGTase added Yield of (unit) cyclotetrasaccharide (%) 0 54.6 2.5 60.1 5 63.1 10 65.2

As evident from the Table 33, it was revealed that the addition of CGTase increased the yield of cyclotetrasaccharide.

EXPERIMENT 29

Preparation of Isomaltose-releasing Enzyme

A liquid medium, consisting of 3.0% (w/v) of dextran, 0.7% (w/v) of peptone, 0.2% (w/v) of dipotassium phosphate, 0.05% (w/v) of magnesium sulfate heptahydrate, and water, was placed in 500-ml Erlenmeyer flasks in a volume of 100 ml each, autoclaved at 121.degree. C. for 20 minutes for sterilization, cooled, inoculated with a stock culture of Arthrobacter globiformis T6 strain (IAM 12103), and incubated at 27.degree. C. for 48 hours under rotary shaking conditions of 230 rpm to obtain a seed culture. About 20 L of a fresh preparation of the same nutrient culture medium as used in the above culture were placed in a 30-L fermentor, sterilized by heating, cooled to 27.degree. C., inoculated with 1% (v/v) of the seed culture, and further incubated for about 72 hours while stirring under aeration-agitation conditions at 27.degree. C. and pH 6.0 to 8.0. After completion of the culture, the resultant culture, having about 16.5 units/ml of isomaltodextranase activity, was centrifuged at 10,000 rpm for 30 min to obtain about 18 L of a supernatant, having about 16 units/ml of the enzyme, in a total enzyme activity of about 288,000 units. The activity of isomaltodextranase was assayed by providing, as a substrate solution, four milliliters of 1.25% (w/v) of an aqueous dextran solution in the form of 0.1M acetate buffer (pH 5.5), adding one milliliter of an enzyme solution, subjecting the mixture to an enzymatic reaction at 40.degree. C. for 20 min, sampling one milliliter of the reaction mixture, adding two milliliters of the Somogyi copper solution to suspend the enzymatic reaction, and quantifying the reducing power of the formed isomaltose by the Somogyi-Nelson's method. One unit activity of isomaltodextranase is defined as the enzyme amount that forms a reducing power corresponding to one micromole of isomaltose per minute. About 18 L of the culture supernatant were concentrated with a UF membrane into about two liters, salted out in an 80% ammonium sulfate solution, and allowed to stand at 4.degree. C. for 24 hours. The resulting precipitate was collected by centrifugation at 10,000 rpm for 30 min, dissolved in 5 mM phosphate buffer (pH 6.8), and dialyzed against a fresh preparation of the same buffer as used in the above to obtain about 400 ml of a dialyzed solution. The solution as a crude enzyme solution thus obtained was fed to ion-exchange chromatography using two liters of "SEPABEADS FP-DA13" gel. The component with isomaltodextranase activity did not adsorb on the gel and it was eluted in non-adsorbed fractions. The non-adsorbed fractions with the desired enzyme activity were collected, pooled, salted out in an 80% ammonium sulfate solution, and allowed to stand at 4.degree. C. for 24 hours. The resulting precipitate was collected by centrifugation at 10,000 rpm for 30 min, dissolved in 5 mM phosphate buffer (pH 6.8), and dialyzed against a fresh preparation of the same buffer as used in the above to obtain about 500 ml of a dialyzed solution having an isomaltodextranase activity of 161,000 units.

EXPERIMENT 30

Preparation of Isomaltose from .alpha.-isomaltosylglucosaccharide and Cyclotetrasaccharide

To a 0.2% aqueous solution of panose, .alpha.-isomaltosylmaltose, .alpha.-isomaltosyltriose, .alpha.-isomaltosyltetraose, or cyclotetrasaccharide was added 100 units/g solid, d.s.b., of an isomaltodextranase specimen, obtained by the method in Experiment 29, where 3,000 units/g solid, d.s.b., of the specimen was also used for the aqueous solution with cyclotetrasaccharide. The mixture was subjected to an enzymatic reaction at 40.degree. C. and pH 5.5 for 24 hours and heated at 100.degree. C. for 20 min to suspend the enzymatic reaction. The saccharide composition of the resulting mixture was analyzed on HPLC using column of "MCIGEL CK04SS", a column commercialized by Mitsubishi Chemical Industries, Ltd., Tokyo, Japan; an inner column temperature of 80.degree. C.; a flow rate of 0.5 ml/min of water as an eluate; and a detector of "RI-8012", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. The results are in Table 34.

TABLE-US-00034 TABLE 34 Saccharide as reaction product Enzyme (peak area (%) on HPLC) Substrate (unit) G1 IM G2 G3 G4 A IMG1 100 35 65 0 0 0 0 IMG2 100 0 51 49 0 0 0 IMG3 100 0 41 0 59 0 0 IMG4 100 0 35 0 0 65 0 Cyclotetrasaccharide 100 0 22 0 0 0 78 3,000 0 100 0 0 0 0 Note: In the table, the symbols "IMG1", "IMG2", "IMG3" and "IMG4" mean panose, .alpha.-isomaltosylmaltose, .alpha.-isomaltoglucotriose, and isomaltoglucotetraose, respectively. The symbols "G1", "G2", "G3" and "G4" mean glucose, isomaltose, maltose, maltotriose, and maltotetraose, respectively. The symbol "A" means an intermediate formed during the formation of isomaltose from cyclotetrasaccharide.

As evident from the results in Table 34, it was revealed that, when isomaltodextranase was allowed to act on .alpha.-isomaltosylglucosaccharides, only glucose and isomaltose were formed from panose as a substrate; only isomaltose and maltose were formed from .alpha.-isomaltosylmaltose as a substrate; only isomaltose and maltotriose were formed from .alpha.-isomaltosyltriose; and only isomaltose and maltotetraose were formed from .alpha.-isomaltosyltetraose as a substrate. It was also found that only isomaltose was formed via the product "A" from cyclotetrasaccharide as a substrate.

Then, the purification and isolation of the above-identified product A were conducted as follows: The product A was subjected to "YMC-PACK ODS-AR355-15S-15 12A", a separatory HPLC column commercialized by YMC Co., Ltd., Tokyo, Japan, for purifying and isolating. Thus, the product A with a purity of at least 98.2% was obtained in a yield of about 7.2% from the reaction product of cyclotetrasaccharide.

The product A was subjected to methyl analysis and NMR analysis in a usual manner. The result on the methyl analysis is in Table 35. While the results on the NMR analyses are respectively in FIG. 46 for .sup.1H-NMR spectrum and in FIG. 47 for .sup.13C-NMR spectrum. The data on assignment of the product A is tabulated in Table 36.

TABLE-US-00035 TABLE 35 Analyzed methyl compound Composition ratio 2,3,4-Trimethyl compound 2.00 2,3,6-Trimethyl compound 0.92 2,3,4,6-Tetramethyl compound 0.88

TABLE-US-00036 TABLE 36 Glucose No. Carbon No. NMR chemical shift (ppm) a 1a 100.7 2a 74.2 3a 75.8 4a 72.3 5a 74.5 6a 63.2 b 1b 102.1 2b 74.3 3b 75.9 4b 72.6 5b 74.2 6b 68.0 c 1c 100.6 2c 72.8 3c 83.0 4c 72.0 5c 73.1 6c 62.9 e 1e 94.9(.alpha.), 98.8(.beta.) 2e 74.1(.alpha.), 76.6(.beta.) 3e 75.8(.alpha.), 78.7(.beta.) 4e 72.1(.alpha.), 72.1(.beta.) 5e 72.6(.alpha.), 76.9(.beta.) 6e 68.3(.alpha.), 68.3(.beta.)

From these results, the product A, formed as an intermediate during the formation of isomaltose from cyclotetrasaccharide by the action of isomaltodextranase, was revealed as a tetrasaccharide in the form of a ring-opened cyclotetrasaccharide, formed as a result of the hydrolysis of any one of the 1,3-linkages of cyclotetrasaccharide, represented by Formula 3, i.e., .alpha.-glucosyl-(1.fwdarw.6)-.alpha.-glucosyl-(1.fwdarw.3)-.alpha.-gluco- syl-(1.fwdarw.6)-glucose (or ring-opened tetrasaccharide). .alpha.-D-Glcp-(1.fwdarw.6)-.alpha.-D-Glcp-(1.fwdarw.3)-.alpha.-D-Glcp-(1- .fwdarw.6)-.alpha.-D-Glcp Formula 3:

Based on these results, it can be concluded that the mechanism of the action of isomaltodextranase on .alpha.-isomaltosylglucosaccharide is as follows:

Isomaltodextranase acts on an .alpha.-isomaltosylglucosaccharide, having a 6-O-.alpha.-glucosyl group at the non-reducing end, as a substrate, and specifically hydrolyzes the .alpha.-1,4 linkage between the isomaltosyl residue at the non-reducing end and the resting glucose or maltooligosaccharide residue to form isomaltose and glucose or a maltooligosaccharide. Then the enzyme also acts on cyclotetrasaccharide as a substrate and hydrolyzes the .alpha.-1,3 linkage for ring-opening to form ring-opened cyclotetrasaccharide as an intermediate, and further acts on the formed ring-opened cyclotetrasaccharide and hydrolyzes the .alpha.-1,3 linkage thereof to form isomaltose.

EXPERIMENT 31

Formation of Isomaltose from Different Substrates

Using different saccharides, the formation mechanism of the action of .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase was examined. Maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, amylose, or "PINE-DEX #100", a partial starch hydrolyzate commercialized by Matsutani Chemical Ind., Tokyo, Japan, was dissolved in water to give a final concentration of five percent. Also, calcium chloride was dissolved in water to give a final concentration of 1 mM. To each of the above aqueous solutions 0.2 unit/g solid, d.s.b., of the purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained in Experiment 7-2, and 100 units/g solid, d.s.b., of an isomaltodextranase specimen obtained by the method in Experiment 29, followed by an enzymatic reaction at 40.degree. C. and pH 5.5. The reaction conditions used were the following two systems:

(1) After contacting the .alpha.-isomaltosylglucosaccharide-forming enzyme with any of the substrates for 65 hours, the enzyme was inactivated by heating, then the isomaltodextranase was allowed to act on the resulting mixture for 65 hours and inactivated by heating.

(2) After contacting the .alpha.-isomaltosylglucosaccharide-forming enzyme and the isomaltodextranase with any of the substrates in combination for 65 hours, the enzymes were inactivated by heating.

The resulting heated reaction mixtures were assayed for isomaltose yield on HPLC. The results are in Table 37:

TABLE-US-00037 TABLE 37 Yield of isomaltose (%) Substrate Sequential use* Combination use** Maltose 6.6 7.0 Maltotriose 15.7 18.7 Maltotetraose 15.8 45.4 Maltopentaose 15.3 55.0 Maltohexaose 10.1 58.1 Maltoheptaose 8.5 63.6 Amylose 4.0 64.9 Partial starch hydrolyzate 3.8 62.7 Note: The symbols "*" and "**" mean that .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase were allowed to act on a substrate in this order and in combination, respectively.

As evident from the results in Table 37, all of the saccharides tested formed isomaltose through the action of .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase. It was revealed that the sequential use of .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase in this order only gave a low yield of isomaltose as low as less than about 15%, while the combination use of the enzymes gave an improved yield of isomaltose, particularly, up to a high yield of 60% or higher of isomaltose when the enzymes were allowed to coact on maltoheptaose, amylose, or partial starch hydrolyzate. The isomaltose formation mechanism by the combination use of .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase is speculated as follows based on their enzymatic reaction properties:

(1) .alpha.-Isomaltosylglucosaccharide-forming enzyme acts on the glucose residue at the non-reducing end of an .alpha.-1,4 glucan chain such as of amylose and partial starch hydrolyzates, and intermolecularly transfers the glucose residue to the hydroxyl group at C-6 of the glucose residue at the non-reducing end of another .alpha.-1,4 glucan chain to form an .alpha.-1,4 glucan chain having an .alpha.-isomaltosyl group at the non-reducing end.

(2) Isomaltodextranase acts on the formed .alpha.-1,4 glucan chain, having an .alpha.-isomaltosyl group at the non-reducing end, and hydrolyzes the .alpha.-1,4 linkage between the isomaltosyl group and the resting .alpha.-1,4 glucan chain to form/release isomaltose and an .alpha.-1,4 glucan chain with a reduced glucose polymerization degree by two.

(3) The released .alpha.-1,4 glucan chain sequentially receives the enzymatic reactions of (1) and (2) and forms another isomaltose.

As explained above, it can be speculated that, when used in combination, .alpha.-Isomaltosylglucosaccharide-forming enzyme and isomaltodextranase repeatedly act on their substrates to form isomaltose and increase the yield.

EXPERIMENT 32

Effect of the Addition of Isoamylase

An aqueous solution of "PINE-DEX #100", a partial starch hydrolyzate, with a final concentration of five percent and 1 mM calcium chloride, was prepared, admixed with 0.2 unit/g starch, d.s.b., of the purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained in Experiment 7-2, 100 units/g starch, d.s.b., of an isomaltodextranase specimen obtained by the method in Experiment 29, and 0 to 250 units/g starch, d.s.b., of an isoamylase specimen of a microorganism of the species Pseudomonas amyloderamosa commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, followed by an enzymatic reaction at 40.degree. C. and pH 5.5 for 65 hours. Thereafter, the resulting mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes. The formed isomaltose was quantified by HPLC. The results are in Table 38.

TABLE-US-00038 TABLE 38 Isoamylase added Yield of isomaltose (unit) (%) 0 62.7 50 65.1 250 71.1

As evident from the results in Table 38, it was revealed that the addition of isoamylase increases the yield of isomaltose.

EXPERIMENT 33

Influence of the Concentration of Partial Starch Hydrolyzate

Eight types of aqueous solutions, having different concentrations of "PINE-DEX #100", a partial starch hydrolyzate, with a DE of about two to about five, having a final concentration of 1 to 40%, and containing 1 mM calcium chloride, were prepared, admixed with 0.2 unit/g starch, d.s.b., of the purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained in Experiment 7-2, 100 units/g starch, d.s.b., of an isomaltodextranase specimen obtained by the method in Experiment 29, and 250 units/g starch, d.s.b., of an isoamylase specimen of a microorganism of the species Pseudomonas amyloderamosa commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, followed by an enzymatic reaction at 40.degree. C. and pH 5.5 for 65 hours. Thereafter, the resulting mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes. The formed isomaltose was quantified by HPLC. The results are in Table 39.

TABLE-US-00039 TABLE 39 Concentration of "PINE DEX 100" Yield of isomaltose (%) (%) 1 73.0 2.5 72.8 5 71.1 10 67.0 15 63.7 20 60.7 30 55.4 40 50.7

As evident from the results in Table 39, it was revealed that the yield of isomaltose increased up to about 73% at a low concentration of one percent of partial starch hydrolyzate, but decreased to about 51% at a concentration of 40% of partial starch hydrolyzate, meaning that the yield of isomaltose varies depending on the concentration of partial starch hydrolyzate as a substrate.

EXPERIMENT 34

Influence of the Degree of Liquefied Starch

A 15% corn starch suspension was prepared, admixed with 0.1% calcium carbonate, adjusted to pH 6.0, and then mixed with 0.2 to 2.0% per gram starch of "TERMAMYL 60L.TM.", an .alpha.-amylase specimen commercialized by Novo Indutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95.degree. C. for 10 min. Thereafter, the reaction mixture was autoclaved at 120.degree. C., promptly cooled to about 40.degree. C. to obtain a liquefied starch solution with a DE of 3.2 to 20.5. The liquefied starch solution was adjusted to give a final starch concentration of 5% and to pH 5.5, and then mixed with 0.2 unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2, 100 units/g solid, d.s.b., of a purified specimen of isomaltodextranase obtained by the method in Experiment 29, and 250 units/g solid, d.s.b., of an isoamylase specimen from Pseudomonas amyloderamosa commercialized by Hayashibara Biochemical laboratories, Inc., Okayama, Japan, followed by an incubation at 40.degree. C. for 65 hours. After completion of the reaction, the reaction mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes. The formed isomaltose was quantified by HPLC. The results are in Table 40.

TABLE-US-00040 TABLE 40 Amount of .alpha.-amylase Yield of per g starch (%) DE isomaltose (%) 0.2 3.2 71.5 0.4 4.8 71.0 0.6 7.8 66.2 1.0 12.5 59.8 1.5 17.3 53.2 2.0 20.5 47.9

As evident from the results in Table 40, it was revealed that the formation of isomaltose by the coaction of .alpha.-isomaltosylglucosaccharide-forming enzyme and isomaltodextranase is influenced by the liquefaction degree of starch, i.e., the lower the liquefaction degree or the lower the DE, the higher the yield of isomaltose from starch increases. On the contrary, the higher the liquefaction degree or the higher the DE, the lower the yield of isomaltose from starch decreases. It was revealed that a suitable liquefaction degree is a DE of about 20 or lower, preferably, a DE of about 12 or lower, more preferably, a DE of about five or lower.

EXPERIMENT 35

Effect of the Addition of CGTase and Glucoamylase

An aqueous solution, containing 20% of "PINE-DEX #100", a partial starch hydrolyzate, and 1 mM calcium chloride, was prepared, mixed with 0.2 unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2, 100 units/g solid, d.s.b., of a purified specimen of isomaltodextranase obtained by the method in Experiment 29, and 0 to 0.5 unit/g solid, d.s.b., of a CGTase specimen from Bacillus stearothermophilus commercialized by Hayashibara Biochemical laboratories, Inc., Okayama, Japan, followed by incubating the mixture at 40.degree. C. and pH 5.5 for 65 hours and heating the resulting mixture at 100.degree. C. for 15 min to inactivate the remaining enzymes. To the mixture thus obtained was added 20 units/g starch, d.s.b., of "XL-4.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated at 50.degree. C. for 24 hours, and heated at 100.degree. C. for 20 min to inactivate the remaining enzyme. The formed isomaltose was quantified on HPLC. The results are in Table 41.

TABLE-US-00041 TABLE 41 Amount of CGTase added (unit/g solid, d.s.b.) Yield of isomaltose (%) 0 60.7 0.1 62.9 0.25 65.0 0.5 66.4

As evident from the results in Table 41, it was revealed that the addition of CGTase to the enzymatic reaction system of isomaltodextranase and .alpha.-isomaltosylglucosaccharide-forming enzyme increased the yield of isomaltose. In the above enzymatic reaction system, the glucoamylase was used to form isomaltose from saccharides, composed of isomaltose linked with one or more D-glucose residues, and to release the D-glucose residue(s) therefrom, resulting in an increased yield of isomaltose.

EXPERIMENT 36

Formation of Isomaltose

About one hundred liters of an aqueous solution of phytoglycogen from corn, commercialized by Q.P. Corporation, Tokyo, Japan, were adjusted to give a concentration of 4% (w/v) and pH 6.0, heated to 30.degree. C., admixed with one unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C11 strain obtained by the method in Experiment 7-2, 10 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from C11 strain obtained by the method in Experiment 7-3, followed by incubating the mixture for 48 hours and heating the resulting mixture at 100.degree. C. for 10 min to inactivate the remaining enzymes. The mixture thus obtained was sampled for quantifying the yield of cyclotetrasaccharide on HPLC, revealing that it had about 84% of cyclotetrasaccharide in terms of sugar composition, where HPLC was carried out using "SHOWDEX KS-801.TM. column", Showa Denko K.K., Tokyo, Japan, at a column temperature of 60.degree. C. and a flow rate of 0.5 ml/min of water, and using "RI-8012.TM.", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. The above mixture was adjusted to pH 5.0 and 45.degree. C., admixed with 1,500 units/g starch, d.s.b. of "TRANSGLUCOSIDASE L AMANO.TM.", an .alpha.-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g starch, d.s.b., of "XL-4.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated for 24 hours to hydrolyze the remaining reducing oligosaccharides, etc. The resulting mixture was adjusted to pH 5.8, kept at 90.degree. C. for one hour to inactivate the remaining enzymes, and filtered to remove insoluble substances. The filtrate was concentrated to give a concentration of about 16% with "HOLLOSEP.RTM. HR 5155PI", a reverse osmotic membrane, Toyobo Co., Ltd., Tokyo, Japan, and in a usual manner decolored, desalted, filtered, and concentrated to obtain about 6.2 kg of a saccharide solution with a solid content of about 3,700 g, d.s.b. The saccharide solution was fed to a column packed with about 225 L of "AMBERLITE CR-1310 (Na.sup.+-form)", a strong-acid cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a column temperature of 60.degree. C. and a flow rate of about 45 L/h. While the saccharide composition of eluate from the column was monitoring by the above-identified HPLC, fractions of cyclotetrasaccharide with a purity of at least 98% were collected, and in a usual manner desalted, decolored, filtered, and concentrated to obtain about 7.5 kg of a saccharide solution with a solid content of about 2,500 g, d.s.b. HPLC analysis for saccharide composition of the solution thus obtained revealed that it contained cyclotetrasaccharide with a purity of about 99.5%. The resulting saccharide solution with cyclotetrasaccharide was concentrated into an about 50% solution by an evaporator, and about five kilograms of which were placed in a cylindrical plastic vessel, cooled from 65.degree. C. to 20.degree. C. over about 20 hours under gentle stirring conditions to crystallize cyclotetrasaccharide. Then, the resulting massecuite was centrifugally separated to collect 1,360 g of crystalline cyclotetrasaccharide by wet weight, and dried at 60.degree. C. for three hours to obtain 1,170 g of a crystalline cyclotetrasaccharide powder. HPLC analysis for saccharide composition of the powder revealed that it had a purity of cyclotetrasaccharide crystal as high as at least about 99.9%.

The above crystalline cyclotetrasaccharide powder was dissolved in deionized water, adjusted to give a concentration of one percent, pH 5.5 and 50.degree. C., admixed with 500 units/g solids, d.s.b., of an isomaltodextranase specimen prepared by the method in Experiment 29, and enzymatically reacted at pH 5.5 and 50.degree. C. for 70 hours. Thereafter, the resulting mixture was heated to and kept at 95.degree. C. for 10 min, cooled, and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 50%. Thus, a high isomaltose content syrup was obtained in a yield of about 95%, d.s.b., to the solid contents. HPLC analysis for saccharide composition of the syrup revealed that it contained 96.1% of isomaltose, 2.8% of ring-opened tetrasaccharide, and 1.1% of other saccharides.

Four hundred grams of the above syrup were in a usual manner placed in an autoclave with 0.1 g/solids, d.s.b., of "N154.TM.", an alkaline-developed Raney nickel catalyst commercialized by Nikki Chemical Co., Ltd., Yokohama, Japan, stirred at 100.degree. C. for four hours while keeping the inner hydrogen pressure at 100 kg/cm.sup.2, and stirred at 120.degree. C. for another two hours to effect hydrogenation. After standing to cool, the hydrogenated products were collected from the autoclave and passed through an activated charcoal layer about 1-cm thick to remove the Raney nickel catalyst. The filtrate was in a usual manner desalted, purified, and concentrated to give a concentration of about 73%. The concentrate was placed in a cylindrical plastic vessel, admixed with 0.1% to the solids, d.s.b., of a crystalline isomaltitol powder as a seed, cooled to 35.degree. C. over about 20 hours under gentle stirring conditions to crystallize isomaltitol. Then, the resulting mixture was separated by a centrifuge to collect isomaltitol crystal, and dried in vacuo at 80.degree. C. for 20 hours to obtain about 168 g of isomaltitol crystal.

The product had an isomaltitol purity of about 99.9% or higher, d.s.b. The results on x-ray powder diffraction pattern, .sup.1H-NMR spectrum, and .sup.13C-NMR spectrum of the product are respectively shown in FIGS. 48 to 50. Based on the data, the product was judged to be isomaltitol.

The following Example A explains isomaltose or saccharides comprising the same and the process for producing isomaltitol and/or saccharides comprising the same; and Example B explains the uses of isomaltitol and/or saccharides comprising the same:

EXAMPLE A-1

About one hundred liter of an aqueous solution of phytoglycogen from corn commercialized by Q.P. Corporation, Tokyo, Japan, was adjusted to give a concentration of 4% (w/v) and pH 6.0, heated to 30.degree. C., and admixed with one unit/g starch of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, and 12 units/g starch of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from Bacillus globisporus N75 strain obtained by the method in Experiment 11-3, followed by an enzymatic reaction for 48 hours and a heat treatment at 100.degree. C. for 10 min to inactivate the remaining enzymes. The mixture thus obtained was sampled for quantifying the yield of cyclotetrasaccharide on HPLC, revealing that it had about 80% of cyclotetrasaccharide in terms of sugar composition, where HPLC was carried out using "SHOWDEX.TM. KS-801 column", Showa Denko K.K., Tokyo, Japan, at a column temperature of 60.degree. C. and a flow rate of 0.5 ml/min of water, and "RI-8012", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. The above mixture was adjusted to pH 5.0 and 45.degree. C., admixed with 1,500 units/g starch, d.s.b. of "TRANSGLUCOSIDASE L AMANO.TM.", an .alpha.-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g starch, d.s.b., of "XL-4.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated for 24 hours to hydrolyze the remaining reducing oligosaccharides, etc. The resulting mixture was adjusted to pH 5.8, kept at 90.degree. C. for one hour to inactivate the remaining enzymes, and filtered to remove insoluble substances. The filtrate was concentrated to give a concentration of about 16% with "HOLLOSEP.RTM. HR 5155PI", a reverse osmotic membrane, Toyobo Co., Ltd., Tokyo, Japan, and in a usual manner decolored, desalted, filtered, and concentrated to obtain about 6.0 kg of a saccharide solution with a solid content of about 3,500 g, d.s.b. The saccharide solution was fed to a column packed with about 225 L of "AMBERLITE CR-1310 (Na.sup.+-form)", a strong-acid cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, and chromatographed at a column temperature of 60.degree. C. and a flow rate of about 45 L/h. While the saccharide composition of eluate from the column was monitoring by the above-identified HPLC, fractions of cyclotetrasaccharide with a purity of at least 80% were collected, and in a usual manner desalted, decolored, filtered, and concentrated into a saccharide solution.

HPLC analysis for saccharide composition of the saccharide solution thus obtained revealed that it contained cyclotetrasaccharide with a purity of about 95.5%. The resulting saccharide solution with cyclotetrasaccharide was concentrated in vacuo into a powder containing cyclotetrasaccharide. The powder was dissolved in deionized water, adjusted to give a concentration of one percent, pH 5.5 and 50.degree. C., and admixed with 80 units/g solids, d.s.b., of an isomaltose-releasing enzyme obtained by the method in Experiment 29, followed by an enzymatic reaction at pH 5.5 and 50.degree. C. for 70 hours. Thereafter, the resulting mixture was sequentially heated to 95.degree. C., kept at the temperature for 10 min, cooled, and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 43.0%. Thus, a high isomaltose content syrup was obtained in a yield of about 95%, d.s.b., to the solid contents. HPLC analysis for saccharide composition of the syrup revealed thus obtained that it contained 43.1% of isomaltose, 37.8% of ring-opened tetrasaccharide, and 13.8% of cyclotetrasaccharide.

The product has a satisfactory moisture-retaining ability, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-2

A powder containing cyclotetrasaccharide, obtained by the method in Example A-1, was dissolved in deionized water, adjusted to give a concentration of one percent, pH 5.5 and 50.degree. C., and admixed with 500 units/g solids, d.s.b., of an isomaltose-releasing enzyme obtained by the method in Experiment 29, followed by an enzymatic reaction at pH 5.5 and 50.degree. C. for 70 hours. Thereafter, the resulting mixture was sequentially heated to 95.degree. C., kept at the temperature for 10 min, cooled, and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 75%. Thus, a high isomaltose content syrup was obtained in a yield of about 90%, d.s.b., to the solid contents. HPLC analysis for saccharide composition of the syrup revealed that it contained 92.8% of isomaltose, 2.7% of ring-opened tetrasaccharide, and 4.5% of other saccharides.

The product has a satisfactory moisture-retaining ability, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-3

An about 20% corn starch suspension was prepared, admixed with 0.1% calcium carbonate, adjusted to pH 6.5, and then mixed with 0.3% per gram starch of "TERMAMYL 60L.TM.", an .alpha.-amylase specimen commercialized by Novo Indutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95.degree. C. for 15 min. Thereafter, the reaction mixture was autoclaved at 120.degree. C. for 20 min, and promptly cooled to about 50.degree. C. to obtain a liquefied starch solution with a DE of about four. To the liquefied solution were added 0.2 unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from N75 strain obtained by the method in Experiment 11-2, 100 units/g solid, d.s.b., of an isomaltodextranase specimen obtained by the method in Experiment 29, 250 units/g solid, d.s.b., of an isoamylase specimen from Pseudomonas amyloderamosa commercialized by Hayashibara Biochemical laboratories, Inc., Okayama, Japan, and 0.5 unit/g starch of a CGTase specimen from Bacillus stearothermophilus commercialized by Hayashibara Biochemical laboratories, Inc., Okayama, Japan, followed by an incubation at 50.degree. C. and pH 5.5 for 65 hours. After completion of the reaction, the reaction mixture was heated at 100.degree. C. for 15 min to inactivate the remaining enzymes. To the resulting mixture was added 20 units/g starch of "XL-4.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated at 50.degree. C. for 24, and heated at 100.degree. C. for 20 min to inactivate the remaining enzyme. The reaction mixture was cooled and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 60%. Thus, a high isomaltose content syrup was obtained in a yield of about 95%, d.s.b., to the solid contents. HPLC analysis for saccharide composition of the syrup revealed that it contained 62.9% of isomaltose, 30.1% of glucose, and 7.0% of other saccharides.

The product has a satisfactory moisture-retaining ability, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-4

A high isomaltose content syrup, obtained by the method in Example A-3, as a saccharide solution, was column chromatographed to increase the concentration of isomaltose using "AMBERLITE CR-1310 (Na.sup.+-form)", a strong-acid cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, in such a manner of packing the above resin to 10 stainless-steel columns equipped with an inner jacket having 12.5 cm in diameter, cascading the columns in series to give a total column bed depth of 16 m, applying the above syrup in a volume of 1.5% (v/v) to the volume of resin, fractionating and purifying the syrup by feeding hot water heated to 40.degree. C. to the columns at a space velocity (SV) of 0.2, collecting fractions rich in isomaltose while monitoring the sugar composition of the eluates, and concentrating the pooled eluates up to give a concentration of 75% to obtain a high isomaltose content syrup, consisting of, on a dry solid basis, 4.3% glucose, 90.5% isomaltose, 3.5% of other saccharides, and 1.7% of trisaccharide or higher, in a yield of about 45%.

The product has a satisfactory moisture-retaining ability, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-5

An isomaltose content syrup, obtained by the method in Example A-1, was hydrogenated in accordance with the method in Experiment 36, and the resulting mixture was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 73%. The concentrate was spray dried in a usual manner to obtain a high isomaltitol content powder, containing 43.3% of isomaltitol, 37.8% of ring-opened tetrasaccharide, 13.8% of cyclotetrasaccharide, and 3.5% of other sugar alcohols, in a yield of about 80%.

The product is substantially a non-reducing saccharide which does not substantially cause the Maillard reaction and substantially has non-hygroscopicity, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, health supplements, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-6

A 20% tapioca starch suspension was prepared, admixed with 0.1% calcium carbonate, adjusted to pH 6.5, and then mixed with 0.3% per gram starch of "TERMAMYL 60L.TM.", an .alpha.-amylase specimen commercialized by Novo Indutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95.degree. C. for 15 min. Thereafter, the reaction mixture was autoclaved at 120.degree. C. for 20 min, and promptly cooled to about 40.degree. C. to obtain a liquefied starch solution with a DE of about four. To the liquefied starch solution were added 0.2 unit/g solid, d.s.b., of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from C9 strain obtained by the method in Experiment 4-2, 100 units/g solid, d.s.b., of a purified specimen of .alpha.-isomaltodextranase obtained by the method in Experiment 29, 250 units/g of an isoamylase specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and 0.5 unit/g of a CGTase specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, followed by an incubation at pH 5.5 and 40.degree. C. for 64 hours. After completion of the reaction, the reaction mixture was sequentially heated at 95.degree. C. for 30 min, cooled to 50.degree. C., admixed with 10 units/g of "GLUCOZYME.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, subjected to an enzymatic reaction for 24 hours, heated to 95.degree. C., incubated at 95.degree. C. for 30 min, cooled, and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 50% (w/v). Thus, a high isomaltose content syrup, containing 11.0% of glucose, 66.5% of isomaltose, 2.4% of disaccharide other than isomaltose, and 20.1% of trisaccharide or higher, was obtained in a yield of about 95%, d.s.b.

The high isomaltose content syrup thus obtained was hydrogenated in accordance with the method in Experiment 36, followed by removing the Raney Nickel catalyst from the mixture. The resulting mixture was decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, concentrated, and dried in vacuo to obtain a high isomaltitol content powder in a yield of about 85%.

The powder contained 12.3% of sorbitol, 66.7% of isomaltitol, and 21.0% of other sugar alcohols.

The product substantially does not have reducibility and does not cause the Maillard reaction, and it has a relatively low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc. Thus the product can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-7

In accordance with the method in Experiment 1, Bacillus globisporus C9 strain (FERM BP-7143) was cultured in a fermentor for 48 hours. Thereafter, the culture was membrane filtered to remove the cells to collect about 18 L of a filtrate which was then concentrated with a UF membrane to yield about one liter of an enzyme concentrate containing 8.8 units/ml of .alpha.-isomaltosylglucosaccharide-forming enzyme and 26.7 units/ml of .alpha.-isomaltosyl-transferring enzyme. While, an about 27% corn starch suspension was prepared, admixed with 0.1% calcium carbonate, adjusted to pH 6.5, and then mixed with 0.3% per gram starch of "TERMAMYL 60L.TM.", an .alpha.-amylase specimen commercialized by Novo Indutri A/S, Copenhagen, Denmark, followed by an enzymatic reaction at 95.degree. C. for 15 min. Thereafter, the reaction mixture was autoclaved at 120.degree. C. for 20 min and promptly cooled to about 40.degree. C. to obtain a liquefied starch solution with a DE of about four. To the liquefied starch solution were added 0.25 ml of the above enzyme solution of .alpha.-isomaltosylglucosaccharide-forming enzyme and .alpha.-isomaltosyl-transferring enzyme, 100 units/g starch of an isomaltodextranase specimen obtained by the method in Experiment 29, 250 units/g starch of an isoamylase specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, and 0.5 unit/g starch of a CGTase specimen commercialized by Hayashibara Biochemical Laboratories, Inc., Okayama, Japan, followed by an incubation at pH 5.5 and 40.degree. C. for 70 hours. After completion of the reaction, the reaction mixture was sequentially heated to 95.degree. C., incubated at 95.degree. C. for 10 min, adjusted to 50.degree. C., admixed with 20 units/g starch of "GLUCOZYME.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, enzymatically reacted for 24, and heated to and incubated at 95.degree. C. for 30 min. The resulting mixture was cooled and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 50%. Thus, a high isomaltose content syrup, containing 32.6% glucose, 59.4% of isomaltose, 1.2% of disaccharide other than isomaltose, 6.8% of trisaccharide or higher, was obtained in a yield of about 95%, d.s.b.

The high isomaltose content syrup thus obtained was hydrogenated in accordance with the method in Experiment 36, followed by removing the Raney Nickel catalyst from the mixture in a usual manner. The resulting mixture was decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, concentrated to give a concentration of about 50%. Thus, a high isomaltitol content syrup was obtained in a yield of about 85%, d.s.b.

The product contained 33.4% of sorbitol, 59.1% of isomaltitol, 6.4% of sugar alcohols other than sorbitol and isomaltitol, and 1.1% of cyclotetrasaccharide. The product substantially does not has reducibility and does not cause the Maillard reaction, and it has a relatively low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc. Thus the product can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-8

A high isomaltose content syrup, obtained by the method in Example A-7, as a saccharide solution, was column chromatographed to increase the content of isomaltose using "AMBERLITE CR-1310 (Na.sup.+-form)", a strong-acid cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, in such a manner of packing the above resin to 10 stainless-steel columns equipped with an inner jacket having 12.5 cm in diameter, cascading the columns in series to give a total column bed depth of 16 m, applying the above syrup in a volume of 1.5% (v/v) to the volume of resin, fractionating and purifying the syrup by feeding hot water heated to 40.degree. C. to the columns at SV 0.2, collecting fractions rich in isomaltose while monitoring the sugar composition of the eluates, and concentrating the pooled eluates up to give a concentration of 55% to obtain a high isomaltose content syrup, consisting of, on a dry solid basis, 4.8% glucose, 88.0% isomaltose, 4.1% of other saccharides, and 3.1% of trisaccharide or higher, in a yield of about 55%.

The high isomaltose content syrup thus obtained was hydrogenated in accordance with the method in Experiment 36, followed by removing the Raney Nickel catalyst from the mixture in a usual manner. The resulting mixture was decolored with an activated charcoal and desalted for purification with ion-exchange resins in H- and OH-forms to obtain a high isomaltitol content syrup, consisting of, on a dry solid basis, 4.9% sorbitol, 88.1% isomaltitol, and 7.0% of other sugar alcohols, in a yield of about 90%.

The high isomaltitol content syrup thus obtained was concentrated to give a concentration of about 73%, and the concentrate was placed in a crystallizer, admixed with a crystalline isomaltitol powder as a seed in an amount of 0.1%, d.s.b., to the solid contents, and allowed to crystallize maltitol at 25.degree. C. for about 20 hours. The mixture was separated by a centrifuge, followed by separately collecting the resulting isomaltitol crystal and syrup. The isomaltitol crystal thus obtained was dried in vacuo at 80.degree. C. for 20 hours to obtain a crystalline maltitol powder in a yield of about 39%, d.s.b. In accordance with the above method, the above syrup was column chromatographed using a strong-acid cation exchange resin, followed collecting high isomaltitol content fractions with an isomaltitol content of about 88%, d.s.b. The fractions were pooled, purified, concentrated, crystallized, and separated to collect isomaltitol crystal which was then aged and dried in vacuo to obtain a crystalline isomaltitol powder in a yield of about 20%, d.s.b. By combining the powder thus obtained and the previously obtained powder, a crystalline isomaltitol powder was obtained in a total yield of about 59%, d.s.b.

The product contained, on a dry solid basis, 0.7% sorbitol, 98.0% isomaltitol, and 1.3% sugar alcohol. The product has non-reducibility, non-hygroscopicity, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, health supplements, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-9

About one hundred liter of an aqueous solution of phytoglycogen from corn commercialized by Q.P. Corporation, Tokyo, Japan, was adjusted to give a concentration of 4% (w/v) and pH 6.0, heated to 30.degree. C., and admixed with one unit/g starch of a purified specimen of .alpha.-isomaltosylglucosaccharide-forming enzyme from Bacillus globisporus N75 strain obtained by the method in Experiment 11-2, and 12 units/g starch of a purified specimen of .alpha.-isomaltosyl-transferring enzyme from Bacillus globisporus N75 strain obtained by the method in Experiment 11-3, followed by an enzymatic reaction for 48 hours and a heat treatment at 100.degree. C. for 10 min to inactivate the remaining enzymes. The mixture thus obtained was sampled for quantifying the yield of cyclotetrasaccharide on HPLC, revealing that it contained about 80% of cyclotetrasaccharide in terms of sugar composition, where HPLC was carried out using "SHOWDEX KS-80.TM. column", Showa Denko K.K., Tokyo, Japan, at a column temperature of 60.degree. C. and a flow rate of 0.5 ml/min of water, and "RI-8012.TM.", a differential refractometer commercialized by Tosoh Corporation, Tokyo, Japan. The above mixture was adjusted to pH 5.0 and 45.degree. C., admixed with 1,500 units/g starch, d.s.b. of "TRANSGLUCOSIDASE L AMANO.TM.", an .alpha.-glucosidase commercialized by Amano Pharmaceutical Co., Ltd., Aichi, Japan, and 75 units/g starch, d.s.b., of "XL-4.TM.", a glucoamylase specimen commercialized by Nagase Biochemicals, Ltd., Kyoto, Japan, incubated for 24 hours to hydrolyze the remaining reducing oligosaccharides, etc. The resulting mixture was adjusted to pH 5.8, kept at 90.degree. C. for one hour to inactivate the remaining enzymes, and filtered to remove insoluble substances. The filtrate was concentrated to give a concentration of about 16% (w/v) with "HOLLOSEP.RTM. HR 5155PI", a reverse osmotic membrane, Toyobo Co., Ltd., Tokyo, Japan, and in a usual manner decolored, desalted, filtered, and concentrated into a saccharide solution. Then, the saccharide solution was adjusted to give concentration of about one percent, pH 5.5, and 50.degree. C., admixed with 80 units/g solids of an isomaltodextranase specimen prepared by the method in Experiment 29, and subjected to an enzymatic reaction at pH 5.5 and 50.degree. C. for 70 hours. Thereafter, the reaction mixture was heated to and incubated at 95.degree. C. for 10 min, cooled, and filtered. The filtrate was in a usual manner decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 43% (w/v). Thus, an isomaltose content syrup was obtained in a yield of about 95%, d.s.b. HPLC analysis for saccharide composition of the syrup revealed that it contained 35.5% of isomaltose. The isomaltose content syrup thus obtained was hydrogenated in accordance with the method in Experiment 36, followed by removing the Raney Nickel catalyst from the mixture in a usual manner. The resulting mixture was decolored with an activated charcoal, desalted for purification with ion-exchange resins in H- and OH-forms, and concentrated to give a concentration of about 40%. The resulting concentrate was column chromatographed using a column packed with about 225 L of "AMBERLITE CR-1310 (Na.sup.+-form)", a strong-acid cation exchange resin commercialized by Japan Organo Co., Ltd., Tokyo, Japan, at a column temperature of 60.degree. C. and a flow rate of about 45 L/h, followed by collecting fractions containing isomaltitol with a purity of at least 50% while monitoring the saccharide composition on the above-identified HPLC. The fractions were pooled, and in a usual manner desalted for purification with ion-exchange resins in H- and OH-forms, decolored, filtered, and concentrated to give a concentration of about 50%, d.s.b. Thus a high isomaltitol content syrup, containing 65.3% of isomaltitol, 13.8% of reduced ring-opened cyclotetrasaccharide, 5.2% of cyclotetrasaccharide, and 15.7% of sugar alcohols such as sorbitol, was obtained in a yield of about 78%, d.s.b.

The product is substantially free of the Maillard reaction, and it has a satisfactory osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, non-fermentability, ability of preventing the retrogradation of starch, etc. Thus the product can be arbitrarily used in various food products, health foods, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE A-10

An high isomaltitol content syrup, consisting of 4.9% sorbitol, 88.1% of isomaltitol, and 7.0% of other sugar alcohols, was concentrated to give a concentration of about 88%. The concentrate was placed in a crystallizer, admixed with crystalline isomaltitol powder in an amount of two percent to the contents, d.s.b., heated to 50.degree. C., incubated for two hours under gentle stirring conditions, transferred to a vat, allowed to stand at 20.degree. C. for four days to crystallize and solidify the contents. The resulting solid product was pulverized by a cutter and dried to obtain a crystalline isomaltitol powder in a yield of about 90%.

The product has non-reducibility, non-hygroscopicity, low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc., it can be arbitrarily used in various food products, health foods, health supplements, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods.

EXAMPLE B-1

Sweetener.

To 0.8 part by weight of a crystalline isomaltitol powder, obtained by the method in Example A-8, were added to homogeneity 0.2 part by weight of "TREHA.RTM.", an .alpha.,.alpha.-trehalose product commercialized by Hayashibara Shoji, Inc., Okayama, Japan, 0.01 part by weight of ".alpha.G SWEET.TM.", an .alpha.-glycosyl stevioside commercialized by Toyo Sugar Refining Co., Ltd., Tokyo, Japan, and 0.01 part by weight of "ASPARTAME.TM." or L-aspartyl phenylalanine methyl ester. The mixture was subjected to a granulator to obtain a granular sweetener. The product, which does not substantially has hygroscopicity but has satisfactory moisture-retaining ability and low sweetness, is a stable sweetener containing isomaltitol free from causing deterioration even when stored at ambient temperature.

EXAMPLE B-2

Hard Candy

To 100 parts by weight of a 55% sucrose solution were added 50 parts by weight of a high isomaltitol content syrup obtained by the method in Example A-7, and the mixture was concentrated by heating under a reduced pressure to give a moisture content of less than two percent. The concentrate was admixed with 0.6 part by weight of citric acid and adequate amounts of a lemon flavor and a color, followed by shaping the resulting mixture into a hard candy. The product, which is only less colored by the Maillard reaction and is satisfactory in biting property, flavor, and taste, is a stable, high quality hard candy free from causing crystallization of sucrose and having lesser hygroscopicity.

EXAMPLE B-3

Chewing Gum

Three parts by weight of a gum base were melted by heating to an extent to be softened and then admixed with two parts by weight of anhydrous crystalline maltitol, two parts by weight of xylitol, two parts by weight of a high isomaltitol content syrup obtained by the method in Example A-7, and one part by weight of hydrous crystalline .alpha.,.alpha.-trehalose, monohydrate, and further mixed with adequate amounts of a flavor and a color. The mixture was in a usual manner kneaded by a roll and then shaped and packed to obtain a chewing gum. The product is a relatively low cariogenic, caloric chewing gum having a satisfactory texture, flavor, and taste.

EXAMPLE B-4

Chocolate

Forty parts by weight of a cacao paste, 10 parts by weight of a cacao butter, and 50 parts by weight of a crystalline isomaltitol obtained by the method in Example A-8 were mixed, and the mixture was fed to a refiner to reduce the granular size and then placed in a conche and kneaded at 50.degree. C. over two days and nights. During the processing, 0.5 part by weight of lecithin was added to the kneaded mixture and well dispersed therein. Thereafter, the resulting mixture was adjusted to 31.degree. C. with a thermo controller, and then poured into a mold just before solidification of the butter, deairated by a vibrator, and solidified by passing through a cooling tunnel kept at 10.degree. C. over 20 min. The solidified contents were removed from the mold and packed to obtain a chocolate.

The product substantially has no hygroscopicity but has satisfactory color, gloss, and internal texture; smoothly melts in the mouth; and has a high quality sweetness and a mild taste and flavor. The product can be useful as a low caloric, cariogenic chocolate.

EXAMPLE B-5

Powdery Peptide

One part by weight of 40% of "HINUTE S.TM.", a peptide solution of edible soy beans commercialized by Fuji Oil Co., Ltd., Tokyo, Japan, was mixed with two parts by weight of a high isomaltitol content syrup obtained by the method in Example A-6, and the resultant mixture was placed in a plastic vat, dried in vacuo at 50.degree. C., and pulverized to obtain a powdery peptide. The product, which is only less colored by the Maillard reaction, is useful as a material for low caloric confectionery and also as a material for controlling intestinal function, health food, and hardly assimilable dietary fiber for oral or tube fed liquid diets.

EXAMPLE B-6

Bath Salt

One part by weight of a peel juice of "yuzu" (a Chinese lemon) was admixed with 10 parts by weight of a crystalline isomaltitol powder obtained in accordance with the method in Example A-10, and 10 parts by weight of anhydrous crystalline cyclotetrasaccharide, followed by crystallizing hydrous cyclotetrasaccharide crystal, penta- or hexa-hydrate, aging the crystal and pulverizing the aged crystal to obtain an isomaltitol and cyclotetrasaccharide powder with a yuzu extract.

To five parts by weight of the powder thus obtained were added 90 parts by weight of roast salt, two parts by weight of hydrous crystalline .alpha.,.alpha.-trehalose, one part by weight of silicic anhydride, and 0.5 part by weight of ".alpha.G HESPERIDIN.TM.", .alpha.-glucosyl hesperidin commercialized by Hayashibara Shoji, Inc., Okayama, Japan, to obtain a bath salt.

The product is a high quality bath salt enriched with yuzu flavor and used by diluting in a bathtub with hot water by 100-10,000 folds, and it moisturizes and smooths the skin and does not make you feel cold after a bath.

EXAMPLE B-7

Cosmetic Cream

Two parts by weight of polyoxyethylene glycol monostearate, five parts by weight of glyceryl monostearate, self-emulsifying, two parts by weight of a high isomaltitol content syrup obtained by the method in Example A-7, one part by weight of ".alpha.G RUTIN.TM.", .alpha.-glucosyl rutin commercialized by Hayashibara Shoji, Inc., Okayama, Japan, one part by weight of liquid petrolatum, 10 parts by weight of glyceryl tri-2-ethylhexanoate, and an adequate amount of an antiseptic were dissolved by heating in a usual manner. The resultant solution was admixed with two parts by weight of L-lactic acid, five parts by weight of 1,3-butylene glycol, and 66 parts by weight of refined water, and the resultant mixture was emulsified by a homogenizer and admixed with an adequate amount of a flavor while stirring to obtain a cosmetic cream. The product exhibits an antioxidant activity and has a relatively high stability, and these render it advantageously useful as a high quality sunscreen, skin-refining agent, and skin-whitening agent.

EXAMPLE B-8

Toothpaste

A toothpaste was obtained by mixing 45 parts by weight of calcium secondary phosphate, 1.5 parts by weight of sodium lauryl sulfate, 25 parts by weight of glycerine, 0.5 part by weight of polyoxyethylene sorbitan laurate, 15 parts by weight of a high isomaltitol content syrup obtained by the method in Example A-2, 0.02 part by weight of saccharine, 0.05 part by weight of an antiseptic, and 13 parts by weight of water. The product has an improved after taste and satisfactory feeling after use without lowering the detergent power of the surfactant.

EXAMPLE B-9

Solid Preparation for Fluid Diet

A composition was prepared by mixing 100 parts by weight of a high isomaltitol content powder obtained by the method in Example A-5, 200 parts by weight of hydrous crystalline .alpha.,.alpha.-trehalose, 200 parts by weight of a high maltotetraose content powder, 270 parts by weight of an egg yolk powder, 209 parts by weight of a skim milk powder, 4.4 parts by weight of sodium chloride, 1.8 parts by weight of potassium chloride, four parts by weight of magnesium sulfate, 0.01 part by weight of thiamine, 0.1 part by weight of sodium L-ascorbate, 0.6 part by weight of vitamin E acetate, and 0.04 part by weight of nicotinamide. Twenty-five gram aliquots of the composition were injected into moisture-proof laminated small bags which were then heat-sealed to obtain the desired product.

The product is a fluid diet having a satisfactory action of improving intestinal function. In use, one bag of the product is dissolved in about 150 to about 300 ml of water into a fluid diet and arbitrarily administered orally or administered intubationally into the nasal cavity, stomach, intestines, etc.

EXAMPLE B-10

Tablet

Fifty parts by weight of aspirin were sufficiently mixed with 14 parts by weight of a crystalline isomaltitol powder obtained by the method in Example A-7, and four parts by weight of corn starch. The resulting mixture was in a usual manner tabletted by a tabletting machine to obtain a tablet, 680 mg and 5.25 mm in thickness.

The tablet, processed by using the filler-imparting ability of isomaltitol, has substantially no hygroscopicity, but has a sufficient physical strength and satisfactory degradability in water.

EXAMPLE B-11

Sugar Coated Tablet

A crude tablet as a core, 150 mg weight, was sugar coated with a first solution, consisting of 40 parts by weight of a crystalline isomaltitol obtained by the method in Experiment 36, two parts by weight of pullulan having an average molecular weight of 200,000, 30 parts by weight of water, 25 parts by weight of talc, and three parts by weight of titanium oxide until the total weight increased to about 230 mg. The resultant tablet was further sugar coated with a second solution, consisting of 65 parts by weight of a powder of hydrous crystalline cyclotetrasaccharide, penta- or hexa-hydrate, one part by weight of pullulan, and 34 parts by weight of water. Then, the resulting tablet was glossed with a liquid wax into a sugar coated tablet having a satisfactory gloss and appearance. The product has a relatively high shock tolerance and retains its initial high quality for a relatively-long period of time.

EXAMPLE B-12

Ointment for Treating Trauma

To 100 parts by weight of a high isomaltitol content syrup, obtained by the method in Example A-7, and 300 parts by weight of maltose were added 50 parts by weight of methanol dissolving three parts by weight of iodine, and further added 200 parts by weight of a 10% (w/v) aqueous pullulan solution to obtain the desired product with an adequate extensibility and adhesiveness. The product is a high-valued ointment in which the volatilization of iodine and methanol is well inhibited by isomaltitol and is relatively less in property change during storage.

Because the product exerts a sterilizing action by iodine and acts as an energy-supplementing agent on living cells due to maltose, it shortens the curing term and well cures the affected parts and surfaces.

INDUSTRIAL APPLICABILITY

As described above, the present invention relates to a novel method for producing isomaltose and isomaltitol, more particularly, to a process for producing isomaltose, which comprises the steps of contacting a saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with one or more .alpha.-isomaltosylglucosaccharide-forming enzymes derived from Bacillus globisporus N75 strain (FERM BP-7591), Arthrobacter globiformis A19 strain (FERM BP-7590) and Arthrobacter ramosus S1 strain (FERM BP-7592) in the presence or the absence of an .alpha.-isomaltosyl-transferring enzyme derived from Bacillus globisporus N75 strain (FERM BP-7591) and/or Arthrobacter globiformis A19 strain (FERM BP-7590) to form .alpha.-isomaltosylglucosaccharides having the .alpha.-1,6 glucosidic linkage as the linkage of non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, and/or to form a saccharide with the structure of cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.}; contacting the resulting mixture with isomaltose-releasing enzyme to form isomaltose; and collecting the produced isomaltose. The present invention also relates to a method for producing isomaltitol, which comprises the steps of contacting a saccharide, having the .alpha.-1,4 glucosidic linkage as the linkage of non-reducing end and a glucose polymerization degree of at least two, with .alpha.-isomaltosylglucosaccharide-forming enzyme to form .alpha.-isomaltosylglucosaccharides having the .alpha.-1,6 glucosidic linkage as the linkage of non-reducing end and the .alpha.-1,4 glucosidic linkage other than the above linkage, and/or to form a saccharide with the structure of cyclo{.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucopyr- anosyl-(1.fwdarw.6)-.alpha.-D-glucopyranosyl-(1.fwdarw.3)-.alpha.-D-glucop- yranosyl-(1.fwdarw.}; contacting the resulting mixture with isomaltose-releasing enzyme to form isomaltose; hydrogenating either the resulting mixture directly or the isomaltose separated from the mixture to form isomaltitol; and collecting the formed isomaltitol. The present invention further relates to saccharide compositions containing isomaltose and/or isomaltitol, and uses thereof. According to the present invention, saccharide compositions containing isomaltose and/or isomaltitol, which are useful in this art, can be produced on an industrial scale, at a relatively low cost and in a relatively high yield. The saccharide compositions of the present invention can be arbitrarily used in various food products, health foods, health supplements, feeds, pet foods including bait for fish, cosmetics, pharmaceuticals, and favorite foods because the compositions, which are substantially free of reducibility and the Maillard reaction, have satisfactory low sweetness, osmosis-controlling ability, filler-imparting ability, gloss-imparting ability, moisture-retaining ability, viscosity-imparting ability, ability of preventing crystallization of other saccharides, insubstantial fermentability, ability of preventing the retrogradation of starch, etc.

The present invention with these outstanding functions and effects is a significant invention that greatly contributes to this art.

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26 Bacillus globisporus al Ser Ser Leu Gly Asn Leu Ile acillus globisporus 2 Ile Asp Gly Val Tyr His Ala Pro Asn Gly 3 Bacillus globisporus 3 Ile Asp Gly Val Tyr His Ala Pro Tyr Gly 4 8 PRT Bacillus globisporus 4 Ile Asp Gly Val Tyr His Ala Pro PRT Bacillus globisporus 5 Asp Ala Ser Ala Asn Val Thr Thr PRT Bacillus globisporus 6 Trp Ser Leu Gly Phe Met Asn Phe PRT Bacillus globisporus 7 Asn Tyr Thr Asp Ala Trp Met Phe PRT Bacillus globisporus 8 Gly Asn Glu Met Arg Asn Gln Tyr PRT Bacillus globisporus 9 Ile Thr Thr Trp Pro Ile Glu Ser 8 PRT Bacillus globisporus Ala Phe Gly Leu Trp Met Ser 9 PRT Bacillus globisporus Val Ser Ala Leu Gly Asn Leu Leu 8 PRT Bacillus globisporus Phe Ser Asn Asn Pro Thr Val 8 PRT Bacillus globisporus Thr Val Asn Ala Pro Ala Ala 8 PRT Bacillus globisporus Glu Ala Glu Ser Ala Glu Leu 6 PRT Bacillus globisporus Trp Trp Met Ser Lys 8 PRT Bacillus globisporus Asp Gly Gly Glu Met Val Trp 8 PRT Bacillus globisporus Ile Tyr Leu Pro Gln Gly Asp Arthrobacter globiformis Pro Leu Gly Val Gln Arg Ala Gln Phe Gln Ser Gly RT Arthrobacter globiformis Thr Leu Asp Gly Val Trp His Asn Pro Tyr Gly Ala Asp Glu Leu Ala Thr Gln 2 PRT Arthrobacter ramosus 2hr Leu Ser Gly Val Phe His Gly Pro 2DNA Bacillus globisporus CDS (877)..(4728) 2ccggt ttttgtgaag tttggcagta ttcttccgat gaatttgaac gcgcaatatc 6ggcgg gaccattggc aacagcttga cgagctacac gaatctcgcg ttccgcattt cgcttgg gacaacaacg tacgactgga atgatgatat tggcggttcg gtgaaaacca cttctac agagcaatat gggttgaata aagaaaccgt gactgttcca gcgattaatt 24aagac attgcaagtg tttacgacta agccttcctc tgtaacggtg ggtggttctg 3gacaga gtacagtact ttaactgccc taacgggagc gtcgacaggc tggtactatg 36gtaca gaaattcact tacgtcaagc ttggttcaag tgcatctgct caatccgttg 42aatgg cgttaataag gtggaatatg aagcagaatt cggcgtgcaa agcggcgttt 48aacac gaaccatgca ggttatactg gtacaggatt tgtggacggc tttgagactc 54gacaa tgttgctttt gatgtttccg tcaaagccgc aggtacttat acgatgaagg 6gtattc atccggtgca ggcaatggct caagagccat ctatgtgaat aacaccaaag 66gacct tgccttgccg caaacaacaa gctgggatac atgggggact gctacgttta 72tcgct gagtacaggt ctcaacacgg tgaaagtcag ctatgatggt accagttcac 78attaa tttcgataac atcgcgattg tagagcaata aaaggtcggg agggcaagtc 84cttaa tttctaatcg aaagggagta tccttg atg cgt cca cca aac aaa 894 Met Arg Pro Pro Asn Lys att cca cgt att ctt gct ttt ttt aca gcg ttt acg ttg ttt ggt 942 Glu Ile Pro Arg Ile Leu Ala Phe Phe Thr Ala Phe Thr Leu Phe Gly cc ctt gcc ttg ctt cct gct ccg cct gcg cat gcc tat gtc agc 99hr Leu Ala Leu Leu Pro Ala Pro Pro Ala His Ala Tyr Val Ser 25 3c cta gga aat ctc att tct tcg agt gtc acc gga gat acc ttg acg r Leu Gly Asn Leu Ile Ser Ser Ser Val Thr Gly Asp Thr Leu Thr 4 cta act gtt gat aac ggt gcg gag ccg agt gat gac ctc ttg att gtt u Thr Val Asp Asn Gly Ala Glu Pro Ser Asp Asp Leu Leu Ile Val 55 6 caa gcg gtg caa aac ggt att ttg aag gtg gat tat cgt cca aat agc n Ala Val Gln Asn Gly Ile Leu Lys Val Asp Tyr Arg Pro Asn Ser 75 8a acg ccg agc gcg aag acg ccg atg ctg gat ccg aac aaa act tgg e Thr Pro Ser Ala Lys Thr Pro Met Leu Asp Pro Asn Lys Thr Trp 9ct gta gga gct acg att aat acg aca gcc aat cca atg acc atc r Ala Val Gly Ala Thr Ile Asn Thr Thr Ala Asn Pro Met Thr Ile act tcc aat atg aag att gag att acc aag aat cca gta cga atg r Thr Ser Asn Met Lys Ile Glu Ile Thr Lys Asn Pro Val Arg Met gtc aag aag gcg gac ggc act acg cta ttc tgg gaa cca tca ggc r Val Lys Lys Ala Asp Gly Thr Thr Leu Phe Trp Glu Pro Ser Gly gga ggg gta ttc tca gac ggt gtg cgc ttc ctt cat gcc aca ggg gat y Gly Val Phe Ser Asp Gly Val Arg Phe Leu His Ala Thr Gly Asp atg tat ggc atc cgg agc ttc aat gct ttt gat agc ggg ggt gac n Met Tyr Gly Ile Arg Ser Phe Asn Ala Phe Asp Ser Gly Gly Asp ctg cgg aat tcg tcc aat cat gcc gcc cat gcg ggt gaa cag gga u Leu Arg Asn Ser Ser Asn His Ala Ala His Ala Gly Glu Gln Gly tcc ggt ggt ccg ctt att tgg agt acg gca gga tat gga cta tta p Ser Gly Gly Pro Leu Ile Trp Ser Thr Ala Gly Tyr Gly Leu Leu 22gat agc gat ggc ggc tac ccc tat aca gat agc aca acc ggt caa l Asp Ser Asp Gly Gly Tyr Pro Tyr Thr Asp Ser Thr Thr Gly Gln 2225 23ag ttt tat tat ggt ggg acc cct cct gag gga cgt cgt tat gcg t Glu Phe Tyr Tyr Gly Gly Thr Pro Pro Glu Gly Arg Arg Tyr Ala 235 24aa caa aac gtg gaa tat tat att atg ctc gga acc ccc aag gaa att s Gln Asn Val Glu Tyr Tyr Ile Met Leu Gly Thr Pro Lys Glu Ile 256cc gac gta ggg gaa atc aca ggg aaa ccg cct atg ctg cct aag t Thr Asp Val Gly Glu Ile Thr Gly Lys Pro Pro Met Leu Pro Lys 265 27gg tcg ctt gga ttc atg aac ttt gag tgg gat acg aat caa acg gag p Ser Leu Gly Phe Met Asn Phe Glu Trp Asp Thr Asn Gln Thr Glu 289cg aat aat gtg gat acg tat cgt gcc aaa aat atc ccc ata gat e Thr Asn Asn Val Asp Thr Tyr Arg Ala Lys Asn Ile Pro Ile Asp 295 33tac gcc ttc gac tat gac tgg aaa aag tac ggg gaa acc aac tat a Tyr Ala Phe Asp Tyr Asp Trp Lys Lys Tyr Gly Glu Thr Asn Tyr 3325 ggt gaa ttc gcg tgg aat acg act aat ttc cct tct gcg tca acg act y Glu Phe Ala Trp Asn Thr Thr Asn Phe Pro Ser Ala Ser Thr Thr 334ta aag tca aca atg gat gct aaa ggc atc aaa atg atc gga att r Leu Lys Ser Thr Met Asp Ala Lys Gly Ile Lys Met Ile Gly Ile 345 35ca aaa ccc cgc atc gtt acg aag gat gct tca gcg aat gtg acg acc r Lys Pro Arg Ile Val Thr Lys Asp Ala Ser Ala Asn Val Thr Thr 367gg acg gac gcg aca aat ggc ggt tat ttt tat cca ggc cat aac 2 Gly Thr Asp Ala Thr Asn Gly Gly Tyr Phe Tyr Pro Gly His Asn 375 389at cag gat tat ttc att ccc gta act gtg cgt agt atc gat cct 2 Tyr Gln Asp Tyr Phe Ile Pro Val Thr Val Arg Ser Ile Asp Pro 395 4tac aat gct aac gaa cgt gct tgg ttc tgg aat cat tcc aca gat gcg 2 Asn Ala Asn Glu Arg Ala Trp Phe Trp Asn His Ser Thr Asp Ala 442at aaa ggg atc gta ggt tgg tgg aat gac gag acg gat aaa gta 2 Asn Lys Gly Ile Val Gly Trp Trp Asn Asp Glu Thr Asp Lys Val 425 43ct tcg ggt gga gcg tta tat tgg ttt ggc aat ttc aca aca ggc cac 2238 Ser Ser Gly Gly Ala Leu Tyr Trp Phe Gly Asn Phe Thr Thr Gly His 445ct cag acg atg tac gaa ggg ggg cgg gct tac acg agt gga gcg 2286 Met Ser Gln Thr Met Tyr Glu Gly Gly Arg Ala Tyr Thr Ser Gly Ala 455 467gt gtt tgg caa acg gct aga acc ttc tac cca ggt gcc cag cgg 2334 Gln Arg Val Trp Gln Thr Ala Arg Thr Phe Tyr Pro Gly Ala Gln Arg 475 48at gcg act acg ctt tgg tct ggc gat att ggc att caa tac aat aaa 2382 Tyr Ala Thr Thr Leu Trp Ser Gly Asp Ile Gly Ile Gln Tyr Asn Lys 49gaa cgg atc aat tgg gct gcc ggg atg cag gag caa agg gca gtt 243lu Arg Ile Asn Trp Ala Ala Gly Met Gln Glu Gln Arg Ala Val 55cta tcc tcc gtg aac aat ggc cag gtg aaa tgg ggc atg gat acc 2478 Met Leu Ser Ser Val Asn Asn Gly Gln Val Lys Trp Gly Met Asp Thr 523ga ttc aat cag cag gat ggc acg acg aac aat ccg aat ccc gat 2526 Gly Gly Phe Asn Gln Gln Asp Gly Thr Thr Asn Asn Pro Asn Pro Asp 535 545ac gct cgg tgg atg cag ttc agt gcc cta acg cct gtt ttc cga 2574 Leu Tyr Ala Arg Trp Met Gln Phe Ser Ala Leu Thr Pro Val Phe Arg 555 56tg cat ggg aac aac cat cag cag cgc cag cca tgg tac ttc gga tcg 2622 Val His Gly Asn Asn His Gln Gln Arg Gln Pro Trp Tyr Phe Gly Ser 578cg gag gag gcc tcc aaa gag gca att cag ctg cgg tac tcc ctg 267la Glu Glu Ala Ser Lys Glu Ala Ile Gln Leu Arg Tyr Ser Leu 585 59tc cct tat atg tat gcc tat gag aga agt gct tac gag aat ggg aat 27Pro Tyr Met Tyr Ala Tyr Glu Arg Ser Ala Tyr Glu Asn Gly Asn 66ctc gtt cgg cca ttg atg caa gcc tat cca aca gat gcg gcc gtc 2766 Gly Leu Val Arg Pro Leu Met Gln Ala Tyr Pro Thr Asp Ala Ala Val 6625 63at tac acg gat gct tgg atg ttt ggt gac tgg ctg ctg gct gca 28Asn Tyr Thr Asp Ala Trp Met Phe Gly Asp Trp Leu Leu Ala Ala 635 64ct gtg gta gat aaa cag cag acg agt aag gat atc tat tta ccg tct 2862 Pro Val Val Asp Lys Gln Gln Thr Ser Lys Asp Ile Tyr Leu Pro Ser 656ca tgg att gac tat gcg cga ggc aat gca ata act ggc ggt caa 29Ser Trp Ile Asp Tyr Ala Arg Gly Asn Ala Ile Thr Gly Gly Gln 665 67cc atc cga tat tcg gtt aat ccg gac acg ttg aca gac atg cct ctc 2958 Thr Ile Arg Tyr Ser Val Asn Pro Asp Thr Leu Thr Asp Met Pro Leu 689tt aaa aaa ggt gcc att att cca aca cag aaa gtg cag gat tac 3 Ile Lys Lys Gly Ala Ile Ile Pro Thr Gln Lys Val Gln Asp Tyr 695 77ggg cag gct tcc gtc act tcc gtt gat gtg gat gtg ttt ccg gat 3 Gly Gln Ala Ser Val Thr Ser Val Asp Val Asp Val Phe Pro Asp 7725 acg acg cag tcg agt ttc acg tac tac gat gat gat ggc gcc agt tat 3 Thr Gln Ser Ser Phe Thr Tyr Tyr Asp Asp Asp Gly Ala Ser Tyr 734at gag agc ggc act tat ttt aag caa aat atg act gct cag gat 3 Tyr Glu Ser Gly Thr Tyr Phe Lys Gln Asn Met Thr Ala Gln Asp 745 75at ggg tca ggc tcg tta agt ttt act tta gga gca aag agt ggc agt 3 Gly Ser Gly Ser Leu Ser Phe Thr Leu Gly Ala Lys Ser Gly Ser 767cg ccg gct ctc caa tcc tat atc gtt aag ctg cac ggt tct gct 3246 Tyr Thr Pro Ala Leu Gln Ser Tyr Ile Val Lys Leu His Gly Ser Ala 775 789ct tct gtt acg aat aac agc gca gct atg aca tct tat gca agc 3294 Gly Thr Ser Val Thr Asn Asn Ser Ala Ala Met Thr Ser Tyr Ala Ser 795 8ttg gaa gca tta aaa gct gct gct ggg gaa ggc tgg gcg act ggg aag 3342 Leu Glu Ala Leu Lys Ala Ala Ala Gly Glu Gly Trp Ala Thr Gly Lys 882tt tat ggg gat gtc acc tat gtg aaa gtg acg gca ggt aca gct 339le Tyr Gly Asp Val Thr Tyr Val Lys Val Thr Ala Gly Thr Ala 825 83ct tct aaa tct att gct gtt aca ggt gtt gct gcc gtg agc gca act 3438 Ser Ser Lys Ser Ile Ala Val Thr Gly Val Ala Ala Val Ser Ala Thr 845cg caa tac gaa gct gag gat gca tcg ctt tct ggc aat tcg gtt 3486 Thr Ser Gln Tyr Glu Ala Glu Asp Ala Ser Leu Ser Gly Asn Ser Val 855 867ca aag gcg tcc ata aac acg aat cat acc gga tat acg gga act 3534 Ala Ala Lys Ala Ser Ile Asn Thr Asn His Thr Gly Tyr Thr Gly Thr 875 88ga ttt gta gat ggt ttg ggg aat gat ggc gct ggt gtc acc ttc tat 3582 Gly Phe Val Asp Gly Leu Gly Asn Asp Gly Ala Gly Val Thr Phe Tyr 89aag gtg aaa act ggc ggt gac tac aat gtc tcc ttg cgt tat gcg 363ys Val Lys Thr Gly Gly Asp Tyr Asn Val Ser Leu Arg Tyr Ala 99gct tca ggc acg gct aag tca gtc agt att ttt gtt aat gga aaa 3678 Asn Ala Ser Gly Thr Ala Lys Ser Val Ser Ile Phe Val Asn Gly Lys 923tg aag tcc acc tcg ctc gct aat ctc gca aat tgg gac act tgg 3726 Arg Val Lys Ser Thr Ser Leu Ala Asn Leu Ala Asn Trp Asp Thr Trp 935 945ca caa tct gag aca ctg ccg ttg acg gca ggt gtg aat gtt gtg 3774 Ser Thr Gln Ser Glu Thr Leu Pro Leu Thr Ala Gly Val Asn Val Val 955 96cc tat aaa tat tac tcc gat gcg gga gat aca ggc aat gtt aac atc 3822 Thr Tyr Lys Tyr Tyr Ser Asp Ala Gly Asp Thr Gly Asn Val Asn Ile 978ac atc acg gta cct ttt gcg cca att atc ggt aag tat gaa gca 387sn Ile Thr Val Pro Phe Ala Pro Ile Ile Gly Lys Tyr Glu Ala 985 99ag agt gct gag ctt tct ggt ggc agc tca ttg aac acg aac cat 39Ser Ala Glu Leu Ser Gly Gly Ser Ser Leu Asn Thr Asn His tgg tac tac agt ggt acg gct ttt gta gac ggt ttg agt gct gta 396yr Tyr Ser Gly Thr Ala Phe Val Asp Gly Leu Ser Ala Val 2ggc gcg cag gtg aaa tac aac gtg aat gtc cct agc gca gga agt 4 Ala Gln Val Lys Tyr Asn Val Asn Val Pro Ser Ala Gly Ser 35 t cag gta gcg ctg cga tat gcg aat ggc agt gca gcg acg aaa 4 Gln Val Ala Leu Arg Tyr Ala Asn Gly Ser Ala Ala Thr Lys 5acg ttg agt act tat atc aat gga gcc aag ctg ggg caa acc agt 4 Leu Ser Thr Tyr Ile Asn Gly Ala Lys Leu Gly Gln Thr Ser 65 t acg agt cct ggt acg aat tgg aat gtt tgg cag gat aat gtg 4 Thr Ser Pro Gly Thr Asn Trp Asn Val Trp Gln Asp Asn Val 8caa acg gtg acg tta aat gca ggg gca aac acg att gcg ttt aaa 4 Thr Val Thr Leu Asn Ala Gly Ala Asn Thr Ile Ala Phe Lys 95 c gac gcc gct gac agc ggg aac atc aac gta gat cgt ctg ctt 423sp Ala Ala Asp Ser Gly Asn Ile Asn Val Asp Arg Leu Leu ctt tca act tcg gca gcg gga acg ccg gtt tct gag cag aac ctg 4275 Leu Ser Thr Ser Ala Ala Gly Thr Pro Val Ser Glu Gln Asn Leu 25 a gac aat ccc ggt ttc gag cgt gac acg agt caa acc aat aac 432sp Asn Pro Gly Phe Glu Arg Asp Thr Ser Gln Thr Asn Asn 4tgg att gag tgg cat cca ggc acg caa gct gtt gct ttt ggc gtt 4365 Trp Ile Glu Trp His Pro Gly Thr Gln Ala Val Ala Phe Gly Val 55 t agc ggc tca acc acc aat ccg ccg gaa tcc ccg tgg tcg ggt 44Ser Gly Ser Thr Thr Asn Pro Pro Glu Ser Pro Trp Ser Gly 7gat aag cgt gcc tac ttc ttt gca gca ggt gcc tat caa caa agc 4455 Asp Lys Arg Ala Tyr Phe Phe Ala Ala Gly Ala Tyr Gln Gln Ser 85 c cat caa acc att agt gtt cct gtt aat aat gta aaa tac aaa 45His Gln Thr Ile Ser Val Pro Val Asn Asn Val Lys Tyr Lys ttt gaa gcc tgg gtc cgc atg aag aat acg acg ccg acg acg gca 4545 Phe Glu Ala Trp Val Arg Met Lys Asn Thr Thr Pro Thr Thr Ala aga gcc gaa att caa aac tat ggc gga tca gcc att tat gcg aac 459la Glu Ile Gln Asn Tyr Gly Gly Ser Ala Ile Tyr Ala Asn 3ata agt aac agc ggt gtt tgg aaa tat atc agc gta agt gat att 4635 Ile Ser Asn Ser Gly Val Trp Lys Tyr Ile Ser Val Ser Asp Ile 45

g gtg acc aat ggt cag ata gat gtt gga ttt tac gtg gat tca 468al Thr Asn Gly Gln Ile Asp Val Gly Phe Tyr Val Asp Ser 6cct ggt gga act acg ctt cac att gat gat gtg cgc gta acc aaa 4725 Pro Gly Gly Thr Thr Leu His Ile Asp Asp Val Arg Val Thr Lys 75 a taaacaaaca accagctctc ccgttaatgg gagggctggt tgtttgttat 4778 Gln gataatccat ctatttagag tggattaaac gttttgaagt gcttgctgaa cttcttgcac 4838 aatggataac gccgcggtgc gggcacttga gaaagcacgt tctgcaagct ctcccttacc 4898 tgtacagccg tctccgcaga agtagaaagg aacgttttcc acgcgtatcg gcagcagatt 4958 attggaagca atgtttttca cgctggaaac catcgctttc ttggaaaccc gtttcacggc 5gacatcg cgccagcctg gataatgttt atcaaataag gcttccattt ggaggttctt 5ttccagg tacgctttgc gctgctcctc gttatcaaag cggtcgctta agtatgcgat 5ttgcagc agctgcccgc cttctggtac tagtgtgtga tc 53869 DNA Bacillus globisporus CDS (2422) 22 tcatcgctac tggcaatcgg attcaaacaa atggctgcag ctcgcacaga cgattgtgga 6aatat ctgatttaac catacggcgg tcgcgattga ttgaatagga ttcgtggccg aatattg aaagggggga tgcgtggagc agcgcatgca cggcgaggaa taactgttgt agcctct aagtcattca tgtttagcaa acaaatttcg gtacgaaagg ggaaatgttt 24at gta agg aat cta aca ggt tca ttc cga ttt tct ctc tct ttt 288 Met Tyr Val Arg Asn Leu Thr Gly Ser Phe Arg Phe Ser Leu Ser Phe ctc tgt ttc tgt ctc ttc gtc ccc tct att tat gcc att gat ggt 336 Leu Leu Cys Phe Cys Leu Phe Val Pro Ser Ile Tyr Ala Ile Asp Gly 2 gtt tat cat gcg cca tac gga atc gat gat ctg tac gag att cag gcg 384 Val Tyr His Ala Pro Tyr Gly Ile Asp Asp Leu Tyr Glu Ile Gln Ala 35 4g gag cgg agt cca aga gat ccc gtt gca ggc gat act gtg tat atc 432 Thr Glu Arg Ser Pro Arg Asp Pro Val Ala Gly Asp Thr Val Tyr Ile 5 aag ata aca acg tgg ccc att gaa tca gga caa acg gct tgg gtg acc 48le Thr Thr Trp Pro Ile Glu Ser Gly Gln Thr Ala Trp Val Thr 65 7 tgg acg aaa aac ggt gtc aat caa gct gct gtc gga gca gca ttc aaa 528 Trp Thr Lys Asn Gly Val Asn Gln Ala Ala Val Gly Ala Ala Phe Lys 85 9c aac agc ggc aac aac act tac tgg gaa gcg aac ctt ggc act ttt 576 Tyr Asn Ser Gly Asn Asn Thr Tyr Trp Glu Ala Asn Leu Gly Thr Phe aaa ggg gac gtg atc agt tat acc gtt cat ggc aac aag gat ggc 624 Ala Lys Gly Asp Val Ile Ser Tyr Thr Val His Gly Asn Lys Asp Gly aat gag aag gtt atc ggt cct ttt act ttt acc gta acg gga tgg 672 Ala Asn Glu Lys Val Ile Gly Pro Phe Thr Phe Thr Val Thr Gly Trp tcc gtt agc agt atc agc tct att acg gat aat acg aac cgt gtt 72er Val Ser Ser Ile Ser Ser Ile Thr Asp Asn Thr Asn Arg Val gtg ctg aat gcg gtg ccg aat aca ggc aca ttg aag cca aag atc aac 768 Val Leu Asn Ala Val Pro Asn Thr Gly Thr Leu Lys Pro Lys Ile Asn tcc ttt acg gcg gat gat gtc ctc cgc gta cag gtt tct cca acc 8Ser Phe Thr Ala Asp Asp Val Leu Arg Val Gln Val Ser Pro Thr aca gga acg tta agc agt gga ctt agt aat tac aca gtt tca gat 864 Gly Thr Gly Thr Leu Ser Ser Gly Leu Ser Asn Tyr Thr Val Ser Asp 2gcc tca acc act tgg ctt aca act tcc aag ctg aag gtg aag gtg 9Ala Ser Thr Thr Trp Leu Thr Thr Ser Lys Leu Lys Val Lys Val 222ag aat cca ttc aaa ctt agt gtg tat aag cct gat gga acg acg 96ys Asn Pro Phe Lys Leu Ser Val Tyr Lys Pro Asp Gly Thr Thr 225 234tt gcc cgt caa tat gac agc act acg aat cgt aac att gcc tgg u Ile Ala Arg Gln Tyr Asp Ser Thr Thr Asn Arg Asn Ile Ala Trp 245 25ta acc aat ggc agt aca atc atc gac aag gta gaa gat cat ttt tat u Thr Asn Gly Ser Thr Ile Ile Asp Lys Val Glu Asp His Phe Tyr 267cg gct tcc gag gag ttt ttt ggc ttt gga gag cat tac aac aac r Pro Ala Ser Glu Glu Phe Phe Gly Phe Gly Glu His Tyr Asn Asn 275 28tc cgt aaa cgc gga aat gat gtg gac acc tat gtg ttc aac cag tat e Arg Lys Arg Gly Asn Asp Val Asp Thr Tyr Val Phe Asn Gln Tyr 29aat caa aat gac cgc acc tac atg gca att cct ttt atg ctt aac s Asn Gln Asn Asp Arg Thr Tyr Met Ala Ile Pro Phe Met Leu Asn 33agc agc ggt tat ggc att ttc gta aat tca acg tat tat tcc aaa ttt r Ser Gly Tyr Gly Ile Phe Val Asn Ser Thr Tyr Tyr Ser Lys Phe 325 33gg ttg gca acc gaa cgc acc gat atg ttc agc ttt acg gct gat aca g Leu Ala Thr Glu Arg Thr Asp Met Phe Ser Phe Thr Ala Asp Thr 345gt agt gcc gcc tcg atg ctg gat tat tat ttc att tac ggt aat y Gly Ser Ala Ala Ser Met Leu Asp Tyr Tyr Phe Ile Tyr Gly Asn 355 36at ttg aaa aat gtg gtg agt aac tac gct aac att acc ggt aag cca p Leu Lys Asn Val Val Ser Asn Tyr Ala Asn Ile Thr Gly Lys Pro 378cg ctg ccg aaa tgg gct ttc ggg tta tgg atg tca gct aac gag r Ala Leu Pro Lys Trp Ala Phe Gly Leu Trp Met Ser Ala Asn Glu 385 39gat cgt caa acc aag gtg aat aca gcc att aat aac gcg aac tcc p Asp Arg Gln Thr Lys Val Asn Thr Ala Ile Asn Asn Ala Asn Ser 44aat att ccg gct aca gcg gtt gtg ctc gaa cag tgg agt gat gag n Asn Ile Pro Ala Thr Ala Val Val Leu Glu Gln Trp Ser Asp Glu 423cg ttt tat att ttc aat gat gcc acc tat acc ccg aaa acg ggc n Thr Phe Tyr Ile Phe Asn Asp Ala Thr Tyr Thr Pro Lys Thr Gly 435 44gt gct gcg cat gcc tat acc gat ttc act ttc ccg aca tct ggg aga r Ala Ala His Ala Tyr Thr Asp Phe Thr Phe Pro Thr Ser Gly Arg 456cg gat cca aaa gcg atg gca gac aat gtg cat aac aat ggg atg p Thr Asp Pro Lys Ala Met Ala Asp Asn Val His Asn Asn Gly Met 465 478tg gtg ctt tgg cag gtc cct att cag aaa tgg act tca acg ccc s Leu Val Leu Trp Gln Val Pro Ile Gln Lys Trp Thr Ser Thr Pro 485 49at acc cag aaa gat aat gat gaa gcc tat atg acg gct cag aat tat r Thr Gln Lys Asp Asn Asp Glu Ala Tyr Met Thr Ala Gln Asn Tyr 55gtt ggc aac ggt agc gga ggc cag tac agg ata cct tca gga caa a Val Gly Asn Gly Ser Gly Gly Gln Tyr Arg Ile Pro Ser Gly Gln 5525 tgg ttc gag aac agt ttg ctg ctt gat ttt acg aat acg gcc gcc aaa p Phe Glu Asn Ser Leu Leu Leu Asp Phe Thr Asn Thr Ala Ala Lys 534gg tgg atg tct aaa cgc gct tat ctg ttt gat ggt gtg ggt atc n Trp Trp Met Ser Lys Arg Ala Tyr Leu Phe Asp Gly Val Gly Ile 545 556gc ttc aaa aca gat ggc ggt gaa atg gta tgg ggt cgc tca aat p Gly Phe Lys Thr Asp Gly Gly Glu Met Val Trp Gly Arg Ser Asn 565 57ct ttc tca aac ggt aag aaa ggc aat gaa atg cgc aat caa tac ccg 2 Phe Ser Asn Gly Lys Lys Gly Asn Glu Met Arg Asn Gln Tyr Pro 589ag tat gtg aaa gcc tat aac gag tac gcg cgc tcg aag aaa gcc 2 Glu Tyr Val Lys Ala Tyr Asn Glu Tyr Ala Arg Ser Lys Lys Ala 595 6gat gcg gtc tcc ttt agc cgt tcc ggc acg caa ggc gca cag gcg aat 2 Ala Val Ser Phe Ser Arg Ser Gly Thr Gln Gly Ala Gln Ala Asn 662tt ttc tgg tcc ggt gac caa gag tcg acg ttt ggt gct ttt caa 2 Ile Phe Trp Ser Gly Asp Gln Glu Ser Thr Phe Gly Ala Phe Gln 625 634ct gtg aat gca ggg ctt acg gca agt atg tct ggc gtt cct tat 22Ala Val Asn Ala Gly Leu Thr Ala Ser Met Ser Gly Val Pro Tyr 645 65gg agc tgg gat atg gca ggc ttt aca ggc act tat cca acg gct gag 2256 Trp Ser Trp Asp Met Ala Gly Phe Thr Gly Thr Tyr Pro Thr Ala Glu 667ac aaa cgt gct act gaa atg gct gct ttt gca ccg gtc atg cag 23Tyr Lys Arg Ala Thr Glu Met Ala Ala Phe Ala Pro Val Met Gln 675 68tt cat tcc gag tct aac ggc agc tct ggt atc aac gag gaa cgt tct 2352 Phe His Ser Glu Ser Asn Gly Ser Ser Gly Ile Asn Glu Glu Arg Ser 69tgg aac gca caa gcg cgt aca ggc gac aat acg atc att agt cat 24Trp Asn Ala Gln Ala Arg Thr Gly Asp Asn Thr Ile Ile Ser His 77ttt gcc aaa tat acg aat acg cgc atg aat ttg ctt cct tat att tat 2448 Phe Ala Lys Tyr Thr Asn Thr Arg Met Asn Leu Leu Pro Tyr Ile Tyr 725 73gc gaa gcg aag atg gct agt gat act ggc gtt ccc atg atg cgc gcc 2496 Ser Glu Ala Lys Met Ala Ser Asp Thr Gly Val Pro Met Met Arg Ala 745cg ctt gaa tat ccg aag gac acg aac acg tac ggt ttg aca caa 2544 Met Ala Leu Glu Tyr Pro Lys Asp Thr Asn Thr Tyr Gly Leu Thr Gln 755 76ag tat atg ttc gga ggt aat tta ctt att gct cct gtt atg aat cag 2592 Gln Tyr Met Phe Gly Gly Asn Leu Leu Ile Ala Pro Val Met Asn Gln 778aa aca aac aag agt att tat ctt ccg cag ggg gat tgg atc gat 264lu Thr Asn Lys Ser Ile Tyr Leu Pro Gln Gly Asp Trp Ile Asp 785 79tgg ttc ggt gct cag cgt cct ggc ggt cga aca atc agc tac acg 2688 Phe Trp Phe Gly Ala Gln Arg Pro Gly Gly Arg Thr Ile Ser Tyr Thr 88ggc atc gat gat cta ccg gtt ttt gtg aag ttt ggc agt att ctt 2736 Ala Gly Ile Asp Asp Leu Pro Val Phe Val Lys Phe Gly Ser Ile Leu 823tg aat ttg aac gcg caa tat caa gtg ggc ggg acc att ggc aac 2784 Pro Met Asn Leu Asn Ala Gln Tyr Gln Val Gly Gly Thr Ile Gly Asn 835 84gc ttg acg agc tac acg aat ctc gcg ttc cgc att tat ccg ctt ggg 2832 Ser Leu Thr Ser Tyr Thr Asn Leu Ala Phe Arg Ile Tyr Pro Leu Gly 856ca acg tac gac tgg aat gat gat att ggc ggt tcg gtg aaa acc 288hr Thr Tyr Asp Trp Asn Asp Asp Ile Gly Gly Ser Val Lys Thr 865 878ct tct aca gag caa tat ggg ttg aat aaa gaa acc gtg act gtt 2928 Ile Thr Ser Thr Glu Gln Tyr Gly Leu Asn Lys Glu Thr Val Thr Val 885 89ca gcg att aat tct acc aag aca ttg caa gtg ttt acg act aag cct 2976 Pro Ala Ile Asn Ser Thr Lys Thr Leu Gln Val Phe Thr Thr Lys Pro 99tct gta acg gtg ggt ggt tct gtg atg aca gag tac agt act tta 3 Ser Val Thr Val Gly Gly Ser Val Met Thr Glu Tyr Ser Thr Leu 9925 act gcc cta acg gga gcg tcg aca ggc tgg tac tat gat act gta cag 3 Ala Leu Thr Gly Ala Ser Thr Gly Trp Tyr Tyr Asp Thr Val Gln 934tc act tac gtc aag ctt ggt tca agt gca tct gct caa tcc gtt 3 Phe Thr Tyr Val Lys Leu Gly Ser Ser Ala Ser Ala Gln Ser Val 945 956ta aat ggc gtt aat aag gtg gaa tat gaa gca gaa ttc ggc gtg 3 Leu Asn Gly Val Asn Lys Val Glu Tyr Glu Ala Glu Phe Gly Val 965 97aa agc ggc gtt tca acg aac acg aac cat gca ggt tat act ggt aca 32Ser Gly Val Ser Thr Asn Thr Asn His Ala Gly Tyr Thr Gly Thr 989tt gtg gac ggc ttt gag act ctt gga gac aat gtt gct ttt gat 3264 Gly Phe Val Asp Gly Phe Glu Thr Leu Gly Asp Asn Val Ala Phe Asp 995 tcc gtc aaa gcc gca ggt act tat acg atg aag gtt cgg tat 33Ser Val Lys Ala Ala Gly Thr Tyr Thr Met Lys Val Arg Tyr tca tcc ggt gca ggc aat ggc tca aga gcc atc tat gtg aat aac 3354 Ser Ser Gly Ala Gly Asn Gly Ser Arg Ala Ile Tyr Val Asn Asn 3acc aaa gtg acg gac ctt gcc ttg ccg caa aca aca agc tgg gat 3399 Thr Lys Val Thr Asp Leu Ala Leu Pro Gln Thr Thr Ser Trp Asp 45 a tgg ggg act gct acg ttt agc gtc tcg ctg agt aca ggt ctc 3444 Thr Trp Gly Thr Ala Thr Phe Ser Val Ser Leu Ser Thr Gly Leu 6aac acg gtg aaa gtc agc tat gat ggt acc agt tca ctt ggc att 3489 Asn Thr Val Lys Val Ser Tyr Asp Gly Thr Ser Ser Leu Gly Ile 75 t ttc gat aac atc gcg att gta gag caa taa aaggtcggga 3532 Asn Phe Asp Asn Ile Ala Ile Val Glu Gln 9agtcc ctcccttaat ttctaatcga aagggagtat ccttgatgcg tccaccaaac 3592 aaagaaattc cacgtattct tgcttttttt acagcgttta cgttgtttgg ttcaaccctt 3652 gccttgcttc ctgctccgcc tgcgcatgcc tatgtcagca gcctagggga aaatctcatt 37cgagtg tcaccggaga taccttgacg ctaactgttg ataacggtgc gccgagtgat 3772 gacctcttga ttgttcaagc ggtgcaaaac ggtattttga aggtggatta tcgtccaaat 3832 agcataacgc cgagcgcgaa gacgccgatg ctggatc 3869 23 499acillus globisporus CDS (466)..(4326) 23 ggtaccggct ttgtcgacgg cttcgatgcg gcaggcgatg cagtgacctt cgacgtatcc 6agcgg ccggcacgta tgcgctcaag gtccggtacg cttccgctgg tggcaacgct cgcgcta tctatgtcaa caacgccaag gtgaccgatc tggcgcttcc ggcaacggcc tgggaca cctgggggac ggcaaccgtc aacgtagcct taaacgccgg ctacaactcg 24ggtca gctacgacaa caccaatacg ctcggcatta atctcgataa cattgcgatc 3agcatt gacagcagga atcttcgcga ggaatgagtt agcgaagagt tcatgcaggc 36ggtta cccataattg taaagcccgg cgcagccagg caccaagtat gcccgggagg 42cggcc ctccctttat ttcaatgatg aaaggcggca tcgat atg ggt cta tgg 477 Met Gly Leu Trp aa cga gtc act cgc atc ctc tcc gta ctc gca gca agc gcg ctg 525 Asn Lys Arg Val Thr Arg Ile Leu Ser Val Leu Ala Ala Ser Ala Leu 5 gc tct acc gta cct tct cta gcg cca cct ccc gct caa gcc cat 573 Ile Gly Ser Thr Val Pro Ser Leu Ala Pro Pro Pro Ala Gln Ala His 25 3g agc gcg ctg ggc aac ctg ctt tcc tcg gcg gtg acc ggg gat acg 62er Ala Leu Gly Asn Leu Leu Ser Ser Ala Val Thr Gly Asp Thr 4 ctc acg ctg acg atc gat aac ggc gcg gaa ccg aat gac gat att cta 669 Leu Thr Leu Thr Ile Asp Asn Gly Ala Glu Pro Asn Asp Asp Ile Leu 55 6t ctg caa gca gtc cag aac ggt att ctg aag gtg gac tac cgg ccg 7Leu Gln Ala Val Gln Asn Gly Ile Leu Lys Val Asp Tyr Arg Pro 7 aac ggt gta gct cca agc gcg gat acg ccg atg ctg gat ccc aat aaa 765 Asn Gly Val Ala Pro Ser Ala Asp Thr Pro Met Leu Asp Pro Asn Lys 85 9gg ccg tcc ata ggc gcc gtt atc aat aca gcc tct aat ccg atg 8Trp Pro Ser Ile Gly Ala Val Ile Asn Thr Ala Ser Asn Pro Met atc aca acg ccg gcg atg aag att gag att gcc aaa aat ccg gtg 86le Thr Thr Pro Ala Met Lys Ile Glu Ile Ala Lys Asn Pro Val ctg acc gtg aaa aaa ccg gac ggc acc gct ctg tta tgg gaa ccc 9Leu Thr Val Lys Lys Pro Asp Gly Thr Ala Leu Leu Trp Glu Pro acc ggc ggc gtc ttc tcg gac ggc gtc cgt ttc ttg cac ggg acg 957 Pro Thr Gly Gly Val Phe Ser Asp Gly Val Arg Phe Leu His Gly Thr gac aat atg tac ggc atc cgc agc ttc aat gct ttt gac agc ggc y Asp Asn Met Tyr Gly Ile Arg Ser Phe Asn Ala Phe Asp Ser Gly ggg gat ctg ctg cgc aac agc tcc acc caa gcc gcc cgt gca ggc gac y Asp Leu Leu Arg Asn Ser Ser Thr Gln Ala Ala Arg Ala Gly Asp ggc aac tcc ggc ggc ccg ctg atc tgg agc aca gcc ggg tac ggg n Gly Asn Ser Gly Gly Pro Leu Ile Trp Ser Thr Ala Gly Tyr Gly 22ctc gtt gac agc gac ggt ggg tat ccg ttc

acg gac gag gct acc l Leu Val Asp Ser Asp Gly Gly Tyr Pro Phe Thr Asp Glu Ala Thr 2225 ggc aag ctg gag ttc tat tac ggc ggc acg cct ccg gaa ggc cgg cgc y Lys Leu Glu Phe Tyr Tyr Gly Gly Thr Pro Pro Glu Gly Arg Arg 234cg aag cag gat gtg gag tac tac atc atg ctc ggc acg ccg aaa r Thr Lys Gln Asp Val Glu Tyr Tyr Ile Met Leu Gly Thr Pro Lys 245 256tc atg tcc ggc gtc ggg gaa att acg ggc aaa ccg ccg atg ctg u Ile Met Ser Gly Val Gly Glu Ile Thr Gly Lys Pro Pro Met Leu 265 27cc aag tgg tcc ctg ggc ttt atg aac ttc gag tgg gat ctg aat gaa o Lys Trp Ser Leu Gly Phe Met Asn Phe Glu Trp Asp Leu Asn Glu 289ag ctc aag aac cat gtg gat acg tac cgg gcc aaa aat att ccg a Glu Leu Lys Asn His Val Asp Thr Tyr Arg Ala Lys Asn Ile Pro 295 3atc gac ggc tat gcg atc gat ttc gat tgg aag aag tac ggc gag aat e Asp Gly Tyr Ala Ile Asp Phe Asp Trp Lys Lys Tyr Gly Glu Asn 332ac ggc gaa ttc gct tgg aat acg gcc aat ttc cct tcc gcc gcc n Tyr Gly Glu Phe Ala Trp Asn Thr Ala Asn Phe Pro Ser Ala Ala 325 334cg gcg ctg aag tcg cag atg gac gcc aag ggc att aaa atg atc r Thr Ala Leu Lys Ser Gln Met Asp Ala Lys Gly Ile Lys Met Ile 345 35gc ata acc aag cct cgc atc gcg acg aag gat ttt tcg aac aat cct y Ile Thr Lys Pro Arg Ile Ala Thr Lys Asp Phe Ser Asn Asn Pro 367tg cag gga acg gac gcg gcg agc ggc ggt tat ttt tat ccg gga r Val Gln Gly Thr Asp Ala Ala Ser Gly Gly Tyr Phe Tyr Pro Gly 375 38at agc gaa tac aag gac tac ttc atc ccg gtc ttt gtg cgc agc atc s Ser Glu Tyr Lys Asp Tyr Phe Ile Pro Val Phe Val Arg Ser Ile 39cct tat aac cct gct gca cgc tcc tgg ttc tgg aac cac tcc aag p Pro Tyr Asn Pro Ala Ala Arg Ser Trp Phe Trp Asn His Ser Lys 44gat gcg ttc gat aaa ggc atc gta ggc tgg tgg aac gac gag acg gat p Ala Phe Asp Lys Gly Ile Val Gly Trp Trp Asn Asp Glu Thr Asp 425 43cg gta tcg tcg gga ggg gcc tcc tac tgg ttc ggc aat ttt acg acc a Val Ser Ser Gly Gly Ala Ser Tyr Trp Phe Gly Asn Phe Thr Thr 445at atg tcc cag gcg ctt tac gag gga cag cgg gca tat acg tcg y His Met Ser Gln Ala Leu Tyr Glu Gly Gln Arg Ala Tyr Thr Ser 455 46ac gcc cag cgc gtc tgg cag aca gcg cgc acg ttc tat ccc ggg gcg n Ala Gln Arg Val Trp Gln Thr Ala Arg Thr Phe Tyr Pro Gly Ala 478gt tat gcg acg acg ctc tgg tcg gga gac atc ggg att cag tat n Arg Tyr Ala Thr Thr Leu Trp Ser Gly Asp Ile Gly Ile Gln Tyr 485 49aag ggg gaa aga atc aac tgg gct gcc ggc atg cag gag cag cgg 2 Lys Gly Glu Arg Ile Asn Trp Ala Ala Gly Met Gln Glu Gln Arg 55gtg atg ctt tct tcg atc aac aac ggc cag gtc aaa tgg gga atg 2 Val Met Leu Ser Ser Ile Asn Asn Gly Gln Val Lys Trp Gly Met 523ca ggc ggc ttc aac cag cag gac ggc acg acg aac aat ccg aat 2 Thr Gly Gly Phe Asn Gln Gln Asp Gly Thr Thr Asn Asn Pro Asn 535 54cg gac ctg tac gcc aga tgg atg cag ttc agc gcg ctg act ccg gtg 2 Asp Leu Tyr Ala Arg Trp Met Gln Phe Ser Ala Leu Thr Pro Val 556gc gtg cat ggc aac aat cac cag cag cgc cag cct tgg tat tat 22Arg Val His Gly Asn Asn His Gln Gln Arg Gln Pro Trp Tyr Tyr 565 578cg aca gcc gag gag gca tcc aag gaa gcg ctc cag ctc cgt tac 2253 Gly Ser Thr Ala Glu Glu Ala Ser Lys Glu Ala Leu Gln Leu Arg Tyr 585 59cc ctg att cct tat atg tat gct tac gaa aga agc gcc tac gag aac 23Leu Ile Pro Tyr Met Tyr Ala Tyr Glu Arg Ser Ala Tyr Glu Asn 66aac gga ctt gtc cgg ccg ctg atg cag gaa tac cct gcc gat gcc 2349 Gly Asn Gly Leu Val Arg Pro Leu Met Gln Glu Tyr Pro Ala Asp Ala 6625 aac gcc aaa aac tat ctc gat gcc tgg atg ttc ggc gat tgg ctg ctg 2397 Asn Ala Lys Asn Tyr Leu Asp Ala Trp Met Phe Gly Asp Trp Leu Leu 634cg cct gtg gtc gag aag cag cag acc tcc aag gaa atc tat ctc 2445 Ala Ala Pro Val Val Glu Lys Gln Gln Thr Ser Lys Glu Ile Tyr Leu 645 656ca ggc act tgg att gac tac aac cgg ggc acg gtg ctc acc ggc 2493 Pro Ala Gly Thr Trp Ile Asp Tyr Asn Arg Gly Thr Val Leu Thr Gly 665 67gc cag aag atc agc tac gcc gtc aat ccc gac acg ctg acg gat att 254ln Lys Ile Ser Tyr Ala Val Asn Pro Asp Thr Leu Thr Asp Ile 689tc ttc att aag aag ggc gcg att atc cct tcg cag aag gtg cag 2589 Pro Leu Phe Ile Lys Lys Gly Ala Ile Ile Pro Ser Gln Lys Val Gln 695 7gac tac gtg ggc cag gct ccc gtc caa acg gtg gat gtg gat gta ttc 2637 Asp Tyr Val Gly Gln Ala Pro Val Gln Thr Val Asp Val Asp Val Phe 772at acg gca caa tcg agc ttt acc tat tat gac gat gac ggc agc 2685 Pro Asn Thr Ala Gln Ser Ser Phe Thr Tyr Tyr Asp Asp Asp Gly Ser 725 734ac aat tat gaa agc gga gct tac ttc aag caa ttg atg acg gct 2733 Ser Tyr Asn Tyr Glu Ser Gly Ala Tyr Phe Lys Gln Leu Met Thr Ala 745 75ag gac aac gga tcc ggt gcg ctg agc ttt acg ctg ggc gcc aaa acc 278sp Asn Gly Ser Gly Ala Leu Ser Phe Thr Leu Gly Ala Lys Thr 767cg tac agc ccc gca ctg caa tcc tat atc gtc aag ctt cac ggg 2829 Gly Thr Tyr Ser Pro Ala Leu Gln Ser Tyr Ile Val Lys Leu His Gly 775 78cc gca ggc gcg tcg gtg aca agc aat ggg gcg gcg ctg gcc tcc tat 2877 Ala Ala Gly Ala Ser Val Thr Ser Asn Gly Ala Ala Leu Ala Ser Tyr 79agc ctg caa gcg ctg aaa gcc tca gcc agt gaa ggc tgg gcc aag 2925 Ala Ser Leu Gln Ala Leu Lys Ala Ser Ala Ser Glu Gly Trp Ala Lys 88ggc aag gac atc tac ggc gat gtc acg tat gtc aag cta tcc gcg ggg 2973 Gly Lys Asp Ile Tyr Gly Asp Val Thr Tyr Val Lys Leu Ser Ala Gly 825 83ca gcg gcg gcc aag gcg att gcc gtc acc ggc aac agc ccg gtc agc 3 Ala Ala Ala Lys Ala Ile Ala Val Thr Gly Asn Ser Pro Val Ser 845cg gat gtg cag tac gaa gcc gaa gaa gct tcg ctg tcc ggc aat 3 Ala Asp Val Gln Tyr Glu Ala Glu Glu Ala Ser Leu Ser Gly Asn 855 86cg aca gca acc aag gcg acc gtg aat acg aac cac gca ggc tac acg 3 Thr Ala Thr Lys Ala Thr Val Asn Thr Asn His Ala Gly Tyr Thr 878gc ggc ttc gtg gat gga ctg agt aat ccg gga gcg gcg gtt acg 3 Ser Gly Phe Val Asp Gly Leu Ser Asn Pro Gly Ala Ala Val Thr 885 89tat ccg aag gtg aaa acg ggc gga gac tac aat gtc tcg ctg cgc 32Tyr Pro Lys Val Lys Thr Gly Gly Asp Tyr Asn Val Ser Leu Arg 99gct aat tcg acg gga gcg gca aag agc gtc agc atc ttc gtt aac 326la Asn Ser Thr Gly Ala Ala Lys Ser Val Ser Ile Phe Val Asn 923ag cgc gtc aaa tcc acg tcg ctg gcg aac ctg ccg aac tgg gat 33Lys Arg Val Lys Ser Thr Ser Leu Ala Asn Leu Pro Asn Trp Asp 935 94cg tgg ggg acg cag gct gag aca ctg ccg ctg acg gcg ggg acg aac 3357 Thr Trp Gly Thr Gln Ala Glu Thr Leu Pro Leu Thr Ala Gly Thr Asn 956tc acc tac aag ttc tac tcg gat gcc gga gat acg ggc tcg gtt 34Val Thr Tyr Lys Phe Tyr Ser Asp Ala Gly Asp Thr Gly Ser Val 965 978tg gac aac atc acg gtg ccc ttc gct ccg gcc atc ggc aaa tac 3453 Asn Leu Asp Asn Ile Thr Val Pro Phe Ala Pro Ala Ile Gly Lys Tyr 985 99ag gcg gag agc gcc gag ctg agc ggc ggc agc acg gtc aac cag 3498 Glu Ala Glu Ser Ala Glu Leu Ser Gly Gly Ser Thr Val Asn Gln aat cat tgg ttc tac agc ggc acg gca ttt gta gat ggc tta acc 3543 Asn His Trp Phe Tyr Ser Gly Thr Ala Phe Val Asp Gly Leu Thr 2gca ccg ggc gcc caa gtc aaa tat acc gtg aac gcc ccg gcc gca 3588 Ala Pro Gly Ala Gln Val Lys Tyr Thr Val Asn Ala Pro Ala Ala 35 c agc tac cag atc gcg ctt cgc tat gcg aac ggc acg ggt gct 3633 Gly Ser Tyr Gln Ile Ala Leu Arg Tyr Ala Asn Gly Thr Gly Ala 5gcg aag acg ctc agc acg tat gtg aac ggg acg aag ctg ggg caa 3678 Ala Lys Thr Leu Ser Thr Tyr Val Asn Gly Thr Lys Leu Gly Gln 65 g gcc ttc gcc agc cct ggc ggc aac tgg aac gtg tgg cag gac 3723 Thr Ala Phe Ala Ser Pro Gly Gly Asn Trp Asn Val Trp Gln Asp 8agc gtg cag acc gtc gcg ctc gcc gcc ggt acg aac acg atc gcg 3768 Ser Val Gln Thr Val Ala Leu Ala Ala Gly Thr Asn Thr Ile Ala 95 c aag tac gat gcc ggc gac agc ggc agc ggc agc gtc aat ctg 38Lys Tyr Asp Ala Gly Asp Ser Gly Ser Gly Ser Val Asn Leu gac cgt ctg ttg ctc tct gcc gca gcg cca ggc gtg ccc gtg tcc 3858 Asp Arg Leu Leu Leu Ser Ala Ala Ala Pro Gly Val Pro Val Ser 25 g cag aac ctg ctc gat aac ggg ggc ttt gaa cgc gat ccg tcg 39Gln Asn Leu Leu Asp Asn Gly Gly Phe Glu Arg Asp Pro Ser 4cag agc agc aac tgg acc gag tgg cat ccg gct tcg cag gcg att 3948 Gln Ser Ser Asn Trp Thr Glu Trp His Pro Ala Ser Gln Ala Ile 55 t tac ggc atc gac agc ggc tcc ggg atg aat ccg cct gaa tcg 3993 Ala Tyr Gly Ile Asp Ser Gly Ser Gly Met Asn Pro Pro Glu Ser 7cca tgg gca ggc gat aag cgc gcc tat ttc tat gcg gca ggc ccg 4 Trp Ala Gly Asp Lys Arg Ala Tyr Phe Tyr Ala Ala Gly Pro 85 t cag caa agc atc cat caa aca gtc agc gtg cct gtc aat aat 4 Gln Gln Ser Ile His Gln Thr Val Ser Val Pro Val Asn Asn gcc aag tac aag ttc gaa gcc tgg gta ttg ctg aag aat aca aca 4 Lys Tyr Lys Phe Glu Ala Trp Val Leu Leu Lys Asn Thr Thr ccg aca acg gcc cgg gtg gag att caa aat tac ggc ggt tcg ccg 4 Thr Thr Ala Arg Val Glu Ile Gln Asn Tyr Gly Gly Ser Pro 3atc ttc acg aac atc agt aaa gac ggc gtc tgg aaa tac atc agc 42Phe Thr Asn Ile Ser Lys Asp Gly Val Trp Lys Tyr Ile Ser 45 c agc gat att cag gtc acg aac ggc caa atc gat att ggc ttc 4263 Val Ser Asp Ile Gln Val Thr Asn Gly Gln Ile Asp Ile Gly Phe 6tat gtg gat tcg ccc gga ggc acc acg ctc cac atc gac gat gtg 43Val Asp Ser Pro Gly Gly Thr Thr Leu His Ile Asp Asp Val 75 g gtc acc aag caa taa tccggtaaca ctagccctcc cccgccttgc 4356 Arg Val Thr Lys Gln caggaggg ctttttgctt ctgtaggttg tgaaggcgat accgagcgat gagaattcga 44gaacag ctcgccctgt gtcctgctaa attcctctcc tccctggcag ggaagccgct 4476 tccacatgtc gaattgggga ggtactatga gaagttagta ctaccgtctg caacggcttt 4536 cgctacaatg gaaccaataa gacatcgcga aggtttggga ggattcggca tgcagagacg 4596 cgaggttaaa gtaataggca cgggcaaata tttgcccgcc catcgagtga ctgcgcagga 4656 gatggaccgg cggctaggag tgcccgacgg atgggtgctg aagaagtcgg atgtggccgt 47tatttc gccggtacgg agaaggcctc ggagatgggg gcgagagcgg ctgaggcggc 4776 gctggcttcc gcaggcctgg ccttcacgga tatcgactgc ctgatgtgcg ccagcgggac 4836 gatggaacag ccgattccat gcacggcggc gctcattcag aaggcgatag gccaaggaca 4896 ctccggagtg ccggcactgg atttgaatac aacctgtctg agctttgtgg cggctctgga 4956 catggtttct tatatggtga cggcgggaag gtacc 49986 DNA Bacillus globisporus CDS (667)..(3948) 24 gagctcggga agaacccgtc cctgcaagct tggacgcagg cggtggagga ggcgggagtc 6cgctt ccgctatggc aggggctggg ggaggtgcat acggcttgat cggccactgc ggagggc tgctggcgtt cgagaccggc cactggctga aggcttgcgg gatgcaggag acgcatc tgttcgtgtc cgggtgcagc ccgccccatc tgctgcaagc gcggccggac 24aacgg gaccatccgg cccggctccg ctccccgatg cctgccggat cgcccaagcg 3gtatgc cttccaggcg cgggccgctg cttgcccggc tgagtgtatt cgccggccgc 36cccgg gcgtgtatgt ggatagtttg gccgaatggg gccgctatac ggcccgcata 42tgttc atattggcga gggcgggcat gcagattggg gacctgatgc agaccgttgg 48attcg tgcaaatgat tgcggagagg gaatattcgt cttcttgaag ccaggtgacc 54taaga tgtcgcacta agctgtatag tttcggaagg gaggtgaggc agagaagcgc 6tgagct gttagcttga cgtttaacgg tcaaaaccaa ttttactttg ggaaggagca 66t atg cat gga aga aac ata ccg aga ccc atc aag ctc att gtt 7His Gly Arg Asn Ile Pro Arg Pro Ile Lys Leu Ile Val tct tgg ctg ctg att ttc ttt tta atg gtg cca agc atc tat gca att 756 Ser Trp Leu Leu Ile Phe Phe Leu Met Val Pro Ser Ile Tyr Ala Ile 5 3gc gta tac cac gcg cct tac ggg atc gac gat ctt tat gag att 8Gly Val Tyr His Ala Pro Tyr Gly Ile Asp Asp Leu Tyr Glu Ile 35 4g gcg acg gag cgc agt ccg aga gac cct gtg gcc ggg gag acg gtg 852 Gln Ala Thr Glu Arg Ser Pro Arg Asp Pro Val Ala Gly Glu Thr Val 5 tat atc aaa atc aca aca tgg ccg atc gag ccc gga cag acg gca tgg 9Ile Lys Ile Thr Thr Trp Pro Ile Glu Pro Gly Gln Thr Ala Trp 65 7g acc tgg acg aaa aac ggc gtc gcc cag ccg gcg gtc ggt gcc gcc 948 Val Thr Trp Thr Lys Asn Gly Val Ala Gln Pro Ala Val Gly Ala Ala 8 tac aag tac aac agc ggc aac aac acc tac tgg gag gcg aac ctg ggc 996 Tyr Lys Tyr Asn Ser Gly Asn Asn Thr Tyr Trp Glu Ala Asn Leu Gly 95 ttc gcc aaa gga gac gta att tcc tac acc gtt cgc ggc aat aag r Phe Ala Lys Gly Asp Val Ile Ser Tyr Thr Val Arg Gly Asn Lys ggt gcc aat gaa aaa acg gcc gga ccg ttc acc ttt acc gta acc p Gly Ala Asn Glu Lys Thr Ala Gly Pro Phe Thr Phe Thr Val Thr tgg gaa tac gtc agc agc atc ggc tcg gtc acg aat aac acg aac p Trp Glu Tyr Val Ser Ser Ile Gly Ser Val Thr Asn Asn Thr Asn gtc ctg ctg aat gcg gtg ccg aac acg ggg acg ctg tcc ccc aag g Val Leu Leu Asn Ala Val Pro Asn Thr Gly Thr Leu Ser Pro Lys aac att tcg ttc acg gcg gac gat gtg ttc cgc gtt cag ctc tcc e Asn Ile Ser Phe Thr Ala Asp Asp Val Phe Arg Val Gln Leu Ser cct acg gga tcg ggg acg ttg agc acg ggc ctg agt aat ttt acc gtc o Thr Gly Ser Gly Thr Leu Ser Thr Gly Leu Ser Asn Phe Thr Val 2gac agt gcg tcc acg gcc tgg atc tct aca tcc aaa tta aag ctg r Asp Ser Ala Ser Thr Ala Trp Ile Ser Thr Ser Lys Leu Lys Leu 222tg gat aag aat ccg ttc aaa ctg agc gtg tac aag ccg gac ggc s Val Asp Lys Asn Pro Phe Lys Leu Ser Val Tyr Lys Pro Asp Gly 225 23cg acg ctg atc gcg cgc cag tat gac agc acg gcc aac cgc aat ctc r Thr Leu Ile Ala Arg Gln Tyr Asp Ser Thr Ala Asn Arg Asn Leu 245gg ctg acc aat ggc agc act gtc atc aat aaa atc gag gac cac a Trp Leu Thr Asn Gly Ser Thr Val Ile Asn Lys Ile Glu Asp His 255 267ac tcg ccg gcg tcc gag gag ttt ttc ggc ttc ggg gag cgc tac e Tyr Ser Pro Ala Ser Glu Glu Phe Phe Gly Phe Gly Glu Arg Tyr 275

28ac aac ttc cgc aag cgc gga acc gac gtg gac acg tat gtc tac aat n Asn Phe Arg Lys Arg Gly Thr Asp Val Asp Thr Tyr Val Tyr Asn 29tac aaa aat caa aac gac cgc acc tat atg gca atc ccc ttc atg n Tyr Lys Asn Gln Asn Asp Arg Thr Tyr Met Ala Ile Pro Phe Met 33aac agc agc ggg tac ggt atc ttc gta aac tcc acg tac tac tcc u Asn Ser Ser Gly Tyr Gly Ile Phe Val Asn Ser Thr Tyr Tyr Ser 323tc cgc ttg gca act gag cgc tcc gat atg tac agt ttt acg gcc s Phe Arg Leu Ala Thr Glu Arg Ser Asp Met Tyr Ser Phe Thr Ala 335 345cc ggg ggc agc gcc aat tcg acg ctg gat tac tac ttt att tac p Thr Gly Gly Ser Ala Asn Ser Thr Leu Asp Tyr Tyr Phe Ile Tyr 355 36gc aat gac ttg aag ggc gtc gtc agc aat tat gcg aac atc aca ggc y Asn Asp Leu Lys Gly Val Val Ser Asn Tyr Ala Asn Ile Thr Gly 378cg gct gct ctg ccc aaa tgg gcg ttt ggc ctc tgg atg tcg gcc s Pro Ala Ala Leu Pro Lys Trp Ala Phe Gly Leu Trp Met Ser Ala 385 39at gag tgg gac cgg caa tcc aaa gta gcg act gcg atc aat aac gcc n Glu Trp Asp Arg Gln Ser Lys Val Ala Thr Ala Ile Asn Asn Ala 44acg aac aac atc ccg gcg acg gcc gtc gtg ctg gag cag tgg agt n Thr Asn Asn Ile Pro Ala Thr Ala Val Val Leu Glu Gln Trp Ser 4425 43ag aat acg ttc tat atg ttc aac gat gcg cag tat acg gcc aaa 2 Glu Asn Thr Phe Tyr Met Phe Asn Asp Ala Gln Tyr Thr Ala Lys 435 44ct ggc ggc agc aca cac tcc tat acg gac tat atc ttc ccg gcg gcc 2 Gly Gly Ser Thr His Ser Tyr Thr Asp Tyr Ile Phe Pro Ala Ala 456gt tgg ccg aat ccg aag caa atg gcg gat aat gta cac agt aac 2 Arg Trp Pro Asn Pro Lys Gln Met Ala Asp Asn Val His Ser Asn 465 47gg atg aag ctg gtg ctg tgg cag gtg ccg att cag aaa tgg acc gcc 2 Met Lys Leu Val Leu Trp Gln Val Pro Ile Gln Lys Trp Thr Ala 489ct cat ctg cag aag gac aac gac gaa agc tat atg atc gcg caa 2 Pro His Leu Gln Lys Asp Asn Asp Glu Ser Tyr Met Ile Ala Gln 495 55tat gcc gta ggc aac ggc agc gga ggc cag tac cgc atc cct agc 2244 Asn Tyr Ala Val Gly Asn Gly Ser Gly Gly Gln Tyr Arg Ile Pro Ser 5525 ggg caa tgg ttt gag aac agc ctg ctg ctg gac ttc acg aac ccg agc 2292 Gly Gln Trp Phe Glu Asn Ser Leu Leu Leu Asp Phe Thr Asn Pro Ser 534aa aac tgg tgg atg tcc aag cgc gcc tat ctg ttt gat ggc gtc 234ys Asn Trp Trp Met Ser Lys Arg Ala Tyr Leu Phe Asp Gly Val 545 55gc atc gac ggg ttc aag acg gac gga ggg gag atg gtc tgg ggc cgc 2388 Gly Ile Asp Gly Phe Lys Thr Asp Gly Gly Glu Met Val Trp Gly Arg 567ac acg ttc gcc aat ggc aaa aaa ggc gat gaa atg cgc aac cag 2436 Trp Asn Thr Phe Ala Asn Gly Lys Lys Gly Asp Glu Met Arg Asn Gln 575 589cg aac gat tac gtg aag gcc tac aac gaa tat gcg cgc tcg aag 2484 Tyr Pro Asn Asp Tyr Val Lys Ala Tyr Asn Glu Tyr Ala Arg Ser Lys 595 6aaa agc gat gcc gtc agc ttc agc cgt tcg ggc acg caa ggg gcg caa 2532 Lys Ser Asp Ala Val Ser Phe Ser Arg Ser Gly Thr Gln Gly Ala Gln 662at cag atc ttc tgg tcc ggt gac cag gaa tcg acg ttc ggt gcc 258sn Gln Ile Phe Trp Ser Gly Asp Gln Glu Ser Thr Phe Gly Ala 625 63tc cag caa gcc gtc cag gcg gga ctg acc gca ggc ttg tcc ggc gtt 2628 Phe Gln Gln Ala Val Gln Ala Gly Leu Thr Ala Gly Leu Ser Gly Val 645at tgg agc tgg gac ttg gct gga ttc acc ggc gct tat ccg tcg 2676 Pro Tyr Trp Ser Trp Asp Leu Ala Gly Phe Thr Gly Ala Tyr Pro Ser 655 667ag cta tat aaa cgc gcg acg gca atg tcg gca ttt gcc ccg att 2724 Ala Glu Leu Tyr Lys Arg Ala Thr Ala Met Ser Ala Phe Ala Pro Ile 675 68tg cag ttc cac tcc gaa gcc aac ggc agt tcc ggc atc aat gag gag 2772 Met Gln Phe His Ser Glu Ala Asn Gly Ser Ser Gly Ile Asn Glu Glu 69tcc ccg tgg aat gct cag gcc cgg act ggc gac aac acg atc atc 282er Pro Trp Asn Ala Gln Ala Arg Thr Gly Asp Asn Thr Ile Ile 77cat ttt gcc aag tat acg aac acc cgg atg aac ctg ctt cct tat 2868 Ser His Phe Ala Lys Tyr Thr Asn Thr Arg Met Asn Leu Leu Pro Tyr 723ac agc gag gct aaa gca gca agc gat act ggc gtg ccg atg atg 29Tyr Ser Glu Ala Lys Ala Ala Ser Asp Thr Gly Val Pro Met Met 735 745cg atg gcg ctg gag tat ccg agc gat acc cag acg tac gga ttg 2964 Arg Ala Met Ala Leu Glu Tyr Pro Ser Asp Thr Gln Thr Tyr Gly Leu 755 76cg cag cag tac atg ttc ggc ggc agc ctg ctg gtg gcg cct gtc ttg 3 Gln Gln Tyr Met Phe Gly Gly Ser Leu Leu Val Ala Pro Val Leu 778aa ggc gag acg aat aag aat atc tac ctt ccg caa gga gat tgg 3 Gln Gly Glu Thr Asn Lys Asn Ile Tyr Leu Pro Gln Gly Asp Trp 785 79tc gac ttc tgg ttc ggc gcg cag cgt ccg ggc ggg cga acg atc agc 3 Asp Phe Trp Phe Gly Ala Gln Arg Pro Gly Gly Arg Thr Ile Ser 88tac gcg ggc gtg gac gat ctt ccc gtc ttc gtg aag tcc ggc agc 3 Tyr Ala Gly Val Asp Asp Leu Pro Val Phe Val Lys Ser Gly Ser 8825 83tg ccg atg aat ctg aac ggg cag tat cag gtt ggc ggc acg atc 32Leu Pro Met Asn Leu Asn Gly Gln Tyr Gln Val Gly Gly Thr Ile 835 84gc aac agc ttg acc gcc tac aac aac ctg acg ttc cgg att tat cca 3252 Gly Asn Ser Leu Thr Ala Tyr Asn Asn Leu Thr Phe Arg Ile Tyr Pro 856gt acg acg acg tac agc tgg aat gat gac atc ggc ggc tcg gtg 33Gly Thr Thr Thr Tyr Ser Trp Asn Asp Asp Ile Gly Gly Ser Val 865 87ag acg att acg tcg aca gag cag tat gga ctg aat aaa gag acg gtg 3348 Lys Thr Ile Thr Ser Thr Glu Gln Tyr Gly Leu Asn Lys Glu Thr Val 889tt ccg gcg atc aac tcg gcg aag acg ctc cag gtg ttc acg acc 3396 Thr Leu Pro Ala Ile Asn Ser Ala Lys Thr Leu Gln Val Phe Thr Thr 895 99ccg tcg tcg gtg acg ctg ggc ggc acg gcc ctc acc gcg cat agc 3444 Lys Pro Ser Ser Val Thr Leu Gly Gly Thr Ala Leu Thr Ala His Ser 9925 aca tta agc gca ttg atc ggc gct tcc tcc ggc tgg tat tac gat acg 3492 Thr Leu Ser Ala Leu Ile Gly Ala Ser Ser Gly Trp Tyr Tyr Asp Thr 934aa aag ctc gcc tat gtg aag ctc ggc gcc agc tca tcg gcg caa 354ln Lys Leu Ala Tyr Val Lys Leu Gly Ala Ser Ser Ser Ala Gln 945 95cc gtc gtg ctt gac ggc gtc aac aag gtc gag tat gag gct gag ttc 3588 Thr Val Val Leu Asp Gly Val Asn Lys Val Glu Tyr Glu Ala Glu Phe 967ca ctt acc ggc gtc acg acc aat acg aat cat gcc ggc tat atg 3636 Gly Thr Leu Thr Gly Val Thr Thr Asn Thr Asn His Ala Gly Tyr Met 975 989cc ggc ttt gtc gac ggc ttc gat gcg gca ggc gat gca gtg acc 3684 Gly Thr Gly Phe Val Asp Gly Phe Asp Ala Ala Gly Asp Ala Val Thr 995 gac gta tcc gtc aaa gcg gcc ggc acg tat gcg ctc aag gtc 3729 Phe Asp Val Ser Val Lys Ala Ala Gly Thr Tyr Ala Leu Lys Val cgg tac gct tcc gct ggt ggc aac gct tca cgc gct atc tat gtc 3774 Arg Tyr Ala Ser Ala Gly Gly Asn Ala Ser Arg Ala Ile Tyr Val 3aac aac gcc aag gtg acc gat ctg gcg ctt ccg gca acg gcc aac 38Asn Ala Lys Val Thr Asp Leu Ala Leu Pro Ala Thr Ala Asn 45 g gac acc tgg ggg acg gca acc gtc aac gta gcc tta aac gcc 3864 Trp Asp Thr Trp Gly Thr Ala Thr Val Asn Val Ala Leu Asn Ala 6ggc tac aac tcg atc aag gtc agc tac gac aac acc aat acg ctc 39Tyr Asn Ser Ile Lys Val Ser Tyr Asp Asn Thr Asn Thr Leu 75 c att aat ctc gat aac att gcg atc gtg gag cat tga cagcaggaat 3958 Gly Ile Asn Leu Asp Asn Ile Ala Ile Val Glu His 9cgagg aatgagttag cgaagagttc atgcaggcag aggggttacc cataattgta 4cccggcg cagccaggca ccaagtatgc ccgggagggc cgccggccct ccctttattt 4tgatgaa aggcggcatc gatatgggtc tatggaacaa acgagtcact cgcatcctct 4tactcgc agcaagcgcg ctgatcggct ctaccgtacc ttctctagcg ccacctcccg 4aagccca tgtgagcgcg ctgggcaacc tgctttcctc ggcggtgacc ggggatacgc 4258 tcacgctgac gatcgataac ggcgcggaac cgaatgacga tattctagtt ctgcaagcag 43gaacgg tattctgaag gtggactacc ggccgaacgg tgtagctcca agcgcggata 4378 cgccgatgct ggatcccaat aaaacctggc cgtccatagg cgccgttatc aatacagcct 4438 ctaatccgat gacgatcaca acgccggcga tgaagattga gattgccaaa aatccggtgc 4498 gcctgaccgt gaaaaaaccg gacggcaccg ctctgttatg ggaacccccg accggcggcg 4558 tcttctcgga cggcgtccgt ttcttgcacg ggacgggcga caatatgtac ggcatccgca 46caatgc ttttgacagc ggcggggatc tgctgcgcaa cagctccacc caagccgccc 4678 gtgcaggcga ccagggcaac tccggcggcc cgctgatctg gagcacagcc gggtacgggg 4738 tgctcgttga cagcgacggt gggtatccgt tcacggacga ggctaccggc aagctggagt 4798 tctattacgg cggcacgcct ccggaaggcc ggcgctatac gaagcaggat gtggagtact 4858 acatcatgct cggcacgccg aaagagatca tgtccggcgt cggggaaatt acgggcaaac 49gatgct gcccaagtgg tccctgggct ttatgaactt cgagtgggat ctgaatgaag 4978 ctgagctc 4986 25 58Arthrobacter globiformis CDS ((4552) 25 ggtacctcgt cgaggagctc ggtgtcgacg gcttcaagac cgacgggagc gaggcgctct 6cgtga cctgatcgtc agcgacgggc gccgcggtga cgagatgcac aacgcctacc acgagta cacctccgcc tacaacgact tcgtgcagga gacgacgggc gccgacggca tcttcag ccgggcgggc acctccggcg gccagagcga atccatcttc tgggccgggg 24gcgtc gacgttcggc gctttccagg aggccgtccg ggccgggcag agcgcgggcc 3gggagt gccgttctgg gcctgggacc tcggcggctt caccgggtcg ttcccaagcg 36ctgta tctgcgctcg accgctcagg cggtgttctc gccgatcatg cagtaccact 42aaggc cgaccccagt ccgtccgagg cgcgcacgcc ctggaacgtg caggcgcgca 48aacac cactgtcgtc cccaccttcg cccgttacgc gaacgtacgg atgaacctcg 54tatct gtacacggag gcggacgaca gcgcgacgac gggtgtgccg atgatgcgcg 6gagcct cgcgttcccc gacgacccgg atgccgcgca gtacgaccag cagtacatgt 66tctca gctgctggtc gcaccgatta cgaaccaggg ccagaccgtg aaagacgtct 72cccgc gggcgagtgg tacgacttct ggaacggcgg acgcgcgagc ggcgagggcg 78atgta cgacgccgga cccgacggca tccccgtata cgctcgcgcc ggagcggtca 84ctcaa cctcaacgac gcgtatgagg tgggcggcac gatcggcaac gacgtggaga 9cgacaa ccttgtgttc cgcgtttacc cctccggtga gagcagctac gagtacttcg 96caagc gaacgcgcac cgccggatcg atgtctcggc cgaccgcgca gcgcgcacgg gaggtgtc tgctcccgcg ctcacgaccg cgagcacctt ccaggtgtcg ggcaccaagc gacaccgt gaccgtcgcg ggctcggcac tgcctgaggt caacagcgtg agcgcgctgg gcatccac cgaggcctgg tactgggatg cgaagcagca gctgacgtac gtgaaggtcg gcgagcac cggcgagcgc acgatcctcc tgctgggcgt cgacaaggcc gggtacgagg gagttcgc gggtcatacg gccgtctcga cgaacgccga ccacccgggc tacaccgggc ggcttcgt cgacggcttc gcgaacgcag gagacgcggt ggagttcgac gtgtgggccg gagaacgg cgcgcaccag ctccgcttcc gctacggaaa cggagcggcg acccccgcca cgcacgat ccgggtcgac ggagcgcctc tgggaacgct gtcgcttccg cccaccgggt tggagttc gtggggcacg gcctcgatcg acgtgaccct cccacccgga cgccacgccg cggatcga gtacgccgga ggcgattccg gcggcgtcaa cctcgacaac ctcgtcctcg cgctgagc gcacacggga aagggagaag aacc atg cct gct ctt ccg tgg cgc t Pro Ala Leu Pro Trp Arg acg acg gcg ctc gcg ctc acc acg gcg gtg acg gcc gcg acc ctg g Thr Thr Ala Leu Ala Leu Thr Thr Ala Val Thr Ala Ala Thr Leu cc gtc ggg gtg aac gac gcc ggt cag gcg gcg gct gct ccc ctg l Ala Val Gly Val Asn Asp Ala Gly Gln Ala Ala Ala Ala Pro Leu 25 3c gtg caa cgc gcg cag ttc cag tcg ggg tcg agc tac ctc gtc gtc y Val Gln Arg Ala Gln Phe Gln Ser Gly Ser Ser Tyr Leu Val Val 4 55 gag gtg ctc gat gac gac ctc gtc cac ttc gag ctg gcc ggg ggc ggc u Val Leu Asp Asp Asp Leu Val His Phe Glu Leu Ala Gly Gly Gly 6 acc gcc ccc ggc acg ggc tcc ccg ctg ttc acg acg cct cag gtc gcg r Ala Pro Gly Thr Gly Ser Pro Leu Phe Thr Thr Pro Gln Val Ala 75 8g cac gac tac gcg gga ccc gac gtg ttc acc cag acc ggg tct gtt s His Asp Tyr Ala Gly Pro Asp Val Phe Thr Gln Thr Gly Ser Val 9ag acc gcg gcg atg cgc atc gag gtc gat ccc gcg gat ctg tgc 2 Gln Thr Ala Ala Met Arg Ile Glu Val Asp Pro Ala Asp Leu Cys acg gcc acc gac atc acc cgc acc ccg aac ctt gta ctg cac gag 2 Thr Ala Thr Asp Ile Thr Arg Thr Pro Asn Leu Val Leu His Glu gcg tgt ccc gcc gac ctc ggc cag gcg tgg aag ggg ctg aac atc acg 2 Cys Pro Ala Asp Leu Gly Gln Ala Trp Lys Gly Leu Asn Ile Thr tcg gcg atg gag aac gcc tac ggt ctc ggg cag cag ttc ttc acg 2 Ser Ala Met Glu Asn Ala Tyr Gly Leu Gly Gln Gln Phe Phe Thr ggc agc gcg gac ggc gac tgg gtg ggc cgc acc cgc acc ccg ggt 22Gly Ser Ala Asp Gly Asp Trp Val Gly Arg Thr Arg Thr Pro Gly acc tac ggc aac gcg atg gtg ttc gac ccc gag aac ggg ccg gtc 225hr Tyr Gly Asn Ala Met Val Phe Asp Pro Glu Asn Gly Pro Val aac acg cag atc ccg gtg ctc ttc gcg gtc ggc gat gac aac gcg 2299 Gly Asn Thr Gln Ile Pro Val Leu Phe Ala Val Gly Asp Asp Asn Ala 22aac tac ggg ctg ttc gtc gat cag ctg tac aag cag gaa tgg aac ctc 2347 Asn Tyr Gly Leu Phe Val Asp Gln Leu Tyr Lys Gln Glu Trp Asn Leu 223gc gac ccg tgg acg gtg cgc atg tgg ggc gac cag gtg cgc tgg 2395 Thr Gly Asp Pro Trp Thr Val Arg Met Trp Gly Asp Gln Val Arg Trp 235 24ac ctc atg agc ggc gac gac ctg ccc gac ctt cgc cac gac tac atg 2443 Tyr Leu Met Ser Gly Asp Asp Leu Pro Asp Leu Arg His Asp Tyr Met 256tg acg ggc acc ccg ccc gtg ccg ccg aag aag gcg ttc ggg ctc 249eu Thr Gly Thr Pro Pro Val Pro Pro Lys Lys Ala Phe Gly Leu 265 27gg gtg tcg gag ttc ggc tac gac aac tgg agc gag gtc gac aat acg 2539 Trp Val Ser Glu Phe Gly Tyr Asp Asn Trp Ser Glu Val Asp Asn Thr 289tc gcg ggc ctg cgc tcg gcc gac ttt ccg gtc gat ggc gcg atg ctc 2587 Ile Ala Gly Leu Arg Ser Ala Asp Phe Pro Val Asp Gly Ala Met Leu 33gta cag tgg ttc ggg ggc gtc acc gcc gac tcg gac gac acc cgc 2635 Asp Val Gln Trp Phe Gly Gly Val Thr Ala Asp Ser Asp Asp Thr Arg 3325 atg ggc acc ctc gat tgg gac acg tcg agg ttt ccc gac cct gcg gga 2683 Met Gly Thr Leu Asp Trp Asp Thr Ser Arg Phe Pro Asp Pro Ala Gly 334tc gcc gac ctc gcc gag gac ggc gtc ggc atc atc ccg atc gag 273le Ala Asp Leu Ala Glu Asp Gly Val Gly Ile Ile Pro Ile Glu 345 35ag tcg tac gtc ggt cgc aac ctg ccg gag cac gcc cgg atg gcg gcg 2779 Glu Ser Tyr Val Gly Arg Asn Leu Pro Glu His Ala Arg Met Ala Ala 367ac ggt tac ctc gtg cgc tcc ggc tgc gct acg tgc ccg ccg gtg tac 2827 Asp Gly Tyr Leu Val Arg Ser Gly Cys Ala Thr Cys Pro Pro Val Tyr 389cg ggg aac ccc tgg tgg ggc aag ggc ggg atg atc gac tgg acg 2875 Leu Thr Gly Asn Pro Trp Trp Gly Lys Gly Gly Met Ile Asp Trp Thr 395 4cag ccg gaa gcc ggc gcc gtc tgg cac gac gag cag cgc cag cat ctc 2923 Gln Pro Glu Ala Gly Ala Val Trp His Asp Glu

Gln Arg Gln His Leu 442ac gag ggc gta ctg ggc cac tgg ctc gat ctc ggc gaa ccg gag 297sp Glu Gly Val Leu Gly His Trp Leu Asp Leu Gly Glu Pro Glu 425 43tg tac gac ccg aac gac tgg acc gcc ggc gtc atc ccc ggc aag cac 3 Tyr Asp Pro Asn Asp Trp Thr Ala Gly Val Ile Pro Gly Lys His 445cg cac gcc gac tat cac aac gcg tac aac ctg ctg tgg gcg cag agc 3 His Ala Asp Tyr His Asn Ala Tyr Asn Leu Leu Trp Ala Gln Ser 467cc gac ggg tac gcc gac aac ggc gtg cag aag cgt ccc ttc atg 3 Ala Asp Gly Tyr Ala Asp Asn Gly Val Gln Lys Arg Pro Phe Met 475 48tg acg cgc gcc gcg gcc gcc ggc atc cag cgt cat ggc gcg ggc atg 3 Thr Arg Ala Ala Ala Ala Gly Ile Gln Arg His Gly Ala Gly Met 49tca gcc gac atc ggg tcg acc atg aag gcg ctc ggg agc cag cag 32Ser Ala Asp Ile Gly Ser Thr Met Lys Ala Leu Gly Ser Gln Gln 55gcg cag atg cac atg tcg atg tcg ggg atc gac tat tac ggc tcc 3259 Asn Ala Gln Met His Met Ser Met Ser Gly Ile Asp Tyr Tyr Gly Ser 523ac atc ggc ggg ttc cgg cgg gag atg gcc gac ggc gac gtg aac gag 33Ile Gly Gly Phe Arg Arg Glu Met Ala Asp Gly Asp Val Asn Glu 545ac acc cag tgg ttc gcc gac agc gcg tgg ttc gac act ccg ctc 3355 Leu Tyr Thr Gln Trp Phe Ala Asp Ser Ala Trp Phe Asp Thr Pro Leu 555 56gg ccg cac acc gac aat ctc tgc aac tgc ctc gag acg agc ccc gac 34Pro His Thr Asp Asn Leu Cys Asn Cys Leu Glu Thr Ser Pro Asp 578tc ggc gac gtc gcg agc aac cgc gag aac ctg gtg cgc cgc tac 345le Gly Asp Val Ala Ser Asn Arg Glu Asn Leu Val Arg Arg Tyr 585 59ag ctg gct ccg tac tac tac tcg ctc gcg cac cgc gct cac cag ttc 3499 Glu Leu Ala Pro Tyr Tyr Tyr Ser Leu Ala His Arg Ala His Gln Phe 66ggc gag ccg ctc gct ccc ccg ctc gtg tac tac tac cag aac gac gac 3547 Gly Glu Pro Leu Ala Pro Pro Leu Val Tyr Tyr Tyr Gln Asn Asp Asp 623tt cgc gag atg ggg cat cag aag atg ctc ggg cgc gac ctg ctg 3595 His Val Arg Glu Met Gly His Gln Lys Met Leu Gly Arg Asp Leu Leu 635 64tc gcg atc gtc gcc gga gag ggc gag cgg gaa cgc gac gtg tac ctt 3643 Ile Ala Ile Val Ala Gly Glu Gly Glu Arg Glu Arg Asp Val Tyr Leu 656cg ggc gag tgg atc gac atc cac acg aac gag cgc atc cag agc 369la Gly Glu Trp Ile Asp Ile His Thr Asn Glu Arg Ile Gln Ser 665 67cg ggt cag tgg atc gac aac gtg ccg ctg tgg cgt gac ggc gtc ttc 3739 Thr Gly Gln Trp Ile Asp Asn Val Pro Leu Trp Arg Asp Gly Val Phe 689cc ctg ccg gcg tac gcc cgg gcg ggg gcg atc atc ccg aag gcc ttc 3787 Thr Leu Pro Ala Tyr Ala Arg Ala Gly Ala Ile Ile Pro Lys Ala Phe 77gac gcc tcc acg aag gac atc acc ggc aag cgc gag gat gcc gcg 3835 Val Asp Ala Ser Thr Lys Asp Ile Thr Gly Lys Arg Glu Asp Ala Ala 7725 gtg cgc aac gag ctg atc gca acc gtt tac gcc gac gac gtc gcg agc 3883 Val Arg Asn Glu Leu Ile Ala Thr Val Tyr Ala Asp Asp Val Ala Ser 734tc acc ctg tac gag gat gac ggc gcg acg acc gca tac gcc gac 393he Thr Leu Tyr Glu Asp Asp Gly Ala Thr Thr Ala Tyr Ala Asp 745 75gg gct gtc agg acc acg cag atc agc caa tcg ctc acg aac ggc gtg 3979 Gly Ala Val Arg Thr Thr Gln Ile Ser Gln Ser Leu Thr Asn Gly Val 767cc acg gtg acg gtg gga gcg gca tct gga acc tac tcc ggt gcg ccc 4 Thr Val Thr Val Gly Ala Ala Ser Gly Thr Tyr Ser Gly Ala Pro 789cc cgt ccc acg gtc gtc gag ctt gtc act gac ggc acg cag gcc 4 Thr Arg Pro Thr Val Val Glu Leu Val Thr Asp Gly Thr Gln Ala 795 8tcg acc gtc tcc ctc ggc agc gtt ccg ctg acg gag cac gcg aac aag 4 Thr Val Ser Leu Gly Ser Val Pro Leu Thr Glu His Ala Asn Lys 882cg ttc gac gcg gcg agc agc ggc tgg tac aac gcc ggc ggg ggg 4 Ala Phe Asp Ala Ala Ser Ser Gly Trp Tyr Asn Ala Gly Gly Gly 825 83tc gtt gtg gcc aag gcg gcg agc agt tcg gtg aac acc gcc aag acc 42Val Val Ala Lys Ala Ala Ser Ser Ser Val Asn Thr Ala Lys Thr 845tc tcg ttc acg ctc ggt gag gag tcg gtc tgg gcg acg ttc tcc tgc 4267 Phe Ser Phe Thr Leu Gly Glu Glu Ser Val Trp Ala Thr Phe Ser Cys 867ac gcc acg acg acc ttc ggt cag tca gtg tac gtc gtc gga aat 43Asn Ala Thr Thr Thr Phe Gly Gln Ser Val Tyr Val Val Gly Asn 875 88tt ccg cag ctc ggc aac tgg tcg ccg gcg gat gcc gtg aag ctc gag 4363 Val Pro Gln Leu Gly Asn Trp Ser Pro Ala Asp Ala Val Lys Leu Glu 89agc gcc tac ccc acc tgg acc ggg gtg gtg cgg aac ctg ccg ccg 44Ser Ala Tyr Pro Thr Trp Thr Gly Val Val Arg Asn Leu Pro Pro 99agc acg gtc gaa tgg aag tgc atc aaa cgt cag gag gcc ggc ctg 4459 Ser Ser Thr Val Glu Trp Lys Cys Ile Lys Arg Gln Glu Ala Gly Leu 923cg aac acg gcg gat gcg tgg gag ccc ggc ggg aac aac atc ctc tcg 45Asn Thr Ala Asp Ala Trp Glu Pro Gly Gly Asn Asn Ile Leu Ser 945ca cct tcc ggc tcg gcg ggg ata acc acc ggc gcc ttc tga 4552 Thr Pro Pro Ser Gly Ser Ala Gly Ile Thr Thr Gly Ala Phe 955 96ccagggggg ctcgatcccg gtcgccagcg caagcgcggc gcccggggtc gacgcgtgtt 46cagtac gcgaaggaac cagccctcta cgacaccggc ctcgaccccg ccgaaggact 4672 ctggcaccgg tcaggctgga tcggacaaca ctgacacgcc ccgacgccat ccactctttt 4732 tggcctacaa cccgttgtcg cacgtgcgcc tcttggcccg ggcacgacga aacccccgcg 4792 atccagggat cggcgggggt ttcggatggc ggtgacggtg ggatttgaac ccacggtagg 4852 gggttaccct acacaacttt tcgagagttg caccttcggc cgctcggaca cgtcaccggg 49agttta cgcgacgttc tcctggcgcg ccaatcggcg gcgccccgcc cgcgagaatc 4972 caggcccgcg ccgagaatcc gcgggcgcct ggattctcag cacggggatg gattctcgcc 5catccga gccccgcggc gagcgggctc agtgctcgtc ctccatgagc atgccgaccg 5tggcgca ggcgtcgccg cgccaggcct cgatgccctc gcgcacggcg aaggcggcga 5tgaggcc ggtgatcgcg tcggcccacc accagcccag gaggctgttg agcacgaggc 52gagcac ggccgccgac aggtaggtgc agatgagggt ctgcttcgag tcggccacgg 5272 cggtggccga tccgagctcg cggccggcgc ggcgctcggc gaacgacagg aacggcatga 5332 tcgccacgct gagcgccgtg atgacgatgc cgagcgtcga gtgctccacg tccgcgccgc 5392 cgacgagggc caggaccgac gtgacggtga cgtacgcggc gagcgcgaag aaggccacgg 5452 cgatgacgcg cagcgtgccg cgctcccagc gctccgggtc gcgccgcgtg aactgccacg 55ggcggc ggccgagagc acctcgatgg tcgagtccag gccgaacgcg acgagcgcgg 5572 ccgacgaggc cgcagctccc gcggcgatcg cgacgaccgc ctcgacgacg ttataggcga 5632 tggtcgcggc gacgatccag cggatgcgcc gctgcaggac ggatcgccga tcggcagacg 5692 cggtggcggt catgcgcagg tgcagctctc tccggcgcag cagccgggct cgacgtacag 5752 gacgacgcgc agcagctcgt cgagcgcggg cgcgaggtgg gcgtcggcca gccggtacc 58 Arthrobacter globiformis CDS ((4646) 26 gcggccgctc cctgggccca ccggtcgtcc tgccaggtct cgaccgcgac ggggatcgcc 6gcgcg ggatgagccc ggcccggaac gcgggctcga agccgaacga ccagtgcgtc gcctccg ccccttcgcc gagcaggatg acgatctccg ggtcgatcag gttcaccacc gccagca cccggccgag caggtgcccg gcctgcgaga actgctgctg cgcggctgga 24gccat cggcgagcgc ctttagggcc gggtagccgt cgccgtcgcc gaggattccc 3cgcggg cgcgccgcac gagcgcttcc tggccgatga gcgcctcgag gcatccgttg 36gcact gacagggcgg accgtcctcc tccaccggga tgtggccgat ctctccggct 42gctgc gtccgcggag cacgcggccc tcggagatga ggccggcgcc ggcacccgtt 48cgtga tcacgagcac gtcctcgtgt ccgcgggcct gaccgtgcag ggcctccgcg 54gaggg cgttgacgtt gttctcgacg aggacgggca ggtcgagctc gcgccggagc 6cgccga gaggcacccg cagccagccc agctccgtcg agtcgaccgt gccgaccgcc 66gtcga cgttgccggg cacgcccacg ccgagaccga gcagggggac gccgtcgccg 72gatga acgagcggag catgtcgctg agcttcgcga tcgcggtgcc cgttccggcg 78gggct cggtgagcga tcgctgcacc cggccgtcga tgctgacctc gaccgcggtg 84gtcgg ctacgacctt aacgccgacg gcacggccgg cgtcggcgac gagaccgagc 9gcgcgg gacgtccacc ctgcgagggg cggtgatcca gctcgaccag caggccatcg 96gagct cccgggtgtg ctgcgtgacg agcgccggcg agacgccgag ctcgcgcgcg gtcggccc gcgaggtcgg gccattgctg ccgatgtggg cgagcatggc cgatcgcgtc atcggtgc gtggattcgg gtgggccaca ggaacccctt tgttcaggac tgaaagaacg agagcaca gcgagggcga acgtcaactc acagcctgat ctcccgggcc gagagcccgg gccgaact cacctcttga cgagcccgtt cgactacgga atattcccgg ctaaatacag ccgaacaa aggggttccc atg tct gat gcc gtc ggc gct ccc cgc gcg ctg t Ser Asp Ala Val Gly Ala Pro Arg Ala Leu aac gat ccg cac cgc tcg cgt ctc gcc gcg ctt ccg cgc cgg acc gcg n Asp Pro His Arg Ser Arg Leu Ala Ala Leu Pro Arg Arg Thr Ala 5 gga ttc gtc ctg gca ctg gtc gcg ggg ctg gtc ttc acg ctg ctc gcc y Phe Val Leu Ala Leu Val Ala Gly Leu Val Phe Thr Leu Leu Ala 3 gct ccg ccc gcc cac gcc aac aca ctc gac ggg gtc tgg cac aac ccg a Pro Pro Ala His Ala Asn Thr Leu Asp Gly Val Trp His Asn Pro 45 5c ggg gcc gac gag ctg tat gcg acg cag ccg acg gag cgt tca cca r Gly Ala Asp Glu Leu Tyr Ala Thr Gln Pro Thr Glu Arg Ser Pro 6 75 cgc gac ccg atg gcc ggc gac aac gtc acc gtc cgg gcg acg acg tgg g Asp Pro Met Ala Gly Asp Asn Val Thr Val Arg Ala Thr Thr Trp 8 ccg gtg gcg ccg ggc cag tcg gtc tgg gtg acg tgg agc gtg aac ggc o Val Ala Pro Gly Gln Ser Val Trp Val Thr Trp Ser Val Asn Gly 95 gtc gcg cag act ccg cgc ggg gca tcc tgg gac tac aac tcc ggc aac l Ala Gln Thr Pro Arg Gly Ala Ser Trp Asp Tyr Asn Ser Gly Asn acg tat tgg aag ctc gat ctg ggt tcc ttc gcc cgg ggc gac gtc n Thr Tyr Trp Lys Leu Asp Leu Gly Ser Phe Ala Arg Gly Asp Val gag tac acg gtt cac gcc gac gtc aac ggc ggt ggg cag cgc agc l Glu Tyr Thr Val His Ala Asp Val Asn Gly Gly Gly Gln Arg Ser tca ggg ccg ttc tcc ttc acc acg aca tcc tgg agc acg gtc acc gac r Gly Pro Phe Ser Phe Thr Thr Thr Ser Trp Ser Thr Val Thr Asp acg tcg gtc gtc gac aac ggc act tcc gtc gac atc gtc acg gga l Thr Ser Val Val Asp Asn Gly Thr Ser Val Asp Ile Val Thr Gly agc gct ggc gat ttc acg ccg aag gtg cgc ttc gcc ttc ccg cgc p Ser Ala Gly Asp Phe Thr Pro Lys Val Arg Phe Ala Phe Pro Arg 2gac ggc ttc gac gtg cag atc gca ccc acg ggc gcg ggg ctc gag u Asp Gly Phe Asp Val Gln Ile Ala Pro Thr Gly Ala Gly Leu Glu 22agc ggg ctg ccg gac tac acc gtc acc gac ggc gcg agc cag gtc u Ser Gly Leu Pro Asp Tyr Thr Val Thr Asp Gly Ala Ser Gln Val 223ag atc gcc acc gac gag ctc gtc ctc cgg atc gac aag aac ccc tat 2 Ile Ala Thr Asp Glu Leu Val Leu Arg Ile Asp Lys Asn Pro Tyr 245tg tcg gtc tac gag ggc gac ggc acg acg ctc atc acc cgc cag 2 Leu Ser Val Tyr Glu Gly Asp Gly Thr Thr Leu Ile Thr Arg Gln 255 26ac gac ccc gcg gtc ttc cgg aac atc ggt tgg gcg agc gac ggc gaa 2 Asp Pro Ala Val Phe Arg Asn Ile Gly Trp Ala Ser Asp Gly Glu 278ct gtg acc cgc atc gag gat cac ttc ctc aca ccc acg ggc gaa 2 Thr Val Thr Arg Ile Glu Asp His Phe Leu Thr Pro Thr Gly Glu 285 29gg ttc gag ggg ttc ggc gaa cgg tac gac cgg ctc gac cac cgg gga 2225 Arg Phe Glu Gly Phe Gly Glu Arg Tyr Asp Arg Leu Asp His Arg Gly 33acc gac gtg cac aac tac gtc tac aac cag tac cag gac cag ggc gcg 2273 Thr Asp Val His Asn Tyr Val Tyr Asn Gln Tyr Gln Asp Gln Gly Ala 323gc cgc acc tac tac tcg gtg ccg tac ttc gcc aac tcc gcc ggc 232rg Arg Thr Tyr Tyr Ser Val Pro Tyr Phe Ala Asn Ser Ala Gly 335 34ac ggc atc cac gtg ccg agc acg cgc tat gcg atc ttc aat ctc gcg 2369 Tyr Gly Ile His Val Pro Ser Thr Arg Tyr Ala Ile Phe Asn Leu Ala 356ac ctc gac gac atg gcc gga ttc acg gtc gac acg gga ggc gcc 24His Leu Asp Asp Met Ala Gly Phe Thr Val Asp Thr Gly Gly Ala 365 37tg gac tcc acg ctg acg tac cag ttc ttc acc ggc gac cag acc gag 2465 Leu Asp Ser Thr Leu Thr Tyr Gln Phe Phe Thr Gly Asp Gln Thr Glu 389tg ctc gac gac ttc acg gcc gag acc ggc cgt ccg ctc ctt ccg ccg 25Leu Asp Asp Phe Thr Ala Glu Thr Gly Arg Pro Leu Leu Pro Pro 44tgg gcg ttt gga ctc tgg ggc tcc gcc aac gag tgg aac aac cag 256rp Ala Phe Gly Leu Trp Gly Ser Ala Asn Glu Trp Asn Asn Gln 4425 gcc gag gtc gag gcc tgg ctc gac cag gtg gag agc tcc ggc atc ccg 26Glu Val Glu Ala Trp Leu Asp Gln Val Glu Ser Ser Gly Ile Pro 434gc gtg ctc gtg ctc gag cag tgg agc gac gag gcg acg ttc tac 2657 His Ser Val Leu Val Leu Glu Gln Trp Ser Asp Glu Ala Thr Phe Tyr 445 45tc tgg aag gac gcg cag tac acc ccc acc gac ggc agc acg ccg ctg 27Trp Lys Asp Ala Gln Tyr Thr Pro Thr Asp Gly Ser Thr Pro Leu 467ag tac gac gac ctc acg ttc ccc agc gga ggt gcg tgg agc gac ccc 2753 Gln Tyr Asp Asp Leu Thr Phe Pro Ser Gly Gly Ala Trp Ser Asp Pro 489ag atg att gcc gag gcg cac gcc cag aac gtc aag gtg ctc ctc 28Gln Met Ile Ala Glu Ala His Ala Gln Asn Val Lys Val Leu Leu 495 5tgg cag att ccg gtg ctg aag gag aac ttc acc tcc aac ccg gcc acg 2849 Trp Gln Ile Pro Val Leu Lys Glu Asn Phe Thr Ser Asn Pro Ala Thr 552cg cag cag cac ctc aac gac aag gcg tat gcg cag gcc cag ggc 2897 Ala Pro Gln Gln His Leu Asn Asp Lys Ala Tyr Ala Gln Ala Gln Gly 525 53ac ctg gtc gac gac ggc gcg ggg cag ccg tac cgc atc ccc acc gga 2945 Tyr Leu Val Asp Asp Gly Ala Gly Gln Pro Tyr Arg Ile Pro Thr Gly 545ag tgg ttt gga gac agc acg gtg ccc gac ttc aca gat gcc gag gcc 2993 Gln Trp Phe Gly Asp Ser Thr Val Pro Asp Phe Thr Asp Ala Glu Ala 567ac tgg tgg atg gac aag cgg cgg tac ctc gtc gag gag ctc ggt 3 Asp Trp Trp Met Asp Lys Arg Arg Tyr Leu Val Glu Glu Leu Gly 575 58tc gac ggc ttc aag acc gac ggg agc gag gcg ctc ttc ggg cgt gac 3 Asp Gly Phe Lys Thr Asp Gly Ser Glu Ala Leu Phe Gly Arg Asp 59atc gtc agc gac ggg cgc cgc ggt gac gag atg cac aac gcc tac 3 Ile Val Ser Asp Gly Arg Arg Gly Asp Glu Met His Asn Ala Tyr 66aac gag tac acc tcc gcc tac aac gac ttc gtg cag gag acg acg 3 Asn Glu Tyr Thr Ser Ala Tyr Asn Asp Phe Val Gln Glu Thr Thr 623gc gcc gac ggc acg atc ttc agc cgg gcg ggc acc tcc ggc ggc cag 3233 Gly Ala Asp Gly Thr Ile Phe Ser Arg Ala Gly Thr Ser Gly Gly Gln 645aa tcc atc ttc tgg gcc ggg gac cag gcg tcg acg ttc ggc gct 328lu Ser Ile Phe Trp Ala Gly Asp Gln Ala Ser Thr Phe Gly Ala 655 66tc cag gag gcc gtc cgg gcc ggg cag agc gcg ggc cag tcg gga gtg 3329 Phe Gln Glu Ala Val Arg Ala Gly Gln Ser Ala Gly Gln Ser Gly Val 678tc tgg gcc tgg gac ctc ggc ggc ttc acc ggg tcg ttc cca agc 3377 Pro Phe Trp Ala Trp Asp Leu Gly Gly Phe Thr Gly Ser Phe Pro Ser 685 69cg gag ctg tat ctg cgc tcg acc gct cag gcg gtg ttc tcg ccg atc 3425 Ala Glu Leu Tyr Leu

Arg Ser Thr Ala Gln Ala Val Phe Ser Pro Ile 77atg cag tac cac tcg gag aag gcc gac ccc agt ccg tcc gag gcg cgc 3473 Met Gln Tyr His Ser Glu Lys Ala Asp Pro Ser Pro Ser Glu Ala Arg 723cc tgg aac gtg cag gcg cgc acc ggg aac acc act gtc gtc ccc 352ro Trp Asn Val Gln Ala Arg Thr Gly Asn Thr Thr Val Val Pro 735 74cc ttc gcc cgt tac gcg aac gta cgg atg aac ctc gtg ccc tat ctg 3569 Thr Phe Ala Arg Tyr Ala Asn Val Arg Met Asn Leu Val Pro Tyr Leu 756cg gag gcg gac gac agc gcg acg acg ggt gtg ccg atg atg cgc 36Thr Glu Ala Asp Asp Ser Ala Thr Thr Gly Val Pro Met Met Arg 765 77cg atg agc ctc gcg ttc ccc gac gac ccg gat gcc gcg cag tac gac 3665 Ala Met Ser Leu Ala Phe Pro Asp Asp Pro Asp Ala Ala Gln Tyr Asp 789ag cag tac atg ttc ggg tct cag ctg ctg gtc gca ccg att acg aac 37Gln Tyr Met Phe Gly Ser Gln Leu Leu Val Ala Pro Ile Thr Asn 88ggc cag acc gtg aaa gac gtc tac ctg ccc gcg ggc gag tgg tac 376ly Gln Thr Val Lys Asp Val Tyr Leu Pro Ala Gly Glu Trp Tyr 8825 gac ttc tgg aac ggc gga cgc gcg agc ggc gag ggc gtg aag atg tac 38Phe Trp Asn Gly Gly Arg Ala Ser Gly Glu Gly Val Lys Met Tyr 834cc gga ccc gac ggc atc ccc gta tac gct cgc gcc gga gcg gtc 3857 Asp Ala Gly Pro Asp Gly Ile Pro Val Tyr Ala Arg Ala Gly Ala Val 845 85tc ccg ctc aac ctc aac gac gcg tat gag gtg ggc ggc acg atc ggc 39Pro Leu Asn Leu Asn Asp Ala Tyr Glu Val Gly Gly Thr Ile Gly 867ac gac gtg gag agc tac gac aac ctt gtg ttc cgc gtt tac ccc tcc 3953 Asn Asp Val Glu Ser Tyr Asp Asn Leu Val Phe Arg Val Tyr Pro Ser 889ag agc agc tac gag tac ttc gaa gac caa gcg aac gcg cac cgc 4 Glu Ser Ser Tyr Glu Tyr Phe Glu Asp Gln Ala Asn Ala His Arg 895 9cgg atc gat gtc tcg gcc gac cgc gca gcg cgc acg gtc gag gtg tct 4 Ile Asp Val Ser Ala Asp Arg Ala Ala Arg Thr Val Glu Val Ser 992cc gcg ctc acg acc gcg agc acc ttc cag gtg tcg ggc acc aag 4 Pro Ala Leu Thr Thr Ala Ser Thr Phe Gln Val Ser Gly Thr Lys 925 93cc gac acc gtg acc gtc gcg ggc tcg gca ctg cct gag gtc aac agc 4 Asp Thr Val Thr Val Ala Gly Ser Ala Leu Pro Glu Val Asn Ser 945tg agc gcg ctg gcc gca tcc acc gag gcc tgg tac tgg gat gcg aag 4 Ser Ala Leu Ala Ala Ser Thr Glu Ala Trp Tyr Trp Asp Ala Lys 967ag ctg acg tac gtg aag gtc ggt gcg agc acc ggc gag cgc acg 424ln Leu Thr Tyr Val Lys Val Gly Ala Ser Thr Gly Glu Arg Thr 975 98tc ctc ctg ctg ggc gtc gac aag gcc ggg tac gag gcc gag ttc gcg 4289 Ile Leu Leu Leu Gly Val Asp Lys Ala Gly Tyr Glu Ala Glu Phe Ala 99 cat acg gcc gtc tcg acg aac gcc gac cac ccg ggc tac acc 4334 Gly His Thr Ala Val Ser Thr Asn Ala Asp His Pro Gly Tyr Thr ggg ctc ggc ttc gtc gac ggc ttc gcg aac gca gga gac gcg gtg 4379 Gly Leu Gly Phe Val Asp Gly Phe Ala Asn Ala Gly Asp Ala Val 25 g ttc gac gtg tgg gcc gag gag aac ggc gcg cac cag ctc cgc 4424 Glu Phe Asp Val Trp Ala Glu Glu Asn Gly Ala His Gln Leu Arg 4ttc cgc tac gga aac gga gcg gcg acc ccc gcc acc cgc acg atc 4469 Phe Arg Tyr Gly Asn Gly Ala Ala Thr Pro Ala Thr Arg Thr Ile 55 g gtc gac gga gcg cct ctg gga acg ctg tcg ctt ccg ccc acc 45Val Asp Gly Ala Pro Leu Gly Thr Leu Ser Leu Pro Pro Thr 7ggg tcg tgg agt tcg tgg ggc acg gcc tcg atc gac gtg acc ctc 4559 Gly Ser Trp Ser Ser Trp Gly Thr Ala Ser Ile Asp Val Thr Leu 85 a ccc gga cgc cac gcc gta cgg atc gag tac gcc gga ggc gat 46Pro Gly Arg His Ala Val Arg Ile Glu Tyr Ala Gly Gly Asp tcc ggc ggc gtc aac ctc gac aac ctc gtc ctc gcg cgc tga 4646 Ser Gly Gly Val Asn Leu Asp Asn Leu Val Leu Ala Arg gcgcacacgg gaaagggaga agaaccatgc ctgctcttcc gtggcgccgc acgacggcgc 47gctcac cacggcggtg acggccgcga ccctggtcgc cgtcggggtg aacgacgccg 4766 gtcaggcggc ggctgctccc ctgggcgtgc aacgcgcgca gttccagtcg gggtcgagct 4826 acctcgtcgt cgaggtgctc gatgacgacc tcgtccactt cgagctggcc gggggcggca 4886 ccgcccccgg cacgggctcc ccgctgttca cgacgcctca ggtcgcgaag cacgactacg 4946 cgggacccga cgtgttcacc cagaccgggt ctgttctgca gaccgcggcg atgcgcatcg 5tcgatcc cgcggatctg tgcgtgacgg ccaccgacat cacccgcacc ccgaaccttg 5tgcacga ggcgtgtccc gccgacctcg gccaggcgtg gaaggggctg aacatcacga 5cggcgat ggagaacgcc tacggtctcg ggcagcagtt cttcacgggc ggcagcgcgg 5gcgactg ggtgggccgc acccgcaccc cgggtggcac ctacggcaac gcgatggtgt 5246 tcgaccccga gaacgggccg gtcggcaaca cgcagatccc ggtgctcttc gcggtcggcg 53caacgc gaactacggg ctgttcgtcg atcagctgta caagcaggaa tggaacctca 5366 ccggcgaccc gtggacggtg cgcatgtggg gcgaccaggt gcgctggtac ctcatgagcg 5426 gcgacgacct gcccgacctt cgccacgact acatggagct gacgggcacc ccgcccgtgc 5486 cgccgaagaa ggcgttcggg ctctgggtgt cggagttcgg ctacgacaac tggagcgagg 5546 tcgacaatac gatcgcgggc ctgcgctcgg ccgactttcc ggtcgatggc gcgatgctcg 56acagtg gttcgggggc gtcaccgccg actcggacga cacccgcatg ggcaccctcg 5666 attgggacac gtcgaggttt cccgaccctg cgggaaagat cgccgacctc gccgaggacg 5726 gcgtcggcat catcccgatc gaggagtcgt acgtcggtcg caacctgccg gagcacgccc 5786 ggatggcggc ggacggttac ctcgtgcgct ccggctgcgc tacgtgcccg ccggtgtacc 5846 tgacggggaa cccctggtgg ggcaagggcg ggatgatcga ctggacgcag ccggaagccg 59cgtctg gcacgacgag cagcgccagc atctcgtcga cgagggcgta ctgggccact 5966 ggctcgatct cggcgaaccg gagatgtacg acccgaacga ctggaccgcc ggcgtcatcc 6gcaagca cgcgcacgcc gactatcaca acgcgtacaa cctgctgtgg gcgcagagca 6ccgacgg gtacgccgac aacggcgtgc agaagcgtcc cttcatgctg acgcgcgccg 6ccgc 6>