α-Isomaltosyltransferase, process for producing the same and use thereof
View Patent ↗The object of the present invention is to provide an α-isomaltosyl-transferring enzyme which forms a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} from a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end; microorganisms which produce the enzyme; process for producing the enzyme; cyclotetrasaccharide or saccharide compositions comprising the same; and uses thereof.
1. An isolated α-isomaltosyl-transferring enzyme which forms a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} from a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end, said enzyme being not activated by Ca 2+ and not inhibited by EDTA.
2. The isolated α-isomaltosyl-transferring enzyme of claim 1 , wherein said saccharide is one or more saccharides selected from the group consisting of panose and isomaltosylmaltose.
3. The isolated α-isomaltosyl-transferring enzyme of claim 1 or 2 , which is derived from a microorganism.
4. An isolated α-isomaltosyl-transferring enzyme of claim 1 which has the following physicochemical properties:
(1) Action
Forming a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} from a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end;
(2) Molecular weight
About 82,000 to about 136,000 daltons when determined on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE);
(3) Isoelectric point (pI)
About 3.7 to about 8.3 when determined on isoelectrophoresis using ampholine;
(4) Optimum temperature
About 45° C. to about 50° C. when reacted at a pH of 6.0 for 30 min;
(5) Optimum pH
About 5.5 to about 6.5 when reacted at 35° C. for 30 min;
(6) Thermal stability
Substantially not inactivated at temperatures of about 45° C. or lower when reacted at a pH of 6.0 for 60 min; and
(7) pH Stability
Substantially not inactivated at pHs about 4.5 to about 9.0 when reacted at 4° C. for 24 hours.
5. The isolated α-isomaltosyl-transferring enzyme of claim 1 , which has one or more amino acid sequences selected from the group consisting of SEQ ID NOs:1 to 8.
6. The isolated α-isomaltosyl-transferring enzyme of claim 1 , which is a purified or crude enzyme.
7. A process for producing α-isomaltosyl-transferring enzyme, of claim 1 , which comprises the steps of:
culturing in a nutrient culture medium a microorganism capable of genus Bacillus or Arthrobacter , capable of producing the α-isomaltosyl-transferring enzyme of claim 1 to produce said enzyme; and
collecting the produced α-isomaltosyl-transferring enzyme from the resulting culture.
8. A method of α-isomaltosyl-transferring reaction, which comprises a step of contacting the α-isomaltosyl-transferring enzyme of claim 1 with a solution comprising a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end.
9. The method of claim 8 , wherein said saccharide is one or more saccharides selected from the group consisting of panose and isomaltosylmaltose.
10. The method of claim 8 , wherein said enzyme is allowed to act on said saccharide to form a saccharide-transferred product in the presence of one or more acceptors selected from the group consisting of D-glucose, D-xylose, L-xylose, D-galactose, D-fructose, D-arabinose, D-fucose, L-sorbose, L-rhamnose, methyl-α-glucopyranoside, methyl-α-glucopyranoside, D-ribose, L-sorbose, D-psicose, sorbitol, xylitol, arabitol, ribitol, erythritol, maltose, maltotriose, maltotetraose, maltopentaose, isomaltose, isomaltotriose, α,α-trehalose, α,α-trehalose, kojibiose, nigerose, cellobiose, gentibiose, isopanose, maltitol, maltotriitol, lactose, sucrose, erlose, isomaltosylglucoside, and L-ascorbic acid.
11. A process for producing a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→} or a saccharide composition comprising the same, which comprises a step of:
contacting the α-isomaltosyl-transferring enzyme of claim 1 with a solution comprising a saccharide having a glucose polymerization degree of at least three and having both the α-1,6 glucosidic linkage as a linkage at the non-reducing end and the α-1,4 glucosidic linkage other than the linkage at the non-reducing end.
12. The process of claim 11 , wherein said saccharide is one or more saccharides selected from the group consisting of panose and isomaltosylmaltose.
13. The process of claim 11 , wherein one or more enzymes selected from the group consisting of α-amylase, α-amylase, glucoamylase, and α-glucosidase are allowed to act on the resulting mixture obtained after the action of α-isomaltosyl-transferring enzyme.
14. The process of claim 11 , wherein one or more purification methods selected from the group consisting of decoloration, desalting, fractionation by column chromatography, separation with a membrane, fermentation treatment using microorganisms, and decomposition and removal by an alkaline treatment are applied to the resulting mixture obtained after the action of α-isomaltosyl-transferring enzyme.
15. The process of claim 11 , wherein said cyclotetrasaccharide or the saccharide composition comprising the same contains at least 10% (w/w), on a dry solid basis, of a cyclotetrasaccharide having the structure of cyclo{→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→6)-α-D-glucopyranosyl-(1→3)-α-D-glucopyranosyl-(1→}.
16. The process of claim 11 , wherein said cyclotetrasaccharide or the saccharide composition comprising the same is in the form of a syrup, massecuite, amorphous powder, amorphous solid, crystalline powder, or crystalline solid.
17. The process of claim 16 , wherein said crystal is prepared by crystallizing in an aqueous solution without using any organic solvent.
18. The process of claim 7 , wherein said microorganism of the genus Bacillus or Arthrobacter is selected from the group consisting of Bacillus globisporus C9, FERM BP-7143; Bacillus globisporus C11, FERM BP-7144, Bacillus globisporus N75, FERM BP-7591; Arthrobacter ramosus S1, FERM BP-7592; and Arthrobacter globiformis A19, FERM EP-7590, which all have been deposited in International Patent Organism Depositary National Institute of Advanced Industrial Science and technology; and mutants thereof.