Modified α-glucosidase and applications of same

Patent metadata

Field	Value
Publication number	US-9493753-B2
Application number	US-201214005076-A
Country	US
Kind code	B2
Filing date	Mar 5, 2012
Priority date	Mar 16, 2011
Publication date	Nov 15, 2016
Grant date	Nov 15, 2016

Abstract

The object is to provide an α-glucosidase in which a transglycosylation activity predominates, and use thereof, and the like. A modified α-glucosidase consisting of an amino acid sequence in which one or two or more of the amino acid(s) is selected from a group of specific amino acids.

First claim

The invention claimed is:

1. A modified α-glucosidase which (a) consists of an amino acid sequence which is identical to SEQ ID NO: 2 except for a substitution at a position corresponding to position 343 of SEQ ID NO: 2, or (b) comprises an amino acid sequence which has 95% or more identity with SEQ ID NO: 2 and comprises a substitution corresponding to position 343 of SEQ ID NO: 2,
wherein the amino acid at position 343 is substituted by a cysteine, aspartic acid, methionine, histidine, alanine, phenylalanine, glycine, threonine, glutamic acid, valine, glutamine, asparagine or isoleucine, and
wherein the modified α-glucosidase has at least one of: improved transglycosylation activity compared to the α-glucosidase of SEQ ID NO: 2, and decreased hydrolysis activity compared to the α-glucosidase of SEQ ID NO: 2.

2. The modified α-glucosidase according to claim 1 , wherein said α-glucosidase consists of the amino acid sequence of SEQ ID No: 20.

3. An enzyme agent comprising the modified α-glucosidase according to claim 2 .

4. A pharmaceutical composition, a composition, a cosmetic composition, a food composition or a feed composition, comprising the modified α-glucosidase according to claim 2 .

5. The α-glucosidase according to claim 2 , having a transglycosylation activity to form α-1,4 or α-1,6 glucosidic linkages.

6. The modified α-glucosidase according to claim 1 , wherein said α-glucosidase (a) consists of the amino acid sequence of SEQ ID No: 20, or (b) comprises an amino acid sequence which has 95% or more identity with the sequence of SEQ ID NO: 20.

7. The modified α-glucosidase according to claim 1 , wherein said α-glucosidase (a) consists of the amino acid sequence of SEQ ID NO.: 20 or (b) comprises an amino acid sequence which has 98% or more identity with the sequence of SEQ ID NO.: 20.

8. The modified α-glucosidase of claim 1 , wherein said modified α-glucosidase comprises an amino acid sequence which has 95% or more identity with SEQ ID NO: 2 and comprises a substitution corresponding to position 343 of SEQ ID NO: 2, wherein the amino acid at position 343 is substituted by a cysteine, aspartic acid, methionine, histidine, alanine, phenylalanine, glycine, threonine, glutamic acid, valine, glutamine, asparagine or isoleucine, wherein the modified α-glucosidase has at least one of: improved transglycosylation activity compared to the α-glucosidase of SEQ ID NO: 2, and decreased hydrolysis activity compared to the α-glucosidase of SEQ ID NO: 2, and wherein said modified α-glucosidase further comprises one or more amino acid substitutions at an amino acid selected from the group consisting of:
(i) an amino acid corresponding to the amino acid at position 452 of SEQ ID NO: 2;
(ii) an amino acid corresponding to the amino acid at position 496 of SEQ ID NO: 2;
(iii) an amino acid corresponding to the amino acid at position 410 of SEQ ID NO: 2;
(iv) an amino acid corresponding to the amino acid at position 495 of SEQ ID NO: 2;
(v) an amino acid corresponding to the amino acid at position 498 of SEQ ID NO: 2;
(vi) an amino acid corresponding to the amino acid at position 499 of SEQ ID NO: 2;
(vii) an amino acid corresponding to the amino acid at position 531 of SEQ ID NO: 2;
(viii) an amino acid corresponding to the amino acid at position 533 of SEQ ID NO: 2;
(ix) an amino acid corresponding to the amino acid at position 579 of SEQ ID NO: 2;
(x) an amino acid corresponding to the amino acid at position 585 of SEQ ID NO: 2;
(xi) an amino acid corresponding to the amino acid at position 662 of SEQ ID NO: 2;
(xii) an amino acid corresponding to the amino acid at position 715 of SEQ ID NO: 2; and
(xiii) an amino acid corresponding to the amino acid at position 721 of SEQ ID NO: 2.

9. The modified α-glucosidase of claim 8 , wherein the modified α-glucosidase comprises a substitution at an amino acid corresponding to the amino acid at position 452 of SEQ ID NO: 2, and the amino acid after the substitution is glycine, aspartic acid or glutamic acid.

10. The modified α-glucosidase of claim 8 , wherein the modified α-glucosidase comprises a substitution at an amino acid corresponding to the amino acid at position 496 of SEQ ID NO: 2, and the amino acid after the substitution is valine, asparagine or glutamine.

11. The modified α-glucosidase of claim 8 , wherein the amino acid corresponding to position 343 of SEQ ID NO: 2 is substituted with aspartic acid, and wherein the amino acid corresponding to position 452 of SEQ ID NO: 2 is substituted with alanine or glycine.

12. The modified α-glucosidase of claim 8 , wherein the amino acid corresponding to position 343 of SEQ ID NO: 2 is substituted with aspartic acid or methionine, and wherein the amino acid corresponding to position 496 of SEQ ID NO: 2 is substituted with isoleucine, arginine, cysteine or threonine.

13. The modified α-glucosidase of claim 8 , wherein the modified α-glucosidase comprises a substitution at an amino acid corresponding to the amino acid at position 495 of SEQ ID NO: 2, and the amino acid after the substitution is glycine, proline or valine.

14. The modified α-glucosidase of claim 8 , wherein the modified α-glucosidase comprises a substitution at an amino acid corresponding to the amino acid at position 498 of SEQ ID NO: 2, and the amino acid after the substitution is leucine or serine.

15. A method for producing an oligosaccharide comprising reacting the modified α-glucosidase of claim 1 with an oligosaccharide or polysaccharide having an α-1,4 glucosidic linkage.

16. A method for producing an oligosaccharide comprising reacting the modified α-glucosidase of claim 13 with an oligosaccharide or polysaccharide having an α-1,6 glucosidic linkage.

Assignees

• Ishihara Satoru
• Amano Enzyme Inc

Inventors

• Ishihara Satoru

Classifications

• C12P19/20

  produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose · CPC title

• C12N9/2428 Primary

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12N9/2408 Primary

  acting on alpha -1,4-glucosidic bonds · CPC title

• C12P19/04

  Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds · CPC title

• Y02P20/52

  using catalysts, e.g. selective catalysts · CPC title