Glucosyltransferase enzymes for production of glucan polymers

• US 9,926,541 B2
• Filed: 02/11/2015
• Issued: 03/27/2018
• Est. Priority Date: 02/14/2014
• Status: Active Grant

• Current Assignee: Nutrition & Biosciences USA 4, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Paullin, Jayme L, Payne, Mark S, Dennes, T Joseph, Brun, Yefim, Nambiar, Rakesh, Scholz, Thomas
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US14/619,193
• Publication Number:
  US 20150232819A1
• Time in Patent Office: 1,140 Days
• CPC Class Codes: C08B 37/0009 alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
• CPC Class Codes: C08B 37/0021 Dextran, i.e. (alpha-1,4)-D-glucan; Derivatives thereof, e.g. Sephadex, i.e. crosslinked dextran
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/18 produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Abstract

Compositions are disclosed herein comprising poly alpha-1,3-1,6-glucan with a weight average degree of polymerization (DP_w) of at least 1000. This glucan polymer comprises at least 30% alpha-1,3 linkages and at least 30% alpha-1,6 linkages. Further disclosed are glucosyltransferase enzymes that synthesize poly alpha-1,3-1,6-glucan. Ether derivatives of poly alpha-1,3-1,6-glucan and methods of using such derivatives as viscosity modifiers are also disclosed.

Claims

1. A reaction solution comprising water, sucrose and an isolated glucosyltransferase enzyme that synthesizes poly alpha-1,3-1,6-glucan, wherein said glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:

2, wherein;

(i) about 30%-40% of the glycosidic linkages of said glucan are alpha-1,3 linkages,

(ii) about 60%-70% of the glycosidic linkages of said glucan are alpha-1,6 linkages, and

(iii) said glucan has a weight average degree of polymerization (DP_w) of 10000 to 15000,

and wherein said glucan is insoluble in water and has no branch points.

2. The reaction solution of claim 1, wherein said glucosyltransferase enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

3. The reaction solution of claim 2, wherein said glucosyltransferase enzyme comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:

2.

4. The reaction solution of claim 3, wherein said glucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO:

2.

5. The reaction solution of claim 1, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 90% identical to SEQ ID NO:

2.

10. The reaction solution of claim 5, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

11. The reaction solution of claim 10, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 98% identical to SEQ ID NO:

2.

12. The reaction solution of claim 1, wherein said glucosyltransferase enzyme consists of the amino acid sequence of SEQ ID NO:

2.

13. The reaction solution of claim 1, wherein a heterologous amino acid sequence is at the N-terminus and/or C-terminus of said glucosyltransferase enzyme.

14. The reaction solution of claim 1, wherein the reaction solution comprises only one of said glucosyltransferase enzyme.

15. The reaction solution of claim 1, wherein the reaction solution is cell-free.

6. A method for producing poly alpha-1,3-1,6-glucan comprising:

contacting at least water, sucrose, and an isolated glucosyltransferase enzyme in a reaction solution, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-1,6-glucan and comprises an amino acid sequence that is at least 90% identical to SEQ ID NO;

2, whereby poly alpha-1,3-1,6-glucan is produced, wherein;

(i) about 30%-40% of the glycosidic linkages of said glucan are alpha-1,3 linkages,

(ii) about 60%-70% of the glycosidic linkages of said glucan are alpha-1,6 linkages, and

(iii) said glucan has a weight average degree of polymerization (DP_w) of 10000 to 15000,

and wherein said glucan is insoluble in water and has no branch points; and

b) optionally, isolating the poly alpha-1,3-1,6-glucan produced in step (a).

7. The method of claim 6, wherein said glucosyltransferase enzyme comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:

2.

8. The method of claim 7, wherein said glucosyltransferase enzyme comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:

2.

9. The method of claim 8, wherein said glucosyltransferase enzyme comprises the amino acid sequence of SEQ ID NO:

2.