Enzymatic production of alpha-1,3-glucan
A method for producing insoluble alpha-1,3-glucan is disclosed. Embodiments of the method comprise providing (i) oligosaccharides that comprise alpha-1,3 and alpha-1,6 glycosidic linkages, or (ii) oligosaccharides derived from a glucosyltransferase reaction; and contacting at least water, sucrose, a glucosyltransferase enzyme, and the oligosaccharides provided in the first step. Glucosyltransferase reaction compositions embodying such a method, and insoluble products thereof, are also disclosed. Yield and other product benefits can be realized when practicing the disclosed subject matter.
1. A method for producing insoluble alpha-1,3-glucan comprising:
(a) providing oligosaccharides produced from a glucosyltransferase reaction that produces insoluble alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a weight-average degree of polymerization (DPw) of at least 100; and
(b) contacting at least water, sucrose, said oligosaccharides, and a glucosyltransferase enzyme that produces insoluble alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a DPw of at least 100, thereby producing said insoluble alpha-1,3-glucan;
wherein:
steps (a) and (b) are repeated one or more times;
the oligosaccharides in each repeated step (a) comprise oligosaccharides that are provided from the products resulting from a preceding step (b);
the glucosyltransferase of the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9;
and the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
2. The method of claim 1 , wherein the oligosaccharides are purified.
3. The method of claim 1 , wherein the oligosaccharides are unpurified.
4. The method of claim 1 , wherein said glucosyltransferase reaction of step (a) produces insoluble alpha-1,3-glucan having at least 80% alpha-1,3 glycosidic linkages, and said glucosyltransferase of step (b) produces insoluble alpha-1,3-glucan having at least 80% alpha-1,3 glycosidic linkages.
5. The method of claim 1 , wherein the oligosaccharides are provided as a soluble fraction of said glucosyltransferase reaction of step (a).
6. The method of claim 5 , wherein the soluble fraction is a portion of, or all of, a filtrate of said glucosyltransferase reaction of step (a).
7. The method of claim 1 , wherein said oligosaccharides are provided in step (b) at an initial concentration of at least about 1 g/L.
8. The method of claim 1 , wherein the yield of said insoluble alpha-1,3-glucan produced is increased compared to the yield of insoluble alpha-1,3-glucan that would be produced if step (b) lacked said oligosaccharides.
9. The method of claim 1 , wherein the viscosity of said insoluble alpha-1,3-glucan produced is decreased compared to the viscosity of insoluble alpha-1,3-glucan that would be produced if step (b) lacked said oligosaccharides, wherein viscosity is measured with alpha-1,3-glucan as mixed or dissolved in a liquid.
10. The method of claim 4 , wherein the glucosyltransferase reaction of step (a) produces insoluble alpha-1,3-glucan having at least 90% alpha-1,3 glycosidic linkages, and said glucosyltransferase of step (b) produces insoluble alpha-1,3-glucan having at least 90% alpha-1,3 glycosidic linkages.
11. The method of claim 1 , wherein the method further comprises isolating the insoluble alpha-1,3-glucan produced in step (b).
12. The method of claim 1 , wherein the oligosaccharides in each repeated step (a) comprise oligosaccharides that are provided from the products resulting from each immediately preceding step (b).
13. The method of claim 1 , wherein the glucosyltransferase of
the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and
the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 95% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
14. The method of claim 13 , wherein the glucosyltransferase of
the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and
the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 96% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
15. The method of claim 14 , wherein the
glucosyltransferase of the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and
the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 97% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
16. The method of claim 15 , wherein the glucosyltransferase of
the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and
the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
17. The method of claim 16 , wherein the
glucosyltransferase of the glucosyltransferase reaction of step (a) comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9, and the glucosyltransferase of step (b) comprises an amino acid sequence that is at least 99% identical to SEQ ID NO:2, 4, 6, 7, 8, or 9.
18. The method of claim 10 , wherein the glucosyltransferase reaction of step (a) produces insoluble alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages, and said glucosyltransferase of step (b) produces insoluble alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages.