Glycosylated beta-galactosidase compositions having improved transgalactosylating activity
The present invention relates to compositions, particularly liquid compositions, comprising polypeptides having beta-galactosidase activity, methods of making said compositions, and uses of the compositions for making e.g. dairy products. The polypeptides having beta-galactosidase activity are modified by glycation of lysine and/or arginine residues by incubating the enzyme in the presence of reducing sugars, optionally combined with a heat treatment. Thereby, transgalactosylating activity is increased.
1. A formulation comprising:
(a) 30-90 wt. % of a reducing sugar, wherein the reducing sugar is fructose, galactose, glucose, glyceraldehyde, ribose, xylose, cellobiose, maltose, or lactose, and
(b) a polypeptide of glycoside hydrolase family 2 and enzyme class EC 3.2.1.23 or 3.2.1.108 comprising at least one lysine and/or arginine residue which is glycated by the reducing sugar, wherein the polypeptide has beta-galactosidase activity and transgalactosylating activity.
2. The formulation of claim 1 , wherein the reducing sugar is fructose, galactose, glucose, glyceraldehyde, ribose, or xylose.
3. The formulation of claim 2 , wherein the reducing sugar is galactose.
4. The formulation of claim 2 , wherein the reducing sugar is fructose.
5. The formulation of claim 2 , wherein the reducing sugar is glucose.
6. The formulation of claim 1 , wherein the reducing sugar is cellobiose or maltose.
7. The formulation of claim 1 , wherein the reducing sugar is lactose.
8. The formulation of claim 1 , wherein at least 1% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.
9. The formulation of claim 1 , wherein at least 3% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.
10. The formulation of claim 1 , wherein at least 5% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.
11. The formulation of claim 1 , wherein at least 10% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.
12. The formulation of claim 1 , wherein at least 20% of the lysine and arginine residues of the polypeptide are glycated by the reducing sugar.
13. The formulation of claim 1 , which comprises 40-90 wt. % of the reducing sugar.
14. The formulation of claim 1 , which comprises 40-65 wt. % of the reducing sugar.
15. The formulation of claim 1 , wherein the polypeptide is from subfamily 5 of glycosyl hydrolase family 2 (GH 2 _ 5 ).
16. The formulation of claim 1 , wherein the polypeptide is from subfamily 6 of glycosyl hydrolase family 2 (GH 2 _ 6 ).
17. The formulation of claim 1 , wherein the polypeptide is a Bacillus polypeptide.
18. The formulation of claim 17 , wherein the polypeptide is a Bacillus circulans polypeptide.
19. The formulation of claim 1 , wherein the polypeptide is a Bifidobacterium polypeptide.
20. The formulation of claim 19 , wherein the polypeptide is a Bifidobacterium bifidum polypeptide.
21. The formulation of claim 1 , wherein the polypeptide is a Kluyvermyces polypeptide.
22. The formulation of claim 21 , wherein the polypeptide is a Kluyvermyces lactis polypeptide.
23. The formulation of claim 1 , wherein the polypeptide has at least 500 amino acid residues.
24. The formulation of claim 1 , wherein the polypeptide has at least 750 amino acid residues.
25. The formulation of claim 1 , wherein the polypeptide has at least 1000 amino acid residues.
26. The formulation of claim 1 , wherein the polypeptide has at least 1250 amino acid residues.
27. The formulation of claim 1 , which has an activity of 200-20,000 LAU(C)/g.
28. The formulation of claim 1 , which is a liquid formulation and has an activity of 200-15,000 LAU(C)/g.
29. The formulation of claim 1 , which is a solid formulation and has an activity of 1,000-20,000 LAU(C)/g.
30. The formulation of claim 1 , which is substantially free of glycerol.
31. The formulation of claim 1 , which further comprises sodium chloride or potassium chloride in an amount of 0.01-5 wt. %.
32. A method for producing galacto-oligosaccharides (GOS) comprising contacting the formulation of claim 1 with lactose at conditions to produce the galacto-oligosaccharides, wherein the conditions are a pH of 5-8, a temperature of 50-80° C., and a time of 3-100 hours.
33. The method of claim 32 , wherein the reducing sugar is fructose, glucose, galactose, or lactose.
34. The method of claim 32 , wherein the conditions are a pH of 6-7, a temperature of 50-70° C., and a time of 15-80 hours.
35. The formulation of claim 1 , wherein the polypeptide has a ratio of transgalactosylating activity to beta-galactosidase activity of at least 1:1.