Mogroside biocatalysis methods and products
Monk fruit extracts containing mogrol glycosides such as mogroside V may be treated with enzymes under specific reaction conditions to shift distribution of the Siamenoside I reaction product. Modified enzymes are also employed to shift Siamenoside I distribution to increase yield of Siamenoside I and reduce reaction contaminants. Methods of purifying bioconversion reaction product are also described. Siamenoside I obtained using these methods is a useful sweetener and flavor enhancer for food and beverage compositions and the like.
1. A modified beta-galactosidase enzyme comprising one or more mutations in the amino acid residues 200 through 212, wherein the beta-galactosidase enzyme having at least 50% identity to Aspergillus oryzae beta-galactosidase comprises one or more mutations selected from G204G, C205R, V208E or combinations thereof.
2. The modified beta-galactosidase enzyme of claim 1 , wherein the beta-galactosidase enzyme having at least 50% identity to Aspergillus oryzae beta-galactosidase comprises the mutations G204G, C205R and V208 E.
3. A modified beta-galactosidase enzyme comprising one or more mutations in the amino acid residues 200 through 212, wherein the beta-galactosidase enzyme having at least 50% identity to Aspergillus oryzae beta-galactosidase comprises one or more mutations in the loop region are selected from E142A, G204G, C205R, and V208E.
4. The modified beta-galactosidase enzyme of claim 3 , wherein the beta-galactosidase enzyme having at least 50% identity to Aspergillus oryzae beta-galactosidase comprises the mutations E142A, G204G, C205R and V208.
5. A method for purifying Siamenoside I from a reaction mixture comprising:
a) providing a mixture of low purity mogrosides and reaction mixture reagents;
b) separating the mogrosides from the reaction mixture reagents by (i) adjusting the pH of the mixture of a) to about 10 or higher, (ii) adding alcohol to provide an alcoholic solution and (iii) filtering the alcoholic solution through a first ultrafiltration membrane to provide a first filtered solution;
c) adjusting pH of the first filtered solution to between about 5 and about 7 and filtering the through a second ultrafiltration membrane to provide a second filtered solution;
d) performing diafiltration on the second filtered solution to concentrate the mogrosides, providing a mogroside mixture, then mixing the mogroside mixture with water/ammonia acetate to provide a mogroside/ammonium acetate solution;
e) contacting the mogroside/ammonia acetate solution with a fractionation column;
f) eluting and collecting fractions containing Siamenoside I; and
g) drying the fractions containing Siamenoside I to obtain high purity Siamenoside I with a Siamenoside content of more than about 60% (w/w).
6. The method of claim 5 , wherein the mogroside mixture of step a) comprises at least 90% Mogroside V.
7. The method of claim 5 , wherein the mogroside mixture of step a) comprises at least 95% Mogroside V.