Production of highly water-soluble genistein -diglucoside using an engineered O--glycoligase with enhanced transglycosylation activity and altered substrate specificity

Genistein is one of isoflavones, showing various biological functions for human health. MalA-D416A, termed O-alpha-glycoligase, is an acid/base catalytic residue-deficient mutant of a alpha-glucosidase from Sulfolobus solfataricus, synthesizing genistein 7-O-alpha-glucoside using alpha-glucosyl fluoride as the donor substrate. Through mutagenesis toward MalA-D416A, an O-alpha-glycoligase variant with two mutations (D416R and Q450S) was identified as a biocatalyst with a 58.8-fold enhanced catalytic efficiency for genistein compared to the parent enzyme. The use of a 2:1 ratio of alpha-glucosyl fluoride and genistein at pH 9 facilitated the synthesis of genistein 7,4'-O-alpha-diglucoside by MalA-D416R/Q450S. The alpha-diglucoside exhibited 2,459-fold improved water solubility compared to genistein itself as well as facile deglycosylation by the intestinal alpha-glucosidase from rat, suggesting the potential of the alpha-diglucoside for improved bioavailability in human intestine. Through molecular docking analyses the modulation of the active site conformation by these mutations was expected for proper binding of both genistein and the monoglucoside.