One-pot synthesis of stable vitamin C analogue using trans-glycosylating enzyme from Aspergillus carbonarius BTCF 5

Biocatalysis using transglycosidases for one-pot glycosylation of small molecules is a greener alternative to chemical glycosylation. The transglycosylation potential of the crude culture filtrate of Aspergillus carbonarius BTCF 5 was evaluated based on the biotransformation of the target molecule, ascorbic acid (Vitamin-C), to its more stable glycosylated analogue -2-O-α-D-glucopyranosyl ascorbic acid (AA2G), using maltose as the glycosyl donor. Media engineering increased the transglycosylation yield by 64%, whereas the use of concentrated culture filtrate increased the transglycosylation yield by 70%. Protein purification studies identified the candidate transglycosylating enzyme to be a GH 31 family CAZyme (α-transglucosidase). A minimally purified enzyme could enhance the transglycosylation yield by 77% with a total AA2G yield of 91.5 mM (10.32 g/L) and improved the overall conversion rate of ascorbic acid and maltose in the reaction by 16% and 6.3% respectively from the base level. A two-step column chromatography helped in product purification with 46% recovery. The structural characterization of purified AA2G provided a detailed insight into the regioselectivity of the enzyme to glycosylate C2 of ascorbic acid and its ability to form AA2G as the predominant isomer, which is also the most preferred one for end applications.