Rapid acting α-transglucosidase from Aspergillus carbonarius: Cloning, characterization, in-silico analyses and synthesis of stable vitamin-C analogue using the recombinant enzyme.

Abstract

The transglycosylation activity of many glycoside hydrolases (GH) can potentially synthesise di, oligo, polysaccharides, or glycosylate various non-carbohydrate moieties, increasing their bioavailability and solubility. While the transglycosylation potential of glycoside hydrolases makes them attractive candidates in glyco-synthesis due to their broad substrate specificity and ability to utilise simple sugar donors, the major challenge is the identification of potent transglucosidases for specific reactions. In this study, a GH 31 α-transglucosidase/glucosidase produced by Aspergillus carbonarius BTCF 5 was cloned and expressed in Komagataella phaffi, and the recombinant enzyme was recognised as a potential catalyst for one-pot glycosylation reactions based on its ability to glycosylate ascorbic acid to 2-o-α-d-glucopyranosyl ascorbic acid (AA2G). The transglucosidase was found to be rapid-acting, with a transglycosylation yield of 13 g/L within 30 min, thus achieving the highest reported overall productivity of 26 g/L/h. Molecular modelling and site-specific docking studies could identify the enzyme subsites for substrate binding and the ability of the enzyme to bind to a selected panel of substrates in the active site region, thus indicating its wider applications in glycosylation reactions. The results identified a rapid-acting transglucosidase with higher productivity for AA2G synthesis at a minimal incubation time compared to several native and recombinant glycoside hydrolases. The GH 31 α-glucosidase/transglucosidase was thus identified as a potential biocatalyst for various one-pot glycosylation reactions and industrial applications.