Enhancing regioselectivity of sucrose phosphorylase by loop engineering for glycosylation of L-ascorbic acid.

Sucrose phosphorylase (SPase) has a remarkable capacity to synthesize numerous glucosides from abundantly available sucrose under mild conditions but suffers from specificity and regioselectivity issues. In this study, a loop engineering strategy was introduced to enhance the regioselectivity and substrate specificity of SPase for the efficient synthesis of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) via L-ascorbic acid (L-AA). P134, L341, and L343 were identified as "hotspots" for modulating the flexibility of loops, which significantly influenced the H-bonding network of L-AA in the active site, as well as the entrance of the substrate channel, thereby altering the regioselectivity and substrate specificity. Finally, the mutant L341V/L343F, with near-perfect control of the selectivity synthesis of the 2-OH group of L-AA (> 99%), was obtained. The AA-2G production by the mutant reached 244 g L-1 in a whole-cell biotransformation system, and the conversion rate of L-AA reached 64%, which is the highest level reported to date. Our work also provides a successful loop engineering case for modulating the regioselectivity and specificity of sucrose phosphorylase. KEY POINTS: • "Hotspots" were identified in the flexible loops of sucrose phosphorylase. • Mutants exhibited improved regioselectivity and specificity against L-ascorbic acid. • Synthesized AA-2G with high yield and regioselectivity by whole-cell of mutant.