Precision Engineering of a Loop-Exchanged Variant of Sucrose Phosphorylase from Streptococcus mutans Enables Regioselective 4″- O -Glucosylation of Epigallocatechin Gallate

Epigallocatechin gallate (EGCG), the most abundant and bioactive catechin in green tea, has limited applications due to its chemical instability, low solubility, and poor bioavailability. Enzymatic glycosylation offers a sustainable method to improve these properties. However, the regioselectivity and catalytic efficiency of enzymatic glycosylation remain challenging. In the present study, a loop-exchanged variant of sucrose phosphorylase from Streptococcus mutans ( Sm SP), designated Sm SP_LoopB, shifted the major product from EGCG-4′- O -α- d -glucopyranoside to EGCG-4″- O -α- d -glucopyranoside. The Sm SP_LoopB I235S/V297A mutant, generated through a tailored, tunnel-focused mutagenesis strategy, exhibited a 140.5-fold increase in catalytic efficiency compared to Sm SP_LoopB. Under optimized reaction conditions (15 g/L EGCG, 336 g/L sucrose, 2 U/mL enzyme), the yield of EGCG-4″- O -α- d -glucopyranoside reached 83.3% (16.9 g/L) within 4 h. The mutant also demonstrated excellent performance and stability in the whole-cell biocatalytic system. This work establishes a synergistic engineering approach that enhances specificity and efficiency, enabling the scalable production of well-defined EGCG glycosides.