Designing a Sucrose Phosphorylase from <i>Leuconostoc mesenteroides</i> for the Highly Selective Production of EGCG-4′- <i>O</i> -α- <scp>d</scp> -Glucopyranoside

The major green tea polyphenol, epigallocatechin gallate (EGCG), has beneficial antioxidant and anti-inflammatory activities but suffers from poor solubility and stability. Its monoglycosylated derivative, (-)-epigallocatechin gallate 4'-<i>O</i>-α-d-glucopyranoside (EGCG-G1), partially overcomes these limitations. In this study, we engineered <i>Leuconostoc mesenteroides</i> sucrose phosphorylase (LmSPase) for efficient EGCG-G1 production. The triple mutant M3 (T219L/E393I/N335G), created via loop engineering, consensus design, and Rosetta Dock design, exhibited 4.09-fold higher transglycosylase activity at 30 °C, a 1.69-fold longer half-life at 45 °C, and significantly improved regioselectivity compared to the wild type. In a fed-batch reaction at 30 °C and pH 6.0, 25 g/L EGCG was converted within 24 h, producing 31.11 g/L (91.92% yield) of EGCG-G1 with 87.27% purity. This semirational design strategy enhanced the key properties of LmSPase and provides an effective biocatalyst for EGCG-G1 production.