Abstract

2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G) is a highly stable glycosylated derivative of vitamin C with broad applications in the food, pharmaceutical, and cosmetic industries. Sucrose phosphorylase (SPase) serves as a promising biocatalyst for the efficient synthesis of AA-2G from sucrose and L-AA. However, its industrial application is often limited by insufficient thermostability. In this study, the thermostability of SPase from Bifidobacterium longum (BlSPase) was significantly improved through an integrated computational semirational design strategy. Using PROSS, DeepDDG, and FireProt, a smart mutant library was constructed, leading to the identification of a positive mutant, G197A, with a half-life extended by 24.75 min and a melting temperature increased by 2.1 °C compared to the wild-type enzyme. Under optimized high-cell-density fermentation in a 10-L bioreactor and whole-cell catalytic conditions, G197A achieved an AA-2G titer of 383.13 g/L and a conversion rate of 80.97%. Furthermore, an anion exchange-based purification process was developed, affording high-purity AA-2G, as confirmed by HPLC and NMR analyses. This work paves the way for the industrial application of SPase by providing a robust computational framework for stability engineering, enabling sustainable and high-yield AA-2G production.