Enhancing the regioselectivity and activity of sucrose phosphorylase by combinational semi‐rational strategies

Abstract Sucrose phosphorylase (SPase) is widely used for the glycosylation of polyhydroxy compounds, while low regioselectivity will limit its application. In this study, the regioselectivity of wild‐type SPase from Limosilactobacillus reuteri for the synthesis of 2‐ O ‐α‐d‐glucosylglycerol (2‐αGG) (84.7% ± 0.5%) was enhanced by combinational semi‐rational strategies. A key residue R137 was identified by sequence alignment analysis of non‐conserved residues in the catalytic pocket, molecular docking analysis, and alanine scanning at the key flexible loops. The regioselectivity of R137M increased to 95.4% ± 0.4%. Iteration mutation, loop engineering, and triple code saturation mutagenesis were used to further increase the 2‐OH selectivity (>97%) and double mutant R137M/L337T (98.0% ± 0.5%) was the best one. Whole‐cell transformation with R137M/L337T produced a higher yield of 2‐αGG with the increased final 2‐OH selectivity (>99%); besides, the amount of byproduct glucose greatly declined, meaning a decreased hydrolysis activity. Hence, the combinational semi‐rational mutagenesis methods are effective for improving the regioselectivity of SPase.