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

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.