Steric hindrance scanning for activity enhancement of β-glucosidase BgMd and its application in glucoside transformation

There is increasing interest in the protein engineering of β-glucosidases for improving transformation of glycosides. In this study, a novel steric hindrance scanning strategy is applied to β-glucosidase BgMd, resulting in six single-point mutants with significantly increased activity. Further iteration results in the optimized mutant E329K/R72N/A94H/T135H showed a remarkable 26.1-fold improvement in activity over the wild type. The time needed for the hydrolysis of 20 g/L polydatin is cut in half, and the conversion rate is increased to 95.93 %. Mutant A94T/V336T hydrolyzes 2 g/L of ginsenoside Rba1 in 48 h to generate CK with a yield increase from 26.94 to 76.38 %. The mutant E329N hydrolyses 2 g/L of astragaloside in 48 h with a yield increase from 30.19 to 96.23 %. BgMd and mutants exhibit optimal activity at 50–60 °C and pH 7.0, with enhanced stability and improved tolerance in select mutants. Molecular docking and molecular dynamics showed that the substrate pocket is split in two, and the catalytic effect of the mutants is largely explained. Molecular dynamics simulations provided evidence of structural changes and reduced interaction energies in the mutants. These results validate the steric hindrance strategy as an effective method to apply β-glucosidase in the biotransformation of glycosides.