β-glucosidase (EC3.2.1.21) can release glucose and corresponding ligands from the substrate by hydrolyzing β-D-glucoside bonds at its non-reducing end, which is one of the key enzymes in the process of cellulose glycosylation.The β-glucosidase from Talaromyces leycetanus JCM12802 (bgl3A) has good thermal stability, but its catalytic activity still needs to be improved urgently.In this study, the structure of the bgl3A-substrate pNPG complex was constructed using the flexible docking module of Discovery Studio 2022.Based on the binding energy prediction module, virtual mutations were performed on those amino acid residues within 6 Å of the catalytic active site of bgl3A, and the binding energy changes of the enzyme to pNPG were calculated before and after the mutation.Expression, purification, and enzymatic property analysis were performed on 20 mutants with binding energy changes less than -0.7 kcal/mol.Compared to bgl3A, the specific enzyme activity and kcat/Km of the mutant bgl3A-N237Y were increased by 22% and 25%, respectively.When bgl3A-N237Y was expressed in Komagataella phaffii, signal peptide substitution and co-expression with molecular chaperones led to 106.6 U/mL of enzyme activity in the shake flask, 75% higher than that before optimization.The highly active bgl3A mutant and its producing strain will improve the industrial application of β-glucosidase.
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