β-葡萄糖苷酶催化活性改造及在毕赤酵母中的高效表达

Catalytic activity modification ofβ-glucosidase and its high-level expression in Komagataella phaffii

β-葡萄糖苷酶(EC 3.2.1.21)能催化底物非还原末端的β-D-葡萄糖苷键水解释放葡萄糖和相应配基,是纤维素糖化过程中的关键酶之一。篮状菌(Talaromyces leycettanus JCM12802)来源的β-葡萄糖苷酶bgl3A热稳定性较好,但催化活性还亟待提高。该研究中,通过Discovery Studio 2022柔性对接模块构建了bgl3A和底物pNPG的复合物结构;基于结合能预测模块对bgl3A催化活性位点6以内的氨基酸残基进行虚拟突变,计算突变前后酶对pNPG的结合能变化;对结合能变化小于-0.7 kcal/mol的20个突变体进行表达、纯化和酶学性质分析。与bgl3A相比,突变体bgl3A-N237Y的比酶活和k_(cat)/K_(m)分别提升了22%和25%。将bgl3A-N237Y在毕赤酵母中表达,通过信号肽替换和共表达分子伴侣使其摇瓶发酵酶活力达106.6 U/mL,较优化前提高75%。研究得到高活性bgl3A突变体及其生产菌将有助于提高其工业化应用效果。

β-glucosidase(EC3.2.1.21)can release glucose and corresponding ligands from the substrate by hydrolyzingβ-D-glucoside bonds at its non-reducing end,which is one of the key enzymes in the process of cellulose glycosylation.Theβ-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 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 k cat/K_(m)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 ofβ-glucosidase.