This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme’s isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/K363E/K420E, was found to be the best with a 7.3-fold increase in half-life (t1/2) at 33 °C compared to that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.

中文翻译：

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通过合理设计提高GH97葡聚糖葡萄糖苷酶的热稳定性

本研究旨在提高葡聚糖葡萄糖苷酶 PspAG97A 的热稳定性，该酶是来自假交替单胞菌属的糖苷水解酶家族 97 的成员。K8。基于表面电荷-电荷相互作用的优化，使用 TKSA-MC 程序选择了总共 9 个赖氨酸残基，并将其突变为谷氨酸以将酶的等电点偏离其最佳 pH 值。三个突变体 K75E、K363E 和 K420E 表现出增强的热稳定性。发现三重突变体 K75E/K363E/K420E 是最好的，与野生型 (WT) 相比，在 33°C 下的半衰期 (t1/2) 增加了 7.3 倍。最重要的是，该突变体显示出与 WT 蛋白相当的酶活性。