Zhang Xiaomin, Chen Feiyun, He Chao, Fang Wei, Fang Zemin, Zhang Xuecheng, Wang Xiaotang, Xiao Yazhong
School of Life Sciences, Anhui University, Hefei, 230601, Anhui, China.
Anhui Key Laboratory of Modern Biomanufacturing, Hefei, 230601, Anhui, China.
Biotechnol Lett. 2020 Nov;42(11):2211-2221. doi: 10.1007/s10529-020-02928-8. Epub 2020 Jun 1.
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 (t) 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.
本研究旨在提高来自假交替单胞菌属K8菌株的糖苷水解酶家族97成员葡聚糖葡萄糖苷酶PspAG97A的热稳定性。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，以将酶的等电点从其最佳pH值偏移。三个突变体K75E、K363E和K420E表现出增强的热稳定性。与野生型（WT）相比，三突变体K75E/K363E/K420E在33°C下的半衰期（t）增加了7.3倍，被发现是最佳的。最重要的是，该突变体显示出与WT蛋白相当的酶活性。结构建模表明，增加的表面电荷-电荷相互作用以及表面疏水和静电接触的优化有助于K75E/K363E/K420E表现出的热稳定性提高。
