Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610064, Sichuan, PR China.
J Biosci Bioeng. 2013 Jun;115(6):623-7. doi: 10.1016/j.jbiosc.2012.12.010. Epub 2013 Jan 16.
In order to study on the relationship between Escherichia coli AppA phytase's thermostability and salt bridges, and indicate an effective technical route of which factor to think about and where to modify at AppA for enhancing its thermostability, a salt bridge subtraction mutant E31Q and a salt bridge addition mutant Q307D were constructed by site-directed mutagenesis. The residual activities of the wild-type AppA phytase, E31Q and Q307D were 31.42%, 17.46%, and 40.57%, respectively, after being heated at 80°C for 10 min. The salt bridge subtraction mutant E31Q showed 13.96% thermostability decreasement, and the salt bridge addition mutant Q307D showed 9.15% thermostability enhancement than the wild-type both without the pH and temperature optimum changed. It proved salt bridges play a key role in E. coli AppA phytase's thermostability and the α/β-domain of AppA may be sensitive to heat. Salt bridges and the α/β-domain of AppA should have high priority to think about to enhance AppA's thermostability for commercial application. Besides, molecular dynamics simulation was used for salt bridges analysis.
为了研究大肠杆菌 AppA 植酸酶的热稳定性与盐桥之间的关系，并指出提高其热稳定性时应考虑哪个因素以及应在 AppA 何处进行修改的有效技术路线，通过定点突变构建了盐桥缺失突变体 E31Q 和盐桥添加突变体 Q307D。野生型 AppA 植酸酶、E31Q 和 Q307D 在 80°C 加热 10 min 后的残余酶活分别为 31.42%、17.46%和 40.57%。盐桥缺失突变体 E31Q 的热稳定性降低了 13.96%，而盐桥添加突变体 Q307D 的热稳定性增强了 9.15%，而最适 pH 和温度均未发生变化。这证明盐桥在大肠杆菌 AppA 植酸酶的热稳定性中起关键作用，并且 AppA 的α/β 结构域可能对热敏感。对于商业应用，应优先考虑盐桥和 AppA 的α/β 结构域以提高 AppA 的热稳定性。此外，还进行了分子动力学模拟以分析盐桥。
