Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122, Jiangsu, China.
J Ind Microbiol Biotechnol. 2020 Dec;47(12):1019-1030. doi: 10.1007/s10295-020-02324-1. Epub 2020 Oct 18.
To improve the thermostability of the lipase (r27RCL) from Rhizopus chinensis through rational design, a newly introduced buried disulfide bond F223C/G247C was proved to be beneficial to thermostability. Interestingly, F223C/G247C was also found to improve the alkali tolerance of the lipase. Subsequently, six other thermostabilizing mutations from our previous work were integrated into the mutant F223C/G247C, leading to a thermo-alkali-stable mutant m32. Compared to the wild-type lipase, the associative effect of the beneficial mutations showed significant improvements on the thermostability of m32, with a 74.7-fold increase in half-life at 60 °C, a 21.2 °C higher [Formula: see text] value and a 10 °C elevation in optimum temperature. The mutated m32 was also found stable at pH 9.0-10.0. Furthermore, the molecular dynamics simulations of m32 indicated that its rigidity was enhanced due to the decreased solvent-accessible surface area, a newly formed salt bridge, and the increased ΔΔG values.
为了通过合理设计提高里氏木霉脂肪酶（r27RCL）的热稳定性，新引入的埋藏二硫键 F223C/G247C 被证明有利于热稳定性。有趣的是，F223C/G247C 还被发现提高了脂肪酶的耐碱性。随后，将我们之前工作中的另外六个热稳定突变整合到突变体 F223C/G247C 中，得到了一个热碱稳定的突变体 m32。与野生型脂肪酶相比，有益突变的协同作用显著提高了 m32 的热稳定性，其半衰期在 60°C 时增加了 74.7 倍，[Formula: see text] 值提高了 21.2°C，最适温度提高了 10°C。突变体 m32 在 pH 值为 9.0-10.0 时也很稳定。此外，m32 的分子动力学模拟表明，由于溶剂可及表面积减少、新形成的盐桥以及 ΔΔG 值增加，其刚性增强。
