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 \(T_{50}^{30}\) 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根霉的脂肪酶（r27RCL）的热稳定性，证明了新引入的掩埋二硫键F223C / G247C有利于热稳定性。有趣的是，还发现F223C / G247C可以改善脂肪酶的耐碱性。随后，将我们先前工作中的其他六个热稳定突变整合到突变体F223C / G247C中，从而产生了热碱稳定的突变体m32。与野生型脂肪酶相比，有益突变的关联效应显示出对m32热稳定性的显着改善，在60°C下的半衰期延长了74.7倍，在2°C时提高了\（T_ {50} ^ {30} \）最佳温度下最高温度升高10°C。还发现突变的m32在pH 9.0-10.0时稳定。此外，M32的分子动力学模拟表示，其刚性得到增强由于减小的溶剂可及表面积，新形成的盐桥，并且增加的ΔΔ ģ值。