Engineering of a Thermo-alkali-stable Lipase from Rhizopus Chinensis by Rational Design of a Buried Disulfide Bond and Combinatorial Mutagenesis

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.