Engineering of a thermo-alkali-stable lipase from Rhizopus chinensis by rational design of a buried disulfide bond and combinatorial mutagenesis

Abstract

Abstract 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 T5030\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$T_{50}^{30}$$\end{document} 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.