Rational design to improve lipase thermostability for ester synthesis

Abstract:

[Background] Candida antarctica lipase B (CALB) exhibits excellent ester synthesis activity and is used widely in biochemical synthesis. [Objective] This study aimed to improve the thermostability of CALB based on retaining its excellent catalytic performance. [Methods] The potential thermostability mutation sites of CALB were calculated by the prediction software PoPMuSiC and FoldX, and further selected according to the spatial position of amino acid residues. Ten single point mutations were introduced into gene calb via the overlap extension PCR and expressed in Pichia pastoris GS115. [Results] The point mutations A146G, A151P and L278M could effectively improve the thermostability of CALB. Based on single point mutations, the thermostability of the combined mutants A146G-L278M and A146G-L278M-A151P was further improved. Compared with the wild-type, the optimum reaction temperature for A146G-L278M and A146G-L278M-A151P were increased by 5 °C, and the Tm value were increased by 3.3 °C and 4.2 °C, respectively. Besides, the kinetic study of the enzymatic reaction of synthesis ethyl caproate shows that mutants A146G-L278M and A146G-L278M-A151P had higher affinity to hexanoic acid and ethanol than the wild-type, and the catalytic efficiency kcatA/KmA to hexanoic acid was 4.1-fold over that of the wild-type. The mechanism of the thermostability of the mutants A146G-L278M and A146G-L278M-A151P were elucidated at molecular level through molecular dynamics simulation. [Conclusion] The rational design strategy adopted in this study is effective for improving the thermostability of CALB. This strategy can also be used as a reference for other industrial enzymes to improve their thermostability.