理性设计提高酯合成催化反应脂肪酶的热稳定性

Rational design to improve lipase thermostability for ester synthesis

【背景】南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,Tm值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率kcatA/Km A是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。

[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 A146 G,A151 P and L278 M could effectively improve the thermostability of CALB.Based on single point mutations,the thermostability of the combined mutants A146 G-L278 M and A146 G-L278 M-A151 P was further improved.Compared with the wild-type,the optimum reaction temperature for A146 G-L278 M and A146 G-L278 M-A151 P 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 A146 G-L278 M and A146 G-L278 M-A151 P had higher affinity to hexanoic acid and ethanol than the wild-type,and the catalytic efficiency kcatA/Km A to hexanoic acid was 4.1-fold over that of the wild-type.The mechanism of the thermostability of the mutants A146 G-L278 M and A146 G-L278 M-A151 P 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.