通过理性设计提高南极假丝酵母脂肪酶B的温度稳定性

Improving the thermostability of Candida antarctica lipase B by rational design

[目的]提高南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的温度稳定性及活性。[方法]基于CALB的结构信息进行了理性设计,最终获得脂肪酶基因calbTm,并构建了pPICZαA-CALBTm重组表达质粒并实现了在毕赤酵母中的高效表达。[结果]突变体酶CALBTm在摇瓶发酵72 h后酶活达到600 U/mL,且在50℃下保温3 h后仍保留74.8%的酶活;此外,中间突变体酶Tm1和Tm2经摇瓶发酵后酶活分别为220 U/mL、170 U/mL,在50℃下保温3 h后酶活分别保留了73.9%、10%。通过对蛋白结构的分析发现,引入疏水性强的氨基酸和增加蛋白质三维空间中的氢键数量,能维持蛋白质构象的稳定,进而提高了脂肪酶的温度稳定性。[结论]成功设计并获得一种新型高活性、耐高温的脂肪酶CALB,其酶活达到了600 U/mL,与野生型CALB相比提高了10倍,同时温度稳定性也显著提升,在50℃保温3 h后酶活保留了74.8%,而野生型CALB在保温3 h后酶活仅保留5%。

[Objective]Improve the thermostability and activity of Candida antarctica lipase B(CALB)to lay the foundation for its industrial production.[Method]Based on the structural information of CALB,rational design was carried out,and finally the lipase gene calbTm was obtained,and the pPICZαA-CALBTm recombinant expression plasmid was constructed and realized high-efficiency expression in Pichia pastoris.[Result]The enzyme activity of the mutant CALBTm reached 600 U/mL after 72 hours of conical flask fermentation,and 74.8%of the enzyme activity remained after 3 hours of holding at 50℃.In addition,the enzyme activity of intermediate mutants Tm1 and Tm2 were 220 U/mL and 170 U/mL respectively after conical flask fermentation,and 73.9%and 10%of the enzyme activity were retained after holding at 50℃for 3 hours,respectively.Through the analysis and comparison of protein structure,it was found that introducing amino acids with strong hydrophobicity and increasing the number of hydrogen bonds in the three-dimensional space of protein could maintain the stability of protein conformation and improve the thermostability of lipase.[Conclusion]A new type of lipase CALB with high activity and high temperature resistance was successfully designed and obtained.Its enzyme activity reached 600 U/mL,which was 10 times higher than that of wild-type CALB.At the same time,the temperature stability was significantly improved.After holding at 50℃for 3 hours,the enzyme activity remained 74.8%,while that of wild-type CALB remained only 5%.