期刊论文

Improving the thermostability of Candida antarctica lipase B by rational design

中文

生物技术

1004-311X

2022

32

03

284-290+296

10.16519/j.cnki.1004-311x.2022.03.0045

国家重点研发计划专项（“医药与食品工业酶创制与催化”，2021YFC2100300）；

本校为第一机构

[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...

[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 remai...