DNA改组技术提高低温脂肪酶Lip98热稳定性  

Improved thermostability of a cold-actvie lipase by DNA shuffling

作　　者：苏宏飞[1,2] 麦志茂[1] 张偲[1] 

机构地区：[1]中国科学院南海海洋研究所中国科学院海洋生物资源可持续利用重点实验室,广东广州510301 [2]中国科学院大学,北京100049

出　　处：《生物技术》2016年第3期293-299,共7页Biotechnology

基　　金：中国科学院战略先导项目(No.XDA10030404);广东省自然科学基金项目(No.2015A030310270)

摘　　要：[目的]提高来源于海洋微生物的低温脂肪酶Lip98的热稳定性。[方法]采用DNA改组技术进行低温脂肪酶的基因改造,连接到表达载体p ET-28a中构建小突变文库。经过活性筛选产酶重组菌株。在96孔板中进行两轮热稳定筛选突变株。[结果]经过2轮筛选,获得了两个突变株P92A和I199F。其突变位点分别是274位的C变成了G和595位的A变成了T。突变株50℃下的半衰期从24 min分别延长到38 min、85 min。[结论]DNA改组有效提高Lip98的热稳定性,半衰期提高到1.5~3.5倍,为酶的分子改造提供理论依据。[ Objective ] It was to improve the thermosbility of cold-active lipase （Lip98） that was cloned from marine microorganism. [ Methods ] The molecular evolution of cold-active lipase using DNA shuffling was carried out, and then the gene was ligated into an expression vector pET-28a to construct mutation library. The activity screening and two rounds of thermostability screening was used to obtain the positive recombinant strains. [ Results] Two mutants （P92A and I199F） were isolated after two rounds of screening. And their half-life under 50 ℃ were improved from 24 min to 38 min and 85 min respectively. [ Conclusion ] Consequently, 1.5 -3.5 folds of thermosbility of Lip98 was improved effectively, which may provide theoretical and constructive foundations for enzymatic molecular modification.

关 键 词：低温脂肪酶 热稳定性 DNA改组 

分 类 号：Q556.1[生物学—生物化学]