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

[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.