基于脯氨酸理论提高枯草芽孢杆菌脂肪酶A 的热稳定性

摘要

以枯草芽孢杆菌脂肪酶 A（LipA）为研究对象，根据从 RCSB 数据库中获取的晶体结构，采用分子动力学模拟和分子生物学实验相结合的方法进行脂肪酶热稳定性位点突变的理性设计。首先，利用分子动力学模拟获得晶体结构中柔性较高的 Loop 区域；进而，结合“脯氨酸理论”，将位于该区域附近的 Gly 残基突变为 Pro，分析引入 Pro 突变对LipA 热稳定性的影响，筛选得到 Gly52和 Gly158两个突变位点；最后，通过定点突变操作对突变株 LipAG52P和 LipAG158P进行热稳定性实验验证。结果显示，突变株 LipAG52P 、LipAG158P 的比活力分别是野生型 LipA 的5.6倍和2.7倍，T m 值分别提高了15℃和7℃，催化效率分别提高了85％和22％。%To improve the thermostability of Bacillus subtilis lipase A(LipA), molecular dynamics simula-tion(MDS)and molecular biology experiments were applied to rational design of the enzyme molecule of LipA. First, molecular dynamics simulation was performed on the crystal structure of LipA to obtain the Loop regions with higher flexibility.Combined with the “proline rule”, Gly52 and Gly158 were selected as mutation targets a-mong several relative glycine residues nearby these Loop regions.Then, the effects of the two mutations on the thermostability of LipA were tested by site-directed mutagenesis and molecular biology experiments.Conse-quently, the mutants LipAG52P and LipAG158P showed a small but significant increase in the enzyme thermostabili-ty.The T m of LipAG52P and LipAG158P were 15 ℃ and 7 ℃ higher than that of the wild type strain LipA.The spe-cific activity of LipAG52P and LipAG158P were 5.6 and 2.7 times of wild type strain LipA while the catalytic effi-ciency of wild type strain LipAG52P and LipAG158P were 1.85 and 1.22 times of wild type strain LipA, respective-ly.