【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,Tm值分别提高了15℃和7℃,催化效率分别提高了85%和22%。更多还原

【Abstract】 To improve the thermostability of Bacillus subtilis lipase A(LipA),molecular dynamics simulation(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 Gly158were selected as mutation targets among 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.Consequently,the mutants LipAG52 Pand LipAG158Pshowed a small but significant increase in the enzyme thermostability.The Tmof LipAG52 Pand LipAG158Pwere 15℃and 7℃ higher than that of the wild type strain LipA.The specific activity of LipAG52 Pand LipAG158Pwere 5.6and 2.7times of wild type strain LipA while the catalytic efficiency of wild type strain LipAG52 Pand LipAG158Pwere 1.85 and 1.22 times of wild type strain LipA,respectively.更多还原