易错PCR法提高土芽孢杆菌ZH1羧酸酯酶的热稳定性

Improving thermal stability of Geobacillus sp. ZH1 carboxylesterase by error-prone PCR

摘要:

摘要:【目的】对土芽孢杆菌(Geobacillus sp.) ZH1的羧酸酯酶基因进行定向进化，筛选得到酶热稳定性提高的突变酶。【方法】利用易错PCR技术向羧酸酯酶基因中随机引入突变，建立酶基因突变文库，筛选获得热稳定性提高的突变体，并对突变酶进行诱导表达、纯化及部分酶学性质研究。【结果】通过筛选，获得羧酸酯酶热稳定性提高的突变菌株65。序列分析表明，突变酯酶65有2个氨基酸发生了改变，包括T113S和M160K。突变酶的三维结构模拟显示，突变T113S位于酶分子的第5个β-折叠上;突变M160K处在酶分子第5个和第6个α-螺旋之间的环结构上，位于酶分子表面，突变后的Lys160与邻近的Thr162形成一个额外氢键。在90 ℃下，突变酶65和亲本酶的半衰期分别为3.1 h 和1.9 h，表明筛选到的突变酶65比亲本酶的热稳定性好。【结论】基于易错PCR技术对Geobacillus sp．ZH1羧酸酯酶的热稳定性进行了定向进化，对改善酶的性质、扩大酯酶的应用范围，以及研究酯酶的结构与功能的关系具有重要意义。

Abstract:

Abstract:［Objective］This study was aimed to improve the thermal stability of carboxylesterase from Geobacillus sp．ZH1 by directed evolution.［Methods］ A library of carboxylesterase mutants was constructed by introducing random mutagenesis using error-prone PCR to screen mutant enzymes with improved thermostability．After induction，expression and purification，the mutant enzyme was characterized.［Results］After screening，one mutant strain 65 was obtained with improved carboxylesterase thermal stability． Sequence analysis revealed two amino acid substitutions，including T113S and M160K． According to homologous modeling，T113S was located on the fifth β-sheet. Another mutant site M160K was located on a loop between the fifth and the sixth α-helix，being on the surface of the mutant enzyme． The mutated Lys160 formed an extra hydrogen bond with nearby Thr162． The half-life of mutant enzyme 65 and the parent enzyme at 90 ℃ was 3.1 h and 1.9 h，respectively． The mutant enzyme 65 had a better thermal stability than the parent enzyme.［Conclusion］Directed evolution by error-prone PCR of Geobacillus sp． ZH1 carboxylesterase gene is effective to improve the thermal stability.