基于易错PCR技术的黏质沙雷氏菌脂肪酶基因LipA的定向进化

Directed Evolution of Serratia marcescens Lipase Gene by Error-prone PCR

利用易错PCR技术对黏质沙雷氏菌脂肪酶基因LipA进行定向进化,经过筛选最终获得一个比活力比野生酶提高了425 U/mg的突变体ep1,测序分析ep1有5个氨基酸发生了突变,与野生酶相比ep1的最适pH值由原来的8.5降低为7.5,Tm值提高3℃,Km值由原来的40 mg/mL降低为12.5 mg/mL。对其三维结构进行分析,推测酶学性质的改变可能与处在活性中心右前方双螺旋发卡结构上的I58A、和处在下部β卷曲折叠拐角处的S375G的突变有关。

Directed evolution to Serratia marcescens Lipase gene-LipA by Error-prone PCR was carried out,and a final mutant ep1 was obtained whose specific activity increased 425 U/mg than the wild type.Sequence analysis showed that ep1 had 5 amino acid mutations.Compared with the wild type enzyme.the optimum pH value of ep1 reduced from 8.5 to 7.5.Tm value increased 3℃ and Km value reduced from 40 mg/mL to 12.5 mg/mL.By analyzing 3D structure,it speculated that the changes of the enzymatic properties may be related with the site I58A and S375G,which respectively located in the double-helix in the right front of the active sites and curled corner of β-sheet.