易错PCR提高华根霉脂肪酶的热稳定性

Enhanced thermostability of Rhizopus chinensis lipase by error-prone PCR

摘要:

为了提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的热稳定性，运用定向进化-易错PCR的方法，经两轮易错PCR引入突变，利用fast-blue RR顶层琼脂法对突变文库进行筛选，第一轮易错PCR后筛选到2株突变菌株，第二轮筛选到4株突变株。第二轮最佳突变株Ep2-4，其中3个氨基酸发生了突变：A129S、P168L和V329A。该突变酶ep2-4在60 ℃下半衰期相对原始酶r27RCL提高5.4倍，T50值提高7.8 ℃。酶学性质研究表明，突变酶ep2-4在热稳定性提高的基础上，仍保有良好的催化活性。蛋白质三维结构模拟显示，突变A129S可以和Gln133形成氢键，增加了酶表面的亲水性和极性；P168L可以与邻近的Leu164形成疏水键，导致突变酶的热稳定性提高。

Abstract:

Directed evolution was conducted to improve the thermostability of lipase from Rhizopus chinensis CCTCC M201021. Mutations were introduced by two rounds of error-prone PCR and mutant lipase was selected by fast-blue RR top agar screening. Two positive variants were selected in the first-round and four in the second-round screening process. Ep2-4 was proved as the most thermostable lipase and its DNA sequencing revealed three amino acid substitutions: A129S, P168L and V329A. Compared with the parent, its half-life at 60 °C was 5.4- times longer and T50 was 7.8 degrees higher. Purified lipase of Ep2-4 was characterized and the result shows that its thermostability improved without compromising enzyme activity. According to the mimicked protein structure, mutation A129S formed a hydrogen bond with Gln133 and improved the thermostability by increasing the hydrophilicity and polarity of protein; mutation P168L by forming a hydrophobic bond with the nearby Leu164.