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

作　　者：王睿[1,2] 喻晓蔚[1] 徐岩 

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]江南大学药学院,江苏无锡214122

出　　处：《生物工程学报》2013年第12期1753-1764,共12页Chinese Journal of Biotechnology

基　　金：国家高科技研究发展计划(973计划)(No.2011CB710800);国家高技术研究发展计划(863计划)(Nos.2012AA022207;2011AA02A209;2011AA02A210);中央高校基本科研业务费专项资金(No.JUSRP11014);国家自然科学基金(No.20802027)资助~~

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。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 ℃ was 5.4- times longer and Ts0 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.

关 键 词：定向进化 易错PCR 华根霉脂肪酶 热稳定性 毕赤酵母 

分 类 号：Q55[生物学—生物化学]