定向进化-易错PCR方法提高华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力

Increasing activity of Rhizopus chinensis CCTCC M201021 lipase by directed evolution-error prone PCR

运用定向进化-易错PCR的方法,提高了华根霉Rhizopus chinensis CCTCC M201021脂肪酶的活力。经过两轮易错PCR和pNPP顶层琼脂法筛选,从第一轮和第二轮突变库中分别筛选获得最佳突变株1-11和2-28,脂肪酶酶活与野生菌株相比分别提高2倍和4倍。基因比对结果表明,突变脂肪酶2-28有4个氨基酸发生了突变:A129S、K161R、A230T、K322R。蛋白质分子空间结构模拟显示,突变A129S、K161R、A230T位于脂肪酶分子表面。突变A230T增强了α-螺旋盖结构的稳定性。突变K322R处在loop上,靠近脂肪酶底物结合区域,与邻近的Asp(带负电)形成盐桥。静电引力将该loop向底物进入酶活性中心的通道口反方向牵引,使底物分子更易进入酶活性中心。酶学性质研究表明,突变株2-28脂肪酶的Km值比出发菌株下降了10%,Kcat值提高为原来的2.75倍。

Directed evolution strategy （error-prone PCR） was conducted to improve the activity of lipase from Rhizopus chinensis CCTCC M201021.Through two rounds of ep-PCR and pNPP top agar screening,two optimum mutant strains 1-11 and 2-28 were obtained with 2 and 4 fold of enzyme activity higher than that of parent strain,respectively.DNA sequencing of mutant lipase 2-28 revealed four amino acid substitutions：A129S,K161R,A230T,K322R.According to the simulated protein structure of Rhizopus chinensis lipase,A129S,K161 R, A230T were located on the surface of the protein. A230T substitution improved the stability of the α-helix loop. K322R, near the catalytic center of lipase, located at a loop, formed a salt-bridge with a nearby aspartic acid （negative charged）. Electrostatic force pulled the loop to the opposite direction of the substrate channel and made it easier for substrate to enter the lipase catalytic domain. Purified lipase was characterized and the result showed that Km of 2-28 lipase decreased by 10% compared with Km of the parent lipase, and gcat was 2.75 fold improved than that of the original lipase.