基于易错PCR的β-甘露聚糖酶体外分子定向进化研究

Study on directed evolution ofβ-mannanase in vitro by error-prone PCR

采用易错PCR方法,对β-甘露聚糖酶进行体外分子定向进化,通过调整dNTP比例和加入不同浓度的Mn2+,向β-甘露聚糖酶中随机引入突变,构建突变文库,并对筛选得到的突变菌株的突变酶酶学性质进行研究.结果表明:在突变文库的构建过程中,当Mn2+浓度为0.01 mmol/L时,阳性转化率最高,达到80%,氨基酸的突变个数为2.0个,符合易错PCR突变文库的构建原则;经过两轮易错PCR构建的突变文库库容量约为4000,从中筛选出的突变毕赤酵母菌X-33/pGAPZαA-GwMan26A-27的突变酶GwMan26A-27具有耐酸、耐高温且酶活力较高的特性;与突变前相比,GwMan26A-27具有较宽的酶促反应温度范围和适宜pH值范围,热稳定性和pH稳定性均有较大提升;在胃蛋白酶液和胰蛋白酶液中水浴2 h,GwMan26A-27的消化酶稳定性较突变前也均有所提升.

Error-prone PCR was used to directed evolutionβ-mannanase in vitro,random introduction of mutations intoβ-mannanase by adjusting dNTP ratio and setting Mn2+of different concentrations,a mutant library was constructed,and the enzymatic properties of the mutant enzymes were studied.The results showed that during the construction of the mutation library,the positive conversion rate could reach the highest 80%and the number of amino acid mutations was 2.0 with the concentration of Mn2+0.01 mmol/L,which was in accordance with the general principles of error-prone PCR mutation libraries;after two rounds of error-prone PCR,a storage capacity of 4000 was constructed,the mutant enzymes GwMan26A-27 of mutant Pichia pastoris X-33/pGAPZαA-GwMan26A-27 with acid resistance,high temperature resistance and high enzyme activity was finally selected;compared with before mutation,GwMan26A-27 has a wider enzymatic reaction temperature before the mutation and a suitable pH range,the thermal stability and pH stability also had been greatly improved;in the pepsin solution and trypsin solution for water bath 2 h,the digestive enzymes stability of GwMan26A-27 had also been improved.