定向进化改善米曲霉中性蛋白酶酶活和最适pH

项目名称： 定向进化改善米曲霉中性蛋白酶酶活和最适pH

项目编号： No.31260389

项目类型： 地区科学基金项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 李燕萍

作者单位： 南昌大学

项目金额： 50万元

中文摘要： 米曲霉中性蛋白酶对蛋白质的利用率、酱油的品质和风味形成起重要作用。偏酸性的发酵环境不利于米曲霉中性蛋白酶更大限度地发挥作用，有必要对其定向进化使其最适pH值下降。本项目拟将米曲霉的中性蛋白酶I基因在毕赤酵母GS115中进行异源表达。利用 SWISS-MODEL 分子模建服务系统同源模建米曲霉中性蛋白酶三维结构。再通过蛋白序列比对和三维结构结果，找出在活性中心附近且非常保守的氨基酸作为待突变位点。接着利用quick change突变试剂盒进行定点突变并将突变酶基因在毕赤酵母中表达，表达产物经纯化后对其酶学性质进行比较。然后将突变的酶基因克隆到含米曲霉 niaD 基因的同源臂和pyrG基因的整合型表达载体上，将其线性化后转化米曲霉pyrG突变株中，以获得能分泌NPI蛋白且在偏酸性条件下能保持高酶活的米曲霉菌株。本项目为提高酱油产品的原料利用率兼顾改进风味，国内酱油企业的国际竞争力增强提供保证。

中文关键词： 米曲霉；中性蛋白酶I；定向进化；定点突变；最适pH

英文摘要： Neutral protease in Aspergillus oryzae plays a crucial role at protein utilization, quality and flavor formation of soy sauce. Because neutral protease in A. oryzae can not effectively catalyze substrate in the acid environment, it is imperative to make protease efficiency work at lower pH. The npI gene was expressed heterogenously in Picha pastoris GS115 with α-signal peptide. Three dimensional structure of neutral protease I was constructed homologously with the SWISS-MODEL system. Then based on dimensional structure and the results of sequence alignment conservative amino acid sites which are all near the active center were chosen as mutant sites. The directed evolution of neutral protease from A. oryzae was carried out by quick change site-directed mutagenesis kit. The mutant genes were expressed in P. pastoris. The expressed mutant neutral protease was purified for compare their characters. One replacing target vectors were constructed vector, which includes two homologous arms of niaD and pyrG and mutant NPI gene, was transformed into mutant pyrG strain to get the mutant A. oryzae. It is useful and important to improve the protein utilization rate and flavor of soy sauce.

英文关键词： Aspergillus oryzae；Neutral protease I；directed evolution；site-specific mutagenesis；optimum pH