一株喜油嗜热芽孢杆菌G1201产高温蛋白酶的性质研究及异源表达初探

Characterization and heterologous expression of a thermophilic protease produced by Geobacillus thermoleovorans G1201

从高温环境中分离出产高温蛋白酶菌株,以脱脂奶粉为唯一碳氮源,通过形态学、生理生化特性和16S rRNA基因序列测定确定菌株种属;硫酸铵沉淀法获得高温蛋白酶的粗酶,并对其进行酶学性质分析;克隆编码高温蛋白酶的基因序列,构建表达载体在大肠杆菌中进行异源表达。结果表明:筛选到1株产高温蛋白酶的菌株G1201,经鉴定为喜油嗜热芽孢杆菌(Geobacillus thermoleovorans),所产高温蛋白酶大小52.5 kD,属丝氨酸蛋白酶家族,最适作用温度和pH分别为70℃和9.0,Ca^(2+)、Mg^(2+)、吐温20和TritonX-100对酶活有促进作用;异源表达结果显示产物以包涵体的形式存在,目的蛋白复性后含量为0.108 mg/mL。该菌株所产高温蛋白酶可耐受饲料、食品加工过程中的高温环境,具有良好的应用潜力。

To select thermophilic-protease-producing strains through screening isolates derive from high temperature habitant.Strains screening were conducted by testing isolates from high temperature environment by using a medium with skim milk as the sole carbon and sole nitrogen sources.The species were determined by morphological,physiological and biochemical characteristics together with 16S rRNA gene sequencing.The crude enzyme preparation,derived from ammonium sulfate precipitation,was used for characterizing its enzymatic conditions.The gene sequence encoding high temperature protease was cloned,and then heterologously expressed in E.coli.A thermophilic protease producing strain G1201 was selected for its thermophilic-protease-producing potential.It was identified as Geobacillus thermoleovorans.The size of the thermophilic protease was 52.5 kD,belonging to serine protease family.The optimal temperature and pH for the enzyme were found at 70℃and pH 9.0,respectively.The enzyme activity was improved with the addition of Ca^(2+),Mg^(2+),Tween 20 and TritonX-100 at proper concentrations,respectively.Heterologous expression in E.coli succeeded with the enzyme produced in the form of inclusion body,and followed renaturation resulted in a heterologous expressed enzyme preparation at 0.108 mg/mL.The thermophilic protease produced by G.thermoleovorans G1201 could tolerate the high temperature processing conditions as found in feed,detergent and food industries processes.Further investigation was deserved thanks to its decent industrial application potential.