苏云金芽孢杆菌chi36基因的克隆、表达和酶活性分析

摘要

通过PCR方法从苏云金芽孢杆菌010菌株克隆出几丁质酶基因chi36,该基因开放阅读框(ORF)的长度为1 083 bp,编码360个氨基酸.氨基酸序列分析表明:Bt 010的Chi36与Bt 15A3、Bc 28-9、Bc 6E1的Chi36相似性分别为99％、99％和96％,其N-端具有27个氨基酸的信号肽序列.该蛋白归类为18家族几丁质酶,属于一种胞外酶.将chi36基因插入到PGEX-KG原核表达载体的表达框中,构建成原核表达载体并转化到大肠杆菌E.coli BL21(DE3)进行蛋白表达,酶活性测定结果表明,表达产物对几丁质有降解作用,在pH值为5.0、温度为55℃时酶活性最高.%child gene was cloned from Bacillus thuringiensis strain 010 by PCR. The nucleotide sequence of chi36 included an open reading frame of 1 083 bp, encoding 360 amino acids. Analysis of its amino acid sequence showed that Chi36 of Bt 010 had 99%, 99% and 96% similarity with Chi36 of Bt 15A3, Bc 28-9 and Bc 6E1 respectively. ScanProsite analysis revealed that Chi36 of Bt 010 belonged to family 18 of glycosyl hydrolases, as an exochitinase with a signal peptide sequence containing 27 amino acids at N terminal. The child gene was inserted into the expression plasmid pGEX-KG and expressed in E. Coli BL21 (DE3). Bioactivity assay indicated that the expressed fusion protein could digest chitin, and with the most high activity at pH5.0 and 55 ℃ respectively.