地衣芽孢杆菌GXN151的纤维素酶基因cel12A的序列分析及其在大肠杆菌中的表达

Sequencing of cellulase gene cel12A of Bacillus licheniformis strain GXN151 and its expression in Escherichia coli

地衣芽孢杆菌菌株GXN151的cel12A基因为783bp,可编码含261个氨基酸的羧甲基纤维素酶Cel12A,Cel12A的N末端第1～28氨基酸具典型的信号肽特征、第9～31氨基酸具跨膜功能域特征、第104～261氨基酸形成家族12糖基水解酶(glycosylhydrolase)功能域。将编码其第73～261氨基酸的DNA序列克隆到大肠杆菌表达载体pET30a(+)得表达质粒pGXN12A,用1mmol/LIPTG诱导处理JM109(DE3)/pGXN12A的培养液6hpGXN12A的表达量达到最高,在JM109(DE3)和BL21(DE3)pLysS中该表达蛋白质可分别占菌体胞内总蛋白的54.3%和20.9%。在含羧甲基纤维素的LA平板上JM109(DE3)/pGXN12A和BL21(DE3)pLysS/pGXN12A表现有较弱的羧甲基纤维素酶活性,说明重组的Cel12A的催化功能域具有独立的催化活性。包含体检测表明pGXN12A在JM109(DE3)中的表达产物大部分形成不溶性包含体。

The cel12A gene of Bacillus licheniformis strain GXN151 was 783 bp, encoding a carboxylmethylcellulase(CMCase) Cel12A with 261 amino acids. The N-minal 28 amino acids of Cel12A was characteristic of signal peptide. Amino acid 9~31 of Cel12A formed transmembrane domain, and amino acid 104~261 of the enzyme formed family 12 glycosyl hydrolase catalytic domain. The DNA sequence encoding amino acid 73~261 of Cel12A was cloned in Escherichia coli expression vector pET-30a(+) to form recombinant expression plasmid pGXN12A The expression level of pGXN12A in Ecoli JM109(DE3) peaked 6 hours after IPTG was added to final concentration of 1 mmol/L to JM109(DE3)/pGXN12A culture The expressed protein of pGXN12A in JM109(DE3) and BL21(DE3)pLysS could account for 543% and 209% of the total bacterial intracellular proteins, repectively JM109(DE3)/pGXN12A and BL21(DE3)pLysS/pGXN12A expressed weak CMCase activity on LA plates containing carboxymethylcellulose, indicating that the recombinant catalytic domain of Cel12A had independent catalytic activity Inclusion body detection indicated that most of the expressed product of pGXN12A in JM109(DE3) formed insoluble inclusion bodies