2个枯草芽孢杆菌源纤维素酶基因的克

发布时间：2018-07-22 11:51

【摘要】：本试验旨在构建不同纤维素酶的融合表达系统及探讨融合纤维素酶的酶学性质。利用PCR技术从实验室前期分离的枯草芽孢杆菌中分别扩增2个纤维素酶基因Cel42和Cel22,设计一段柔性接头(GSGGGS),通过酶切连接将2个纤维素酶基因构建在一个开放阅读框(ORF)内,插入到pET32a(+)中构建重组表达载体pET32a(+)-Cel42-Cel22,转化大肠杆菌BL21(DE3)进行诱导表达,并对其酶学性质进行研究。结果表明:本试验成功克隆了2个纤维素酶基因Cel42和Cel22,并构建了重组表达系统BL21(DE3)/pET32a(+)-Cel42-Cel22,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)估计其分子质量约为101 ku,粗酶液中葡聚糖内切酶活性为57.62 U/mL,葡聚糖外切酶活性为32.57 U/mL。试验所得融合纤维素酶Cel42-Cel22的最适反应温度为50℃,最适反应pH为6.0,温度在30~70℃范围内时可维持70%以上的纤维素酶活性,pH在4.0~9.0范围内时可保持75%以上的纤维素酶活性,除Mn~(2+)外的其他金属离子对纤维素酶的活性均具有一定的抑制作用,其中Hg~(2+)和Cu~(2+)对的抑制作用较明显。由此可见,本试验在大肠杆菌BL21(DE3)中成功表达出了融合纤维素酶Cel42-Cel22,且该酶具有一定的活性,可适应较宽广的温度和pH范围,对金属离子敏感。
[Abstract]:The purpose of this study was to construct fusion expression systems of different cellulases and to investigate the enzymatic properties of the fusion cellulases. Two cellulase genes Cel42 and Cel22 were amplified by PCR from Bacillus subtilis isolated in early laboratory, and a flexible joint (GSGGGS) was designed. The two cellulase genes were constructed into an open reading frame (ORF) by enzyme ligation. The recombinant expression vector pET32a () -Cel42-Cel22 was inserted into pET32a (), and transformed into E. coli BL21 (DE3) to induce the expression, and its enzymatic properties were studied. The results showed that two cellulase genes Cel42 and Cel22 were cloned successfully, and the recombinant expression system BL21 (DE3) / pET32a () -Cel42-Cel22 was constructed. SDS-PAGE estimated its molecular weight to be about 101 ku. The activity of endoglucinase was 57.62 U / mL, and that of dextran was 32.57 U / mL. The optimum reaction temperature and pH of the fusion cellulase Cel42-Cel22 were 50 鈩，

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