2个枯草芽孢杆菌源纤维素酶基因的克隆、r融合表达及其酶学性质分析

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Mn2+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Hg2+和Cu2+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.%The aim of this experiment was to construct fusion expression system based on different cellulases and studied the enzymatic properties of fusion cellulase. The two different cellulase genes Cel42 and Cel22 were amplified from Bacillus subtilis isolated in previous researches in our laboratory using PCR method, re-spectively. The two genes were linked with a flexible polypeptide ( GSGGGS) , which could form a complete ORF, and the fusion gene was inserted into vector pET32a(+) to construct the recombinant expression vector pET32a(+)-Cel42-Cel22, which was transformed into Escherichia coli BL21( DE3) . The recombinant strain was induced to express the fusion cellulose, and the the enzymatic properties of fusion cellulose were studied. The results showed that the two cellulase genes Cel42 and Cel22 were cloned successfully in this experiment, and obtained the recombinant expression system BL21/pET32a(+)-Cel42-Cel22. Sodium dodecyl sulfate polyacrylamide gel electrophoresis ( SDS-PAGE) indicated that the molecular weight of the fusion protein was about 101 ku. Enzymatic activity of the crude enzyme liquid indicated that the endo-1,4-β-D-glucanases activi-ty was 57.62 U/mL, and the exo-1,4-β-D-glucanase activity was 32.57 U/mL. The reaction optimal tempera-ture of the fusion cellulase Cel42-Cel22 was 50 ℃, and could still maintain above 70% cellulase activity when temperature was from 30 to 70℃. The optimal pH of the fusion cellulase Cel42-Cel22 was 6.0, and could still maintain over 75% cellulase activity in a pH range of 4.0 to 9.0. In addition to Mn2+, other metal ions had in-hibitory effects on the activity of fusion cellulase Cel42-Cel22, especially Hg2+and Cu2+. In conclusion, the fu-sion fusion cellulase Cel42-Cel22 is realized to efficiently express in Escherichia coli BL21( DE3) , has a high-er activity within a wide range of temperature and pH, and is sensitive to metal ions.