动物营养学报2017，Vol.29Issue(8)：2725-2733,9.DOI:10.3969/j.issn.1006-267x.2017.08.015
2个枯草芽孢杆菌源纤维素酶基因的克隆、 融合表达及其酶学性质分析
Cloning, Fusion Expression of Two Cellulase Genes from Bacillus subtilis and Its Enzymatic Properties
摘要
Abstract
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.关键词
纤维素酶/枯草芽孢杆菌/克隆/融合表达/酶学性质Key words
cellulase/Bacillus subtilis/clone/fusion expression/enzymatic properties分类
农业科技引用本文复制引用
丁轲,张春杰,邱静静,罗伟光,李旺,李元晓,曹平华,何万领,赵龙妹,王玉琴..2个枯草芽孢杆菌源纤维素酶基因的克隆、 融合表达及其酶学性质分析[J].动物营养学报,2017,29(8):2725-2733,9.基金项目
国家自然科学基金项目(31101744) （31101744）
河南省科技厅重大科技攻关项目(131100110300) （131100110300）
