Molecular Nutrition

Cloning, Fusion Expression of Two Cellulase Genes from Bacillus subtilis and Its Enzymatic Properties

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  • 1. Hongxiang Biological Feed Laboratory, Henan University of Science and Technology, Luoyang 471023, China;
    2. Key Laboratory of Animal Disease and Public Health of Henan Province, Luoyang 471023, China;
    3. Research Center of Breeding Engineering Technology for Meat Sheep of Henan Province, Luoyang 471023, China

Received date: 2017-02-08

  Online published: 2017-08-02

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, respectively. 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 activity was 57.62 U/mL, and the exo-1,4-β-D-glucanase activity was 32.57 U/mL. The reaction optimal temperature 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 inhibitory effects on the activity of fusion cellulase Cel42-Cel22, especially Hg2+ and Cu2+. In conclusion, the fusion fusion cellulase Cel42-Cel22 is realized to efficiently express in Escherichia coli BL21(DE3), has a higher activity within a wide range of temperature and pH, and is sensitive to metal ions.

Cite this article

DING Ke, QIU Jingjing, LUO Weiguang, LI Wang, LI Yuanxiao, CAO Pinghua, HE Wangling, ZHAO Longmei, WANG Yuqin, ZHANG Chunjie . Cloning, Fusion Expression of Two Cellulase Genes from Bacillus subtilis and Its Enzymatic Properties[J]. Chinese Journal of Animal Nutrition, 2017 , 29(8) : 2725 -2733 . DOI: 10.3969/j.issn.1006-267x.2017.08.015

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