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Expression, purification and molecular characterization of a novel endoglucanase protein from Bacillus subtilis SB13

文献类型： 外文期刊

作者： Guan, Xuefang 1 ; Chen, Penglian 1 ; Xu, Qingxian 1 ; Qian, Lei 1 ; Huang, Juqing 1 ; Lin, Bin 1 ;

作者机构： 1.Fujian Acad Agr Sci, Inst Agr Engn Technol, Fuzhou 350003, Fujian, Peoples R China

关键词： Bacillus subtilis;Endoglucanase;Cloning;Expression;Homology modeling;Optimization

期刊名称：PROTEIN EXPRESSION AND PURIFICATION （ 影响因子：1.65； 五年影响因子：1.548 ）

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收录情况： SCI

摘要： Bacillus subtilis strain SB13 which is isolated in our previous work was confirmed to produce endoglucanase. In this study, a novel endoglucanase gene (accession number: 10{576676) was identified and cloned from SB13. Compared with other consensus sequence of reported endoglucanase genes in the GenBank database, this gene displays five differences (including T740C,A874G,A983G, T1210G and T1301C), which leading to five amino acid changes. Homology modeling has indicated that these five changes were located in the alpha-helix and random coil regions of the glycosyl hydrolase family 5 (GH5) domain, the random coil and beta-sandwich of the type 3 carbohydrate-binding module (CBM3) domain, and the random coil domain. Aprokaryotic expression vector pET30a-endoglucanase was constructed and the endoglucanase was induced to express. The expressed endoglucanase was confirmed by liquid chromatography tandem mass spectrometry (LC-MSMS) and detected via reaction with carboxymethyl cellulose. In order to obtain the highest expression level of endoglucanase, the expression conditions including IPTG concentration, temperature and pH were optimized. The recombinant endoglucanase protein was purified using a Ni-NTA column, and the 6 x His-tag was removed with thrombin. The results showed that both the modified and unmodified purified endoglucanase had high activity (7.65 +/- 0.35 U and 15.05 +/- 1.81 U, respectively), thus demonstrating the potential use of this enzyme in various industrial applications. The substitutions of L247P,N292D, F404V and L434P might contribute to the activity of the endoglucanase, and the insertion of a 6 x His-tag at the N-terminal of the endoglucanase might also affect its activity. (C) 2017 Elsevier Inc. All rights reserved.

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