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A novel salt-tolerant endo-β-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil

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Abstract

Although cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported in the Vibrio genus until now. In this report, a novel endo-β-1,4-glucanase gene, cel5A, 1,362 bp in length, was cloned from a newly isolated bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The GH5 domain shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5–7.5 and 50°C, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of total activity even after treatment in pH 5.5–10.5 for 1 h, indicating high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towards β-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose. As an alkali-activated and salt-tolerant enzyme, Cel5A is an ideal candidate for further research and industrial applications.

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Acknowledgments

This work was supported by the Science and Technology Foundation of State Oceanic Administration HE 09302(1)

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Authors and Affiliations

  1. The State Key Lab of Microbial Technology, Marine Biotechnology Research Center, Shandong University, Jinan, 250100, People’s Republic China

    Zhaoming Gao, Xiulan Chen & Yuzhong Zhang

  2. Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration (SOA), No. 178 Daxue Road, Xiamen, 361005, People’s Republic China

    Zhaoming Gao, Lingwei Ruan & Xun Xu

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  1. Zhaoming Gao
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  2. Lingwei Ruan
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  3. Xiulan Chen
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  4. Yuzhong Zhang
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  5. Xun Xu
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Correspondence to Xun Xu.

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ESM 1

Processivity analysis of Cel5A using filter paper as substrate. (DOC 202 kb)

ESM 2

Phylogenetic analysis of Cel5A and the cellulase enzymes reported by Watson et al. (Fig. 5; J. Bacteriol. 191, 5697–5705 (2009)) (DOC 183 kb)

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Gao, Z., Ruan, L., Chen, X. et al. A novel salt-tolerant endo-β-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil. Appl Microbiol Biotechnol 87, 1373–1382 (2010). https://doi.org/10.1007/s00253-010-2554-y

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