Abstract
A gene, umcel5N, was isolated from a metagenomic library constructed from the contents of buffalo rumen. Its putative product belongs to the glycosyl hydrolase family 5 and is most closely related to an endoglucanase (ABN54006.1) from Clostridium thermocellum with 44% identity and 60% similarity. Gene umcel5N was heterologously expressed in Escherichia coli. The purified recombinant Umcel5N hydrolyzed carboxymethyl cellulose with a rapid decrease in the viscosity of the solution but with little release of reducing sugars, suggesting an endo mode of action. The enzyme exhibited optimal activity toward p-nitrophenyl β-d-cellobioside at pH 5.5 and 55°C, and had a Km of 1.56 mM and a Vmax of 285.6 U/mg. Two glutamic acids (E144 and E285) of the wild-type Umcel5N were predicted as a proton donor and a nucleophile, respectively. Site-directed mutagenesis confirmed that they were required for the enzyme’s activity.
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Acknowledgments
This work was supported by Program for New Century Excellent Talents in Universities of China (NCET-05-0752), the National Natural Science Foundation of China (30560003), Hi-tech Research and Development Program of China (863 Program 2007AA021307) and the Open Project of Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization (SB0708).
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Liu, L., Feng, Y., Duan, CJ. et al. Isolation of a gene encoding endoglucanase activity from uncultured microorganisms in buffalo rumen. World J Microbiol Biotechnol 25, 1035–1042 (2009). https://doi.org/10.1007/s11274-009-9983-8
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DOI: https://doi.org/10.1007/s11274-009-9983-8