Abstract
Cytophaga hutchinsonii is an abundant aerobic cellulolytic bacterium that rapidly digests crystalline cellulose in a contact-dependent manner. The different roles of various predicted glycoside hydrolases and the detailed mechanism used by C. hutchinsonii in cellulose digestion are, however, not known. In this study, an endoglucanase belonging to glycoside hydrolase family 5 (GH5) named as ChCel5A was isolated from the outer membrane of C. hutchinsonii. The catalytic domain of ChCel5A exhibited typical endoglucanase activity and was capable of hydrolyzing insoluble cellulose with cellobiose and cellotriose as the predominant digestion products. Site-directed mutagenesis identified two aromatic amino acids in ChCle5A, W61 and W308, that dramatically decreased its hydrolytic activity toward filter paper while causing only a slight decrease in carboxymethylcellulase (CMCase) activity. Disruption of chu_1107 encoding ChCel5A caused no drastic effect on the growth parameters on cellulose for the resulting mutant strain with negligible reduction in the specific CMCase activities for intact cells. The demonstration of targeted gene inactivation capability for C. hutchinsonii has provided an opportunity to improve understanding of the details of the mechanism underlying its efficient utilization of cellulose.
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Acknowledgments
We are grateful for Dr. MJ McBride for providing the plasmids and strains and critical reading of this manuscript. This work is supported by grants from the National Basic Research Program (2011CB707402), the National Natural Science Foundation of China (31170762), New Century Excellent Talents in University (NCET-10-0546), Shandong Provincial Funds for Distinguished Young Scientists (JQ201108), and Independent Innovation Foundation of Shandong University, IIFSDU.
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Yongtao Zhu and Hong Zhou contributed equally to this work.
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Zhu, Y., Zhou, H., Bi, Y. et al. Characterization of a family 5 glycoside hydrolase isolated from the outer membrane of cellulolytic Cytophaga hutchinsonii . Appl Microbiol Biotechnol 97, 3925–3937 (2013). https://doi.org/10.1007/s00253-012-4259-x
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DOI: https://doi.org/10.1007/s00253-012-4259-x