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Gene cloning and characterization of an exocellobiohydrolase (Cbh B) from Cellulomonas fimi

University of British Columbia

The objective of this study was to characterize a cellulose-binding polypeptide, Cbpl20, from Cellulomonas fimi. The gene cbpllO was cloned, its nucleotide sequence determined and the amino acid sequence it encodes deduced. The gene encodes a polypeptide of 1090 amino acids. Mature protein is 1037 amino acids long with a predicted molecular weight of 109,765. Cbpl20 is non-glycosylated as judged by lack of reaction with the periodic acid-Schiff reagent. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, followed by three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose binding domain of 104 amino acids. The catalytic domain is in family 48 of glycoside hydrolases. Cbpl20 is a cellobiohydrolase (cellobiohydrolase B; CbhB) based on its ability to hydrolyze bacterial microcrystalline cellulose (BMCC), viscometric analysis of carboxymethylcellulose (CMC) hydrolysis, and the release of cellobiose as the major product from insoluble cellulose. CbhB catalyzes hydrolysis with inversion of anomeric carbon configuration. It removes cellobiose units from the reducing end of the cellulose molecule. It has an optimum pH of 7.0 for catalytic activity. CbhB binds to cellulose with similar affinity to other well-characterized C. fimi cellulases such as CenA and Cex. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases. Furthermore, both the C. fimi and fungal cellulase systems contain cellobiohydrolases that hydrolyze the cellulose molecule from either the reducing end or the non-reducing end. The exo-cellobiohydrolases from C. fimi, CbhA, CbhB and the xylanase/exoglucanase Cex, all have low but detectable endoglucanase activity by the CMC-Congo red plate assay. It is thus clear that there is not a clear distinction between endo- and exoglucanases.

Thesis/Dissertation

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2009-02-18

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http://hdl.handle.net/2429/4761

Microbiology and Immunology

University of British Columbia

UBCV

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