UBC Theses and Dissertations
A gene fusion of Cex from Cellulomonas fimi and CbhI from Trichoderma reesei Driver, Diane Patricia
The proteins Cex from C. fimi and CbhI from T. reesei are exo-glucanases which are composed of separate catalytic and cellulose binding domains. When separated from one another by treatment with a protease, these domains retain their specific functions. Using polymerase chain reaction, a gene fusion was constructed which encodes a polypeptide containing the catalytic domain of Cex and the cellulose binding domain of Cbhl. During DNA sequencing of the fusion clones, an error was detected in the published Cbhl DNA sequence (Shoemaker et al. 1983b). The corrected sequence codes for two prolines, as Fagerstam (1981) determined during his earlier amino acid sequencing of Cbhl. This fusion protein, expressed in E. coli, was active on the substrates p-nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-lactoside, carboxymethyl cellulose and xylan, as is Cex, and was able to bind to microcrystalline cellulose but not to chitin. It can be eluted from cellulose with 8M guanidine-HC1. The fusion protein was found in the culture supernatant of E. coli cultures and presumably leaks from the periplasm in the same manner as Cex (Guo et al. 1988). Adsorption assays were conducted for the Cex/Cbhl fusion on bacterial microcrystalline cellulose (BMCC).
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