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Characterization of double binding domain derivatives of CenA from Cellulomonas fimi

University of British Columbia

Derivatives of CenA and of CBD.PT from C. fimi with tandemly repeated binding domains were constructed. A sensitive assay using 14C-labeled proteins was developed to measure adsorption affinities for microcrystalline cellulose at low protein concentrations. It was found that the CBDs in the double binding domain derivatives could function in tandem to increase the overall adsorption affinity of CenA, provided that the catalytic domain was present and that the CBDs were separated by a full Pro-Thr linker. The catalytic domain was found to contribute to the overall adsorption affinity of the enzyme, as the affinity of the isolated binding domain was reduced compared to that of CenA. Furthermore, the nature of the linker separating domains in wild-type CenA was important for adsorption affinity of the enzyme. The activities of the double binding domain derivatives of CenA on microcrystalline and soluble substrates were unchanged compared to the activity of the wild type enzyme. Furthermore, double binding domain derivatives of CBD.PT released more small particles from intact cotton fibres than did CBD.PT, and this effect was independent of the adsorption affinity of the proteins.

Thesis/Dissertation

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2009-01-05

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http://hdl.handle.net/2429/3375

Microbiology and Immunology

University of British Columbia

UBCV

DSpace