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The construction and characterization of a Pro-Thr box deletion of a Cellulomonas fimi endoglucanase (Cen A)

The catalytic domain is separated from the cellulose-binding domain in Cellulomonas fimi endoglucanase CenA by a proline-threonine rich sequence called the Pro-Thr box. To study the function of the Pro-Thr box region, a deletion mutant, cenAAPT, was made from cenA by an oligonucleotide directed in vitro mutagenesis. The truncated enzyme, CenAAPT, was purified to homogeneity by affinity chromatography on cellulose and characterized. Comparing CenAAPT to CenA, the following characteristics were observed: 1) the Pro-Thr box affected the migration of CenA on SDS-PAGE; 2) the deletion of the Pro-Thr box altered the high affinity interaction with cellulose; 3) the truncated enzyme showed 40-50% reduction in catalytic activity towards both microcrystalline and amorphous cellulose; 4) the truncated enzyme was as sensitive as CenA to a C.fimi protease, and both enzymes were cleaved at the same site adjacent to the binding domain. The Pro-Thr box is not essential for the catalytic activity of CenA or its binding to cellulose, but it does contribute to both functions. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Identiferoai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/28744
Date January 1990
CreatorsShen, Hua
PublisherUniversity of British Columbia
Source SetsUniversity of British Columbia
LanguageEnglish
Detected LanguageEnglish
TypeText, Thesis/Dissertation
RightsFor non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

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