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UBC Theses and Dissertations

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


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.

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