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UBC Theses and Dissertations

The molecular cloning of Cellulomonas fimi cellulase genes Whittle, Daniel Joseph


Recombinant DNA techniques were used to clone and isolate Cellulo- monas fimi cellulase genes. A sensitive and simple immunoassay was developed to screen Escherichia coli transformed with recombinant plasmids carrying cellulase genes. The screening procedure is based on binding cellulases and other proteins released from lysed clones to CNBr-acti- vated paper. The paper is treated with anti-cellulase antibody and the antigen-antibody complex is detected by autoradiography using ¹²⁵I-labeled protein A from Staphylococcus aureus. This- immunoassay, was used to identify recombinant plasmids containing strains, carrying at least two different cellulase genes. The enzymes present in extracts of E. coli cellulase clones were active in catalysing the hydrolysis of carboxymethy1 eel 1ulose as indicated by the production of reducing sugars. Osmotic shock treatment of one E. coli cellulase clone revealed that the majority of the cellulase enzyme synthesized by this clone was transported to the periplasmic space. Cellulase encoding plasmids were characterized by the presence of either a 6.6 or a 5.0 kilobase C. fimi DNA gene fragment.

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