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Enzyme immobilization using the cellulose-binding domain of the Cellulomonas Fimi exoglucanase

University of British Columbia

A new strategy employing molecular genetic techniques to produce fusion polypeptides was used for enzyme immobilization. The cellulose-binding domain (CBD) of the Cellulomonas fimi exoglucanase (Cex) was used as an affinity "tag" by fusing the sequence encoding CBDcex to the 3'-ends of the genes encoding two β-glucosidases, Abg and Cbg, from a mesophilic Agrobacterium sp. and a thermophilic bacterium, Caldocellum saccharolyticum, respectively. The resulting fusion polypeptides, Abg-CBDcex and Cbg-CBDcex, were purified to >95% homogeneity in a single step by affinity chromatography on cellulose using water as the desorbing agent. Protein recovery varied from 58-70%. The matrix, pH and temperature affected the reversibility of binding. These parameters can be selected to facilitate either purification or stable immobilization of the polypeptide. Fusion of the affinity "tag" appeared not to affect the conformation of the fusion partners significantly, since their specific activities were not altered appreciably. Kinetic analyses revealed that the increase in apparent Km of the immobilized Abg-CBDcex was not directly related to the fusion, but rather, to external mass transfer resistance. The percentage of activity retained by the immobilized enzyme was inversely related to the amount of fusion polypeptide adsorbed to cellulose. This was affected by the presence of an extra 57 amino acid residues between the fusion partners. Abg-CBDcex was stably adsorbed to cellulose between 4° to 50°; pH 3 to 8.5; and in the presence of up to 1 M NaCl. The immobilized Abg-CBDcex column exhibited long-term operational stability (at least 15 days at 37°) and gave 50-70% substrate conversion at the flow rates employed. At 50° Abg-CBDcex lost activity but remained bound to cellulose. Immobilized Cbg-CBDcex was stable at 70° for at least 3 days with no apparent desorption of fusion polypeptide from the column. The use of a more crystalline cellulosic support improved the stability of binding of both fusion polypeptides. A plasmid that expressed the gene encoding CBDcex alone was also constructed to facilitate biochemical analysis of the binding mechanism. CBDcex was purified from culture supernatant to > 98% homogeneity in a single step by batch affinity chromatography on cellulose. Unlike the fusion polypeptides, it was not desorbed with water. The presence of xylan in the CBDcex preparation affected its susceptibility to cleavage by pepsin. CBDcex bound stably to both amorphous and crystalline cellulose, and under a wide range of temperatures, pHs and detergent concentrations.

Thesis/Dissertation

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2008-12-18

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http://hdl.handle.net/2429/3076

Microbiology

University of British Columbia

UBCV

DSpace