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The purification and characterization of two cellulose-binding, glycosylated cellulases from the bacterium Cellulomonas fimi Langsford, Maureen Lynn


Cellulomonas fimi secretes several cellulase activities as well as protease activity into the culture medium. In contrast, few activities are bound to the cellulose in the culture. To characterize the cellulase system and to Identify cloned gene products, it was necessary to purify native, intact cellulases. We hypothesized that the cellulose-bound cellulases would be protected from proteolysis, and therefore represent the intact enzymes. Two cellulases were purified from Cellulomonas fimi. Avicel was recovered from cultures and the proteins were eluted from it with guanidine-HCl (Gdn-HCl). The Gdn-HCl extract was fractionated by Concanavalin A-Sepharose affinity column chromatography and by Mono Q anion exchange column chromatography. The cellulases purified by this procedure were an endoglucanase, EngA, and an exoglucanase, Exg. The purified enzymes were characterized. EngA has Mr 57,000, pi 8.2, and is 10 % mannose by weight. Exg has Mv 56,000, pi 5.8, and is 8 % mannose by weight. Two recombinant DNA plasmids were identified as encoding EngA and Exg. The recombinant gene products were not glycosylated. The role of glycosylation was studied by comparing some properties of the recombinant EngA and Exg with the native EngA and Exg. Both glycosylated and unglycosylated forms bound to Avicel. Sensitivity to the C. fimi protease was also compared. The glycosylated enzymes were protected from proteolysis when bound to cellulose. In contrast, the unglycosylated forms were processed to yield active, truncated products with greatly reduced affinity for cellulose. The cleavage site was predicted based on size of the products and reactivity with anti-PT serum. The N-terminal region of EngA and the C-terminal of Exg show 50 % conservation of sequence (Warren et al., 1986). This region appears to be the cellulose-binding domain and is not required for the hydrolysis of soluble substrates. The C. fimi protease can partially degrade glycosylated EngA when it is not bound to cellulose. Some of the multiple CMCase activities in culture supernatants are derived from EngA by partial proteolysis.

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