MOLECULAR-CLONING, EXPRESSION, AND CHARACTERIZATION OF A NEW ENDOGLUCANASE GENE FROM FIBROBACTER-SUCCINOGENES-S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) of Fibrobacter succinogenes S85 was isolated from a pUC18 gene library in Escherichia coli JM109. The CMCase gene was present as a single copy in the F. succinogenes S85 genome and was found in all the other F. succinogenes strains tested. The gene was expressed from an endogenous promoter in E. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane of E. coli. Zymogram analysis and S-35 labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30-degrees-C. The enzyme was active on CMC, barley beta-glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)-beta-D-glucanase.