Purification and characterization of endoglucanase C from Clostridium cellulolyticum. Catalytic comparison with...

An Escherichia coli clone was constructed to overproduce endoglucanase C (CelCCC) from Clostridium cellulolyticum. This construction made it easier to isolate the enzyme but, as observed in the case of endoglucanase A (CelCCA) from the same organism, the purification led to the isolation of two forms of the cellulase differing in their molecular masses, 48 kDa and 41 kDa. N-terminal sequence analysis of both purified enzymes showed that the shorter form was probably the result of partial proteolysis near the COOH-extremity. The difference in mass indicated that the shorter protein lacks the C-terminal reiterated domains (20-24-amino-acid twice-repeated sequences). These particular domains are characteristic of clostridial cellulases acting on cellulose by the mean of cellulosomal particles. Biochemical and enzymic studies were performed on each form of CelCCC, and revealed that their temperature and pH optima were identical, but their catalytic parameters were quite different. Furthermore, the differences of enzymic behavior observed between the two forms of CelCCC are almost identical to those already noted in the case of the two forms of CelCCA. The stereoselectivity of the reaction catalysed by CelCCC and CelCCA was determined using proton NMR spectroscopy; CelCCC acts by configuration inversion, whereas CelCCA acts by configuration retention. The degradation patterns on cellodextrins (ranging from cellotriose to cellohexaose) and chromophoric cellodextrins (from p-nitrophenyl-cellobiose to p-nitrophenyl-cellopentaose) were also investigated in both forms of CelCCC and CelCCA. It emerged that the natural cellodextrins degradation patterns of CelCCC and CelCCA were very similar but the utilization of p-nitrophenyl-cellodextrins showed the existence of considerable differences between these two endoglucanases in terms of cleavage-site position and catalytic parameters. CelCCC and CelCCA were found not to act synergistically on the tested substrates.