Purification and specificity of cellobiose phosphorylase from Clostridium thermocellum.

A nonspecific cellobiose phosphorylase from Clostridium thermocellum, which has been purified over loo-fold, is active on 10 different glucosyl acceptors, D-glucose, Z-deoxyglucose, 6-deoxyglucose, D-glucosamine, D-mannose, D-a&rose, L-galactose, L-fucose, D-arabinose, and D-xylose. Following electrophoresis on polyacrylamide gels, the activity with various acceptors occurs at the location of the major protein component, indicating that a single enzyme catalyzes these reactions. The pH optimum is about 6.5 with D-xylose as an acceptor. The relative V,, and apparent K, values are 202 and 9.2 rnr.r for 6-deoxyglucose, 100 and 35 mu for D-XylOSe, 53 and 73 mu for 2-deoxyglucose, 31 and 9.5 rnhr for D-glucosamine, 22 and 85 mrd for D-mannose, 9 and 240 mu for D-arabinose and 8 and 160 rnhr for L-fucose. The Ki for D-glucose is 1.2 mM. The apparent K, value is 7.3 mM for cellobiose and 2.9 mM for Pi.