Purification and Specificity of Cellobiose Phosphorylase from Clostridium thermocellum

Abstract

Abstract A nonspecific cellobiose phosphorylase from Clostridium thermocellum, which has been purified over 100-fold, is active on 10 different glucosyl acceptors, d-glucose, 2-deoxyglucose, 6-deoxyglucose, d-glucosamine, d-mannose, d-altrose, 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 Vmax and apparent Km values are 202 and 9.2 mm for 6-deoxyglucose, 100 and 35 mm for d-xylose, 53 and 73 mm for 2-deoxyglucose, 31 and 9.5 mm for d-glucosamine, 22 and 85 mm for d-mannose, 9 and 240 mm for d-arabinose and 8 and 160 mm for l-fucose. The Ki for d-glucose is 1.2 mm. The apparent Km value is 7.3 mm for cellobiose and 2.9 mm for Pi.