Purification and Properties of β-1,4-Oligoglucan: Orthophosphate Glucosyltransferase from Clostridium thermocellum

Abstract

Abstract β-1,4-Oligoglucan:orthophosphate glucosyltransferase from Clostridium thermocellum has been purified nearly 450-fold by a procedure which removes all known interfering enzymes. The enzyme catalyzes the phosphorolysis of cellotriose, cellotetraose, cellopentaose, and cellohexaose. Although cellobiose is an excellent glucosyl acceptor, the enzyme is unable to catalyze either the synthesis or the phosphorolysis of this disaccharide. The product formed in a reaction mixture with α-d-glucose-1-P as the glucosyl donor and cellobiose as the glucosyl acceptor has been isolated and identified as cellotriose. The enzyme is active on the following glucosyl acceptors: cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, cellobiitol, 4-O-β-d-glucosyl-d-altrose, 4-O-β-d-glucosyl-2-deoxy-d-glucose, 4-O-β-d-glucosyl-d-mannose, 4-O-β-d-glucosyl-d-xylose, and laminaritriose. The enzyme catalyzes the arsenolysis of cellotriose; however, no exchange occurs between glucose-1-P and Pi or arsenate. The apparent Km values are 1.2 mm for cellobiose and 4.7 mm for glucose-1-P. The apparent Km value is about 1.0 mm for cellotriose, cellotetraose, and cellopentaose, and the apparent Km value is 0.37 mm for cellohexaose. The apparent Km value for Pi, determined during the phosphorolysis of different cellodextrins, is 0.13 mm with cellotriose, 0.19 mm with cellotetraose, 0.24 mm with cellopentaose, and 0.26 mm with cellohexaose. The pH optimum for the synthesis of cellotriose is about 7.5. The purified enzyme is inactive in the absence of thiol compounds such as cysteine or dithiothreitol. Sodium sulfite activates the enzyme, but it is not as effective as cysteine or dithiothreitol. Pyridoxal 5-phosphate, adenine nucleotides, sugar phosphates, and d-glucose had no effect on the activity of the enzyme. There appears to be no requirement for any dissociable cofactors.