Characterization of a cellobiose phosphorylase from a hyperthermophilic eubacterium, Thermotoga maritima MSB8

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80degreesC. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70degreesC in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and alpha-D-glucose-1-phosphate. The K-m for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the k(cat) was 5.4 s(-1). In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-beta-D-glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s(-1)) k(cat) followed by 6-deoxy-D-glucose (17 s(-1)) and 2-deoxy-D-glucose (16 s(-1)). The natural substrate, D-glucose with the k(cat) of 8.0 s(-1) had the highest (1.1x10(4) M-1 s(-1)) k(cat)/K-m compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, alpha-D-glucose-1-phosphate, at higher concentrations.