Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium, Thermotoga maritima MSB8(Biochemistry & Molecular Biology)

概要

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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 80℃. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70℃ in the pH range of 6-8. The enzyme phos- phorolyzed 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 α-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-β-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 (1.1×10^4 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, α-D-glucose-1-phosphate, at higher concentrations.
社団法人日本農芸化学会の論文
2002-12-23