A method was developed for purification and crystallization of creatinase [creatine amidinohydrolase, EC 3.5.3.3] from Pseudomonas putida var. naraensis C-83. The purified preparation appeared homogeneous on disc electrophoresis and ultracentrifugation and had a molecular weight of 94,000. It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37 degrees. SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47,000. Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme.
Creatinine was found to be rapidly decomposed by several strains belonging to Pseudomonas, one of which was assigned to P. putida. Analyses of the metabolites indi cated that creatinine was converted to sarcosine via creatine and further to glycine. The enzymes involving in a metabolic pathway of creatinine, i.e. creatinine amidohydrolase (EC 3.5.2), creatine amidinohydrolase (EC 3.5.3.3) and sarcosine dehydrogenase (EC 1.5.99.1), were found to be inducibly formed and accumulated in the bacterial cells. A novel assay method for sarcosine dehydrogenase activity was also described.
A sarcosine dehydrogenase was purified to homogeneity from cell free extract of Pseudo monas putida aerobically grown in a medium containing creatinine or betaine as the carbon and nitrogen sources. The enzyme catalyzed dehydrogenation of N-methyl derivatives of some amino acids but was inert toward dimethylglycine, betaine and choline. Phenazine me thosulfate, 2, 6-dichlorophenol indophenol, methylene blue, meldora blue, nile blue and potas sium ferricyanide served as electron carriers. The maximal activity was observed at pH 8.0-9.0. The Km and Vmax values for sarcosine were 29 mm and 1.2 µmol/min/mg, respectively. The molecular weight was estimated to be about 170,000, presumably composed of four sub units. Spectrophotometric and fluorometric analyses indicated that the enzyme was a flavoprotein.
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