Creatinine deiminase (EC 3.5.4.21) from bacterium BN11: purification, properties and applicability in a serum/urine creatinine assay

Creatinine deiminase (EC 3.5.4.21) from the anaerobic microorganism BN11 has been purified to homogeneity by ammonium sulfate fractionation, gel filtration on Sephacryl-S-300 superfine and chromatography on DEAE-Sepharose C1 6B. The final enzyme preparation had a specific activity of 78 units per mg protein. Analysis of creatinine deiminase by polyacrylamide gradient gel electrophoresis and fast-flow-liquid-chromatography gave a relative molecular mass of 285 kDa and 288 kDa, respectively. By treatment with sodium dodecylsulfate and 2-mercaptoethanol creatinine deiminase was dissociated yielding one polypeptide with a relative molecular mass of 47.5 kDa. The enzyme was entirely specific for creatinine and showed a Km value of 0.15 mM. Creatinine deiminase was used to determine the concentration of creatinine in serum and urine using a manual method and an automated system.