Purification and Characterization of Creatinine Amidohydrolase of Alcaligenes Origin

Abstract

Creatinine amidohydrolase (creatininase) from Alicaligenes sp. nov. was purified to electrophoretic homogeneity by adsorption on diethylaminoethyl-cellulose, affinity chromatography on creatinyl-AH-Sepharose, gel filtration on Sephadex G-200 and hydroxyapatite chromatography. The molcular weight of the enzyme was estimated to be approximately 160000 by gel filtration on Sephadex G-200 and 80000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the enzyme was assumed to consist of two identical subunits. The enzyme showed maximum activity at pH 7-8 and was stable in the pH range of 8-11.5. The enzyme catalyzed interconversion between creatinine and creatine, and the Km values for creatinine and creatine were 60.9 mM and 162 mM, respectively. Though the enzyme was markedly inactivated by ethylenediamine-tetraacetate (EDTA), N-bromosuccinimide, Zn2+, Cu2+, Ni2+ or Co2+, activation of the enzyme was only observed in the presence of Mn2+. Furthermore, the reactivation of EDTA-treated inactive enzyme was observed on the addition of Mn2+ to the reaction mixture.

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