Purification and characterization of creatine amidinohydrolase of Alcaligenes origin.

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Abstract
Extracellular creatine amidinohydrolase (creatinase, EC 3.5.3.3) produced by Alcaligenes was purified to electrophoretic homogeneity by ion exchange chromatography on diethyl-aminoethyl-cellulose, gel filtration on Sephadex G-75 and hydrophobic chromatography on phenyl-Sepharose CL-4B. The molecular weight of the enzyme was estimated to be 51000 by gel filtration on Sephadex G-200 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed the maximum activity at pH 8 and was stable at pH 5-9. The pI value was 4.7 as determined by isoelectric focusing. The enzyme catalyzed hydrolysis of creatine to sarcosine and urea, and the Km and Vmax values for creatine were 17.2 mM and 105 .mu.mol/min/mg protein, respectively. The enzyme was markedly inactivated by p-chloromercuribenzoate (PCMB). Besides PCMB, the enzyme was inactivated by N-bromosuccinimide, Zn2+. Cu2+ or Hg2+.

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