A high activity creatine amidinohydrolase (creatinase) from Arthrobacter nicotianae 02181 (a strain newly isolated from soil which may utilize creatinine as the unique organic source) was purified, characterized and the creatinase gene was cloned and analyzed in this study. Cells were cultivated under optimized condition for enzyme yield and creatinase was purified by the DEAE-cellulose and hydroxylapatite (HA) chromatography. The creatinase was found to be a dimmer formed by two identical subunit of 46.4 kDa, and the specific activity of the purified creatinase reached 124.44 U/mg protein, which was about 13 folds of the maximum value ever reported. The enzyme was found to be most active at 37 degrees C (pH 7.0), and it was found to be relatively stable bellow 45 degrees C around pH 7.0 by fluorescence spectroscopy and circular dichroism (CD) analysis. The activity of this creatinase could be significantly inhibited by Cu2+, Hg2+, Fe3+ and SDS, and it could be improved by Ca2+ and NaN3. The creatinase gene was cloned by the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) PCR and the genome walking method, Nucleotide sequence analysis of this gene revealed an open reading frame (ORF) of 1254 base pair (bp) encoding a 417 amino acid (aa) protein. The primary amino acid sequence alignment search in the database revealed a moderate homology between the deduced amino acid sequence and other creatinase. The sequence has been submitted to Genbank with the accession number EU004199. (C) 2008 Elsevier Ltd. All rights reserved.