J. Biochem. 86, 1109-1117 (1979) Creatinine Amidohydrolase (Creatininase) from Pseudomonas putida Purification and Some Properties 1 Kaoru RIKITAKE, Imao OKA, Makoto ANDO, Tadashi YOSHIMOTO, and Daisuke TSURU Faculty of Pharmaceutical Sciences, Nagasaki University, Bunkyo-machi, Nagasaki, Nagasaki 852 Received for publication, April 11, 1979 Creatinine amidohydrolase (EC 3.5.2.-; creatininase] was purified in an overall yield of 11 from cell-free extract of Pseudomonas putida var. naraensis, strain C-83, by column chro matographies on sarcosine-HM-Sepharose and DEAE-cellulose, and gel filtration on Sephadex G-200. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the reversible conversion of creatinine to creatine with an optimal pH of 7-9. Km values for creatinine and creatine were 26 mM and 0.13 M, respectively. The enzyme was also active toward glycocyamidine, though the reaction rate was quite low, but it was completely inert toward hydantoin and its derivatives. The molecular weight of the enzyme was estimated to be 175,000 by ultracentrifugal analysis and the subunit molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 23,000, suggesting that the enzyme is composed of eight subunit monomers. The isoelectric point was 4.7 as judged by iso electric focusing experiments. The enzyme was markedly inactivated by heavy metal ions, N-bromosuccinimide, ethoxyformic anhydride, and dye-sensitized photooxidation, and also partially by metal chelators. The enzyme was found to contain about one gram atom of zinc per subunit monomer. The metal-free, inactive enzyme was prepared and could be reactivated by the addition of Mn2+, Co2+, Mg2+, Fe2+, Ni2+, and Zn2+ in that order of de- creasing effectiveness. These results indicate that metal is intimately involved in the creatini nase activity of P. putida. In order to establish a new enzymatic method for terium belonging to Pseudomonas putida was diagnostic analysis of creatinine and creative in isolated from soil in Nara Prefecture and designated serum (1, 2), microorganisms capable of rapidly as P. putida var. naraensis C-83 (3). The strain was catabolizing creatinine were screened. A bac- found to catabolize creatinine as follows: CreatinineA?CreatineB•¨Urea+Sarcosine C•¨ Glycine +FormaldehydeD•¨ Formic acid a This work was supported by a grant from the Ministry of Education, Science and Culture of Japan. Abbreviations: BPB, Bromphenol Blue; HM, hexamethylenediamine; EDTA, ethylenediaminetetraacetic acid; DFP, diisopropylphosphorofluoridate; DTNB, 5, 5•Œ-dithiobis-(2-nitrobenzoic acid) [Ellman reagent]; PCMB, ƒÏ-chloromercuribenzoate; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl sulfate; NBS, N-bromosuccinimide. Vol. 86, No. 4, 1979 1109 1110 K. RIKITAKE, I. OKA, M. ANDO, T. YOSHIMOTO, and D. TSURU The enzymes involved are creatinine amido of the mixtures were subjected to electrophoresis hydrolase (creatininase) [A], creatine amidino in 7.5 % gels at a constant current of 8 mA per gel hydrolase (creatinase) [B], sarcosine dehydrogenase for 3 h. The gels were stained with 0.25%. Cooma [C], and formaldehyde dehydrogenase [D]. Of ssie Brilliant Blue R-250 and destained by washing these four enzymes, the latter three have already overnight with a mixture of acetic acid-methanol- been purified to homogeneity in our laboratory, water (35 : 105: 315). The molecular weight of and their enzymatic properties were characterized creatininase was estimated by comparison with in detail (4-7). We also attempted to purify the parallel runs of marker proteins, bovine serum remaining enzyme, creatinine amidohydrolase albumin (68,000), catalase (58,000), aldolase (creatininase), and succeeded in obtaining a disc (40,000), chymotrypsinogen (25,700), and horse electrophoretically homogeneous preparation of heart muscle cytochrome c (11,700). the enzyme. The present paper deals with the Estimation of molecular weight by ultracentri purification and characterization of this enzyme fugal analysis and gel filtration: The purified from P. putida C-83. This is the first report enzyme was dialyzed against 20 mm Tris-HCl describing the properties of a homogeneous prepa buffer, pH 7.0, containing 0.15 M NaCl at 4•Ž for ration of creatininase in detail. 48 h, during which time the outer solution was changed every 12 h. The dialyzed solution was MATERIALS AND METHODS subjected to ultracentrifugal analysis using a Beckman model E ultracentrifuge equipped with Materials-Rabbit muscle aldolase, beef liver schlieren and interference optics at 25•Ž.. The catalase, and bovine pancreas chymotrypsinogen molecular weight of the enzyme was estimated were purchased from Boehringer Mannheim, by the meniscus depletion sedimentation equilib Germany. Sephadex G-100, G-200, Sepharose 4B, rium method (10) at 12,000 rpm with a 12 mm and DEAE-Sephadex A-50 were from Pharmacia double sector cell after 24 and 26 h. Fine Chemicals, Sweden, and DEAE-cellulose was The molecular weight of the enzyme was also from Brown Co., U.S.A. Diisopropylphosphoro estimated by the gel filtration method of Andrews fluoridate (DFP), phenylmethanesulfonyl fluoride (11), using a column (2•~110 cm) of Sephadex (PMSF), 5, 5•Œ-dithio-bis-(2-nitrobenzoic acid) G-200 equilibrated with 50 mm phosphate buffer,' [Ellman reagent] were obtained from Sigma pH 7.4, containing 0.15 M KCl. Chemical Co., U.S.A., and creatinine was from Isoelectric focusing run: This was done by Tokyo Kasei Co., Tokyo. Sarcosine-hexamethyl the method of Vesterberg and Svensson as described enediamine-Sepharose (sarcosine-HM-Sepharose) by Matsuo and Horio (12) using an LKB apparatus. was prepared as described by Yoshimoto et ƒ¿l. The purified enzyme (1.5 ml) was dialyzed over- (5). Other chemicals were products of Nakarai night against 0.5 % carrier ampholyte, pH 3.5-10.0, Kagaku Co., Kyoto. applied to a column (110 ml) containing a sucrose Analytical Methods-Disc gel electrophoresis density gradient, and then subjected to isoelectric and SDS polyacrylamide gel electrophoresis: Disc focusing at 300 volts and 9•Ž for 72 h. gel electrophoresis was carried out using a 7.5 Metal analysis: Zinc, manganese, magne gel of pH 8.4 according to Davis (8). The enzyme sium, and calcium contents of the enzyme prepa preparations (20-100 peg) were loaded on gels and rations were measured using a Hitachi 208 atomic a current of 2 mA per tube was applied for 2 h. absorption spectrophotometer. Cobalt was deter- The gels were stained with 1 % Amidoblack B and mined by the ƒ¿-nitroso-ƒÀ-naphthol method (13). destained with 7 % acetic acid. The enzyme was dialyzed for 2 days against 10 mm SDS-polyacrylamide gel electrophoresis was Tris-HCl buffer, pH 7.2, from which metal ions performed by the method of Weber and Osborn had been removed by extraction with 0.2% dith (9). Samples of about 100 ƒÊ1 (50-100 ƒÊg as pro izone in carbon-tetrachloride. tein) were mixed with an equal volume of 20 mm Amino acid composition and sugar content: phosphate buffer, pH 7.0, containing 50%. glycerol, 2 % SDS, 0.1 % BPB, and 1 % 2-mercaptoethanol. 2 Potassium phosphate buffer was used throughout the After incubation at 70•Ž for I h, 100 ƒÊl aliquots present experiments, unless otherwise stated. J. Biochem. CREATININASE FROM Pseudomonas putida 1111 The amino acid composition of the enzyme was Photooxidation: A mixture of the enzyme and determined by the method of Spackman et ƒ¿l. (14) 0.01% Rose Bengal in 50 mm Tris-HCl buffer, with a Nippon Denshi JLC-6AH automatic amino pH 7.1, was placed at a distance of 20 cm from the acid analyzer. The contents of half-cystine and front lens of a 300W slide projector and illuminated tryptophan were determined by the method of at 15•Ž. Aliquots of the reaction mixture were Moore (15) and Goodwin and Morton (16), periodically withdrawn and the residual activities respectively. Sugar content of the enzyme was were assayed in the dark (20). measured by the phenol-sulfuric acid method (17) Cultivation of the Microorganism-Pseudo and expressed as glucose. monas putida C-83 was aerobically grown at 28•Ž Enzyme activity and protein concentration: for 16 h in a medium containing 5% soybean cake The enzyme activity was assayed by the following extract, 5% dextrin, 1% KH2PO4, 0.02% each of two methods. (A) Creatinine formed from creatine KCl and MgSO4.7H20, pH 7.0, and then inocu by the enzyme reaction was measured using the lated into 8 liters of a creatinine-enriched medium Jaffe reaction. The enzyme solution (0.1 ml) was (3) in a Miniferm MF-1 14 jar fermenter (NBS Co., added to 0.9 ml of 50 mm Tris-HCl buffer, pH 7.0, U.S.A.). The cells were aerobically grown at 28•Ž containing 0.1 M creatine at 37•Ž, and after incu for 12 h, harvested by centrifugation (7,000 rpm, bation for 10 min, a 0.1 ml aliquot of the reaction 20 min) and then washed twice with 50 mm phos mixture was withdrawn and mixed with 1 ml each phate buffer, pH 7.4 (buffer A). This buffer A of 1 N NaOH and 1% picric acid and 0.9 ml of was used throughout the purification of the native deionized water. The absorbance at 520 nm was enzyme. For the preparation of metal-free, inactive measured after 20 min against the blank, in which enzyme, on the other hand, buffer B (50 mM Tris- heat-denatured enzyme had been used. One unit HCl buffer, pH 7.1, containing 2.7 mM EDTA) of the activity was defined as the amount of en was used unless otherwise stated. zyme that converts I ƒÊmol of creatine to creatinine per min under the conditions used, and the activity RESULTS AND DISCUSSION units per ml of the original enzyme solution were calculated from the following equation: Preparation of Cell free Extract and Purifica tion of Creatininase-All the procedures were Units/ml =ƒ¢E520nm•~6.45•~dilution factor carried out in the cold.