Fermentation, Purification And Characteristics Of Creatinase From An Isolated Bacterium

The paper deals with the isolation and identification of a creatinase-producing bacterium from soil, the conditions for production of creatinase, purification methods of creatinase and the purified enzyme characteristics. The results were reported as follows: 1 Isolation and identification of stain producing creatinase Seven strains of bacteria (D-BK A1K EK F3^ WBK WHK WB2) which could produce creatinase were isolated from soil. Compared the temperature stability, stain WB1 was selected for further study. It was identified and temporarily named as Pseudomonas stutieri based on its morphological and physiological characteristics and G+C Mol % of DNA.2 Culture conditions of stain WB1 for producing creatinase The optimal conditions for production of creatinase from strain WB1 were as follows. The pH and temperature were 7.5-8.0 and 30*C. The best nitrogen sources were corn liquor and yeast extract, the ratio of them was 2:3 and the concentration of nitrogen sources is 1.6%. When other carbon sources were added to the medium, they can promote the stain to produce creatinase stably. The optimal volume of medium in 100ml flask was 15ml. It is the optimal time for subjecting creatine to the medium when cultured to 12h and the concentration of creatine was 0.75%. Creatine, sarcosine and choline chloride could induce the creatinase production and creatine was the optimal inducer, but creatinine and urea could not induce the creatinase production.3 Purification of creatinase The process of creatinase purification was performed as follows: First the enzyme was completely precipitated in the range of 40-80% of saturation with ammonia sulfate fraction precipitation. Then enzyme was purified with a DEAE-Cellulose(5.5X50cm) column, a Toyopearl HW-65 (5.5 X 50cm) column and a Sephadex G-200(5.5 X 80cm) column. Finally , the enzyme was purified for 10 folds with the recovery of 17.4%. PAGE showed a single band for the purified creatinase. By SDS-PAGE, the molecular weight of the enzyme was estimated to be 48,OOODa. When the enzyme incubated in the buffer of 0.1M, pH 7.5 HEPESNa containing 0.2M MgCl and 30% polyethylene glycol 400 at room temperature, crystals appeared in 60 days.4 The enzyme was most active at pH 7.5-8.0, and was stable in the range of pH6.0~8.5 at 25 癈 for 22h. The enzyme was most active at 30-35 "C, and was stable below 45'C. The Km of creatinase was 24.6mM at 37"C with the creatine as substrate. Cu2+, Hg2+ and Ag+ could completely inhibit the enzyme activity , and Co2+ ,Zn2*could markedly inhibit it, but Li+, Mn2+, Ca2+, Pb2+, Ba2+, Fe2+, Fe3+, Mg2+ and Cr2* did not affected the activity. EDTA, o-phenanthroline and NaNa did not inhibit the activity at all. Tween 20 had strong inhibitory effect to the enzyme activity, and Tween 80 and SDS had no such function.