Studies On The Isolation Of A Creatininase-producing Bacterium And The Characteristics Of The Creatininase

Creatinine is an important product of human metabolism, which is excreted by renal metabolism. In clinical diagnosis, the concentration of creatinine in serum is an important parameter for the evaluation of kidney function. At present the most commonly used assay of creatinine is the Jaffe reaction, which is based on the chemical reaction. However, this reaction is subject to a lot of interferences and lacks specificity. So that it is expected to develop the enzymatic determination methods to replace the Jaffe reaction in order to increase the specificity.In this study, a strain of bacteria capable of producing creatininase was isolated and identified as Arthrobacter sp.. The conditions for producing creatininase, the methods of purification and the characteristics of creatininase were studied. The results were reported as follows:1. The isolation and identification of the creatininase-producing strain: Seven strains of bacteria capable of producing creatininase were isolated from soil, which were strain 42-1,42-2,30-1, An, SAO-C, SAO-I, SAO-S. The creatininase productivities from these seven strains were studied and the enzyme assay showed that strain 42-1 produced higher yield of creatininase that was more heat-resistant than that of others. Considering of these facts, strain 42-1 was selected as the best creatininase-producing strain for further study. Based on the morphological, physiological and biochemical characteristics studies and the result of sequences alignment of 16S rRNA, strain 42-1 was identified as Arthrobacter sp..2. The fermentation conditions: Based on the studies of conditions for producing creatininase, the best fermentation conditions were established. The composition of fermentation medium was: creatine 4g, yeast extract 7.2g, corn-steep liquor 4.8g, glucose 10g, K2HPO4 2g, KH2PO43H2O 0.5g, MgSO47H2O 0.1g, KCl 0.005g, MnCl2 0.1g, H2O 1000mL, pH 7.5-which was added 15 ml into a 100 ml flask and incubated on a shaker of 120 rpm at 20℃ for 36h.3. The purification of creatininase: The specific activity of a crude, cell-free extract of creatininase was increased 145-fold by a series of purification stepsincluding heat treatment at 55℃, ammonium sulfate precipitation, DEAE-Cellulose ion-exchange, Toyopearl HW-65C hydrophobic chromatography. The purified enzyme was homogeneous as judged by polyacrylamide gel electroph-oresis (PAGE). The subunit molecular mass was estimated to be 33 700Da by SDS-PAGE.4. The characteristics of creatininase: The optimum pH for creatininase was 7.5 and the optimal temperature was 60℃. Creatininase was stable between pH 6.0-9.0 and at the temperature below 60℃. The Km values for creatinine and creatine were 21.14mmol/L and 40.8mmol/L, respectively. The enzyme was markedly inactivated by incubation with Immol/L of Hg2+, Ag2+, Li+, Cu2+ and 20mmol/L of 1,11-Phananthroline. Activation of creatininase activity was observed with Immol/L of Co2+ and Mn2+. 20mmol/L of NaN3, and 0.1% of Tween 20 and Tween 80 almost had no effect on creatininase activity.