Identification Of An Arginine Deiminase-producing Strain And Purification Of Arginine Deiminase

Objective:As we known, many metabolites originated from Ocean microorganisms have many potential bioactive functions. So, development of these microorganisms attracts more and more research attention. Arginine deiminase (ADI) has been reported that can inhibit proliferation of different malignant cells. Many microorganisms can produce ADI, however, clinical application of ADI in hepatocellular carcinoma and melanomas was mainly extracted from mycoplasma. Its safety has been disputed. Hence, an increasing research have been focused on development of the ADI source. Base on the location advantage of South Ocean, in this study, a marine bacterial strain producing arginine deiminase (ADI) were selected from ocean ship rust, then identified and characterized. Its optimum fermentation conditions were determined. After purification, properties of ADI were tested. This study will provide more resource to produce bioactive ADI.Methods:Single colony was obtained by dilution method step by step. Selective medium with arginine was applied to separate colonies which producing ADI. The strain was identified on the basis of its morphological, physiological, biochemical characterization and the gene sequence of16S rDNA. Then a series of tests, including single factor experiments of carbon source, nitrogen source, arginine and their concentrations, inoculation amount, liquid volume, incubation temperature, initial pH, shaker speed, fermentation time and orthogonal experiments, were carried out to optimize the fermentation conditions of producing ADI. Next, enzyme activity of ADI was determined through detected the content of citrulline by Fearon reaction. ADI enzyme was purified gradually by salting out, dialysis, ion exchange chromatography and gel chromatography and determined by SDS-PAGE on its molecular weight. Then, the optimal temperature, optimal pH and Km of ADI reaction were detected by Fearon action, indirectly.Results:After inoculating24h, the color of the medium containing with arginine converts from yellow to red. The results of oxidase, arginine dihydrolase, denitrification and gelatin liquefaction were exhibited positive, while amylohydrolysis and PHB accumulation appear negative. The strain could not produce pyocyanin or fluorescent. At the same time, the strain can been observed at the temperature of41?, while not at4?. Taken together, it is suggested that the physiological and biochemical characterization of the strain were in accord with Pseudomonas alcaligenes. Moreover,16S rDNA gene sequence and phylogenetic analysis displayed the strain was belong to Pseudomonas alcaligenes. Condition Optimization assay of fermenting ADI enzyme reveal that sucrose and peptone were the optimal carbon and nitrogen source, and the optimum concentration were1Og/L and8g/L, respectively. And the optimum conditions were as follow:inoculating amount4%, liquid volume30%, temperature33?, initial pH8.5, shaker speed170r/min and fermentation time28h. As for the obtained ADI is concerned, the specific enzyme activity reaches11.80U/mg and the molecular weight is about45kDa. The optimal reaction temperature, pH and Km of this ADI were70?,7and0.459mmol/L, respectively.Conclusion:In our study, a novel strain, which was determined can produce ADI, was discovered from a special environment of ocean ship rust and belong to Pseudomonas alcaligenes on the classification. Under the optimal fermentation conditions, the enzyme activity reaches the highest point and the total amount of424.43U. During the purification, both of specific activity and purification fold of ADI were exhibited arose.