Screening Of Strains Producing Hydantoinase And Their Enzyme Production Conditions

Hydantoinases are valuable enzymes for the production of optically pure D- and L-amino acids. The hydantoinase process is an economically attractive method for the production of many unnatural amino acids, which are components of potential Pharmaceuticals. As soon as these pharmaceuticals enter the commercial market there will be an augmented demand for these amino acids, which will in turn increase the importance of the hydantoinase process in the future.The screening and deteminant of microbial hydantoinase-producing, the fementationconditions of the B.cereus BP-6 were studied systematically, the following results were:Based on studies and comparisons among methods of screening microbial hydantoinase, a rapid, simple and efficient Screening Modle for microbial hydantoinase-producing was developed. More than thirty soil samples from all kinds of abundant nitrogen. An efficient and rapid plating method for determinant of microbial hydantoinase activity by observing the color-changing on the culture was adopted. This was preliminary screening and 62 strains obtained. After culturing in a triangle bottle on the shake and ninhydrin reaction is used to determine their hydantoinase activity and concentration of L-phenylalanine, and investigated the stereospecificity of obtained strains. This was secondary screening and 11 fine strains were selected, also detecting the enantiom excess of production using capillary electrophoresis. The highest activity of strain B.cereus BP-6 was 1.98U/g,ee was 0.73.Isolation and purification the strain BP-6, and through detection of its appearance, physiological and biochemical characteristics, growth characters, it was identified Bacillaceae, Bacillus, B.cereus according to Bergeysmanual of Determiutue Bacteriology(8th), and named B.cereus BP-6.Using B.cereus BP-6 abtained in lab, which can produce hydantoinase, the optimum composition of the fermentation medium was screened in individual factor.Experiment results showed that the best carbon source was glucose, the best nitrogenis peptone combined with beef extract. Ca2+ was the positive ion which had the best effect on enzyme synthesis. Benzylhydantoin was the best inducer. Next the fermentation medium was optimized using response surface analysis (RSA). The optimal medium was: glucose 2.0%, peptone 1.4%, beef extract 0.7%, NaCl 0.3%, KH2PO4 0.2%, CaCl2 0.02%, benzylhydantoin 0.2%. The optimum fermentation conditions as following: the optimum fermentation temperature was 32 , the optimum fermentation time was 16-18h, the optimum fermentation shaking frequency was 150rpm, the optimum fermentation pH was pH7.2. In doing so B.cereus BP-6 hydantoinase activity was 2.49U/g, enlarging 1.26 times.Finally the influence factors including temperature, pH, the quantity of hydantoinase of the conversion were studied in detail. The results showed that the optimal temperature of B.cereus BP-6 hydantoinase reaction was 38~40 , pH8.0~ 8. 5, adding organic solvent had no evident influence, the optimal concentration of substrate was 10g/L, the quantity of hydantoinase was 4 times than that of substrate, keeping reaction for 48h 31% benzylhydantoin was transformed to phenylalanine.