Screeming Of D-pantolactone Hydrolase Producing Strains And Optimizing Of Technology Conditions

The thesis started with forty-three kinds of Streptomyces and five kinds of yeast collected. We preliminary screened the strains through an intuitive, simple, indicator color-circle method. We obtained eight strains which maybe produce D-pantolactone hydrolase according to determine whether the strain produce enzyme and the ability of enzyme production by observing the diameter ratio of color-circle and colony. This eight strains were respectively cultivated in liquid medium, then used the myceliums obtained as the enzyme to hydrolyze 10% DL-pantolactone solution in a certain condition. The specific rotation of mixture reaction solution were measured by the polarimeter. The specific rotation is positive value and the larger of positive value, the stronger the ability of the hydrolysis. In order to further identify the product and the conversion rate, we measured and analyzed the mixture reaction solution by the HPLC. The results showed that six strains can produce D-pantolactone hydrolase. We ultimately determined a strain as the starting strain who had the higher activity and good specificity by re-screening. After a initial identification, we confirmed that this strain was Fusarium oxysporum. After mutation breeding, a more productive mutant fungus was abtained: Fusarium oxysporum CZ-437. Through six generation experiments, we found that Fusarium oxysporum CZ-437 not only Highly produced enzyme but also had good stability in producing enzyme.We studied the conditions for enzyme formation of Fusarium oxysporum CZ-437 including the nutritional composition of the fermentation medium and the optimize fermentation conditions. According the research, we determined the suitable carbon source of fermentation medium were glucose and glycerol; the suitable nitrogen source of fermentation medium were peptone, corn steep liquor and yeast extract. The best inorganic salt was CaCO₃(5g/L). The Optimal fermentation conditions are as follows: The optimum temperature: 28℃; initial pH for enzyme formation: pH7.0; Liquid volume: 50mL/250mL; inoculum size: 8%; speed: 200r/min. When the organism was cultured in the shake flask under the optimum conditions for 72h, about 7.08g dry cells/L and 7.03U/L were obtained.Firstly, we obtained the myceliums of Fusarium oxysporum CZ-437 in optimum fermentation conditions. Secondly, we studied the conditions for enzyme hydrolyze of Fusarium oxysporum CZ-437. According the research, we determined the suitable conditions for enzyme hydrolyze: substrate concentration: 20%; wet cell concentration: 14%; speed: 200r/min; hydrolysis temperature: 28℃. During the hydrolysis process, pH should be Regularly adjust to 7.5 with NH3. In optimize condition, after the substrate hydrolyzed 16h, the specific rotation of mixture reaction solution can reach to +3.7°. Substrate hydrolysis rate was more than 30%.