| In nowadays, microbial productions of L(+)-tartaric acid have superceded chemical synthesize due to ecological and economical advantages. In the process of production, cis-epoxysuccinate (ES) was converted into L(+)-tartaric acid through microbial cells containing cis-epoxysuccinate hydrolase (ESH). From application angle, we obtained two high producing straits through screening, and the fermentation characteristics of aquired strait was analyzed in this paper, based on which, the culture medium was optimized. Furthermore, we built a faster method for measuring ESH activity. The research in this paper mainly experimentized in the following aspects:1. The screening of Nocardia tartaricans strains with higher activitiesAfter the strains from the factory were separated and purified, pick 50 well- grown strains and then determine their abilities of growing and producing ESH, so that we could obtain strains with higher activities. The strains obtained from screening were subcultured and then rescreened. After several screening and the stability testing, 2 strains with the activity 21-30% higher than original strain were obtained.2. The fermentation process analysis of Nocardia tartaricansThe changes of the principal controls parameters in current fermentation process, such as carbon, nitrogen, pH and cell mass, enzyme activities, specific activities was investigated. It found that glucose was utilized as a priority by Nocardia tartaricans when both glucose and propylene glycol were contained in the fermentation culture medium. In the course of fermentation, pH decreased as Nocardia tartaricans using glucose and coproduct organic acid accumulating. When glucose in the culture was used up, following with the addition of ES, pH began to ascensused. The starting and major phase of ESH production was situated in the period of logarithmic phase. L(+)-tartaric acid was not utilized in the entire fermentation period, about 40h. The cell concentration of Nocardia tartaricans could reach 30 g/L, enzyme activity 515 U/g.3.The establishment of a faster menthod for measuring ESH activity On the basis of ultrasonication cells whose permeability was increased, the thesis conducted several groups of experiments with the orthogonal experimental design, and discussed the effect of the above-mentioned parameters on the ESH's activity and concluded an extraordinarily faster method for measuring ESH activity, which was to put 1 g wet cells into 30 mL 0.2 mol/L ES aqueous solution under the condition of 37℃for 10 min to biotransform and then to measure the concentration of L(+)-Tartaric acid with HPLC. This method possesses the advantages of fast speed, high accuracy and small quantities of cells.4.The optimization of Nocardia tartaricans culture mediumType and concentration of carbon,concentration and addition time of ES and concentration of tartaric acid were analysed, with the results that glucose was the best carbon enhancing biomass of cells, and sucrose was the best when cell activity is considered, and the optimal concentrations both are 5 g/L; ES did not stunt the growth of Nocardia tartaricans if its concentration was below 40 g/L, and the optimal concentration is 20 g/L; The activity of cells could be raised when ES was added in the logarithmic growth time of microbial cell; Tartaric acid could postponed the utilization rate of ES, with the result of ehancing expression capability of ESH. With the medium optimized, the total activity of cells could be 22.8% more than that from the original medium. |
