Studies On Producing L-Tryptophan By Direct Fermentation

According to the theory of metabolic control fermentation and metabolic network analysis, the dissertation focuses on the producing L-tryptophan by direct fermentation and its separation and purification. The main research contents and results are as follows:(1) The quantitative determination conditions of improved colorimetry were decided. The maximum absorption was at 600nm. The linear scale is 0～100μg/mL, and the linear equation of standard curve is C=23.56A, C represents tryptophan concentration and A represents absorbency. Index of correlation R² is 0.9972. The determination of L-tryptophan concentration with improved colorimetry is more accurate and faster than with classical colorimetry.(2) The batch fermentation conditions of strain TQ2223 in shake flask were studied in this dissertation. The seed medium was optimized by orthogonal design experiment and the seed culture conditions were also studied. The fermentation medium was optimized by pattern recognition and the fermentation condition was also studied. Under the optimum condition, strain TQ2223 could produce L-tryptophan 8.6g/L by flask-shaking batch fermentation, which is higher by 25.4% than under the original conditions.(3) Pathway analysis for production of tryptophan from glucose by Corynebacterium glutamicum TQ2223 in steady state was conducted, theoretical yield and flux distribution for optimal pathways was determined;The metabolic network was proposed for L-tryptophan producing strain. Using this model, the metabolic flux distribution during the middle and late period were calculated by linear program of MATLAB software. Through compared practical with calculated metabolic flux distribution, the strategies of reducing NH₄⁺ concentration, increasing PO₄^3- concentration and improving DO were indicated which provide theory guide for the fermentation process. Otherwise, the strategy of genetic engineering strain was put forward.(4) Based on the metabolic network analysis, the batch fermentation was performed with strain TQ2223 in 5-liter fermentor. The effect of NH₄⁺ and PO₄^3- concentration on L-trp fermentation was studied. 62mL NH₃.H₂O(NH₃.H₂O was added at 16h, 28h, 40h and 52h, 15.5mL each time) and 10g/L KH₂PO₄ were propitious to accumulation of L-trp. According to the change of DO in 5L fermentor and dynamics analysis during fermentation process, two-stage oxygen supply (0～ 36h 30%;after 36h 20%) was provided and it has been experimentally verified that thestrain could produce L-tryptophan 10.13g/L after fermentation for 64 hours. The L-tryptophan batch fermentation kinetics was studied by neural network based on the experimental data from 5-liter fermentor's batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.(5) Fed-batch fermentation with TQ2223 in shake flask was studied. The optimum initial glucose and amino sulfrate concentration and the amount of them were determined in shake flask fed-batch fermentation. The effect of feeding nutrition and precursor on fed-batch fermentation in shake flask was also studied. Based on the fed-batch fermentation in shake flask and the batch fermentation, fed-batch fermentation in 5-liter fermentor was performed. The optimum strategy of feeding glucose, nutrition and precursor were determined. Because of the technology of low glucose, and feeding bean cake hydrolysis liquid, production of L-tryptophan reached I3.4g/L after fermentation for 72 hours. The production of L-tryptophan reached 15.3g/L by adding aminobenzoic acid.(6) The optimum conditions of membrane filtration, decolour and separation of L-tryptophan were determined. The plot test was conducted in Tian an medicine corporation according to the conditions determined. The total recoveris rate was 63.4%, the extraction rate was 98% and the optical rotation was-30.5-32.5° , which comply with the require of Chinese Pharmacopeia year 2000 edition.