Breeding Of L-Glutamine Hyper-Producer And Optimization Of Its Fermentation

The dissertation firstly investigated the method of qualitative and quantitative determination of L-glutamine in broth. Then according to the metabolic control principles, the study investigated on the breeding of L-glutamine-producing strain and the suitable fermentation condition of shaking flask and so on. The main research contents and results were as follows:(1) The concentrations of L-glutamine in broth were determined with paper chro- matography and spectrophotometer. Then it was determined the composition of exhibi- tion layer system in paper chromatography. The best ratio of the exhibition layer agent was n-propyl alcohol: ammonia (V/V) =5:2. Under this condition, the glutamine and the glutamic acid can be separated very well. It was also found that the best wavelength at 510 nm of the 721 spectrophotometer, and the best elution time was 30min. Furthermore, it was carried out the repeated test to validate the precision and value recovery rate of the sample. It was found that using the method to analyse L-glutamine had better repeatability and recovery rate. These results signified that the method had higher reliability, which provided favorable base for the further study.(2) The L-glutamine-producing strain was derived from the original strain (Coryne- bacterium acetoacidophilum) YG13 (MSOR) and mutated by the methods of ultraviolet (UV), diethyl sulfate(DES) and N-methyl-N'-nitro-N-nitrosoguanidine (NTG). Then the plate screening method using analogues: sulfaguanidine (SG), high concentration (NH4)2SO4, meanwhile, succinate and L-glutamic acid as sole carbon source was used a strain of YL012 (MSOR, SGR, (NH4)2SO4R, L-GluG, SucG) which could accumulate L-glutamine was obtained. The concentration of L-glutamine in the culture was enhanced from 18.7 g/L to 34.3 g/L by using this strain.(3) The optimization of medium component and fermentation conditions for YL012 was conducted. The optimum seed medium contains(g/L): glucose 25, (NH4)2SO4 5, corn steep liquor 40, KH2PO4 1, MgSO4·7H2O 0.5, CaCO3 20, pH 7.0; and the optimum medium volume was the 40 mL/250 mL, culturing at 30℃on reciproca- ting shaker, with shaking at 100r/min,inoculated time 9 h. The optimum fermentation medium contains (g/L): glucose 123.4, (NH4)2SO4 47.8, corn steep liquor 4.3, KH2PO4 1.0, MgSO4·7H2O 0.6, MnSO4·4H2O 0.005, FeSO4·7H2O 0.01, ZnSO4·7H2O 0.005, CaCO3 30. The optimum fermentation conditions were the 15 mL/250 mL, original pH 7.5, seed volume 8 %, at 30℃with shaking at 100 r/min for 72 h. In the optimized medium the concentrations of L-glutamine in the culture was enhanced up to 40.6 g/L.