The Breeding Of L-histidine High Productive Strain

In order to improve the production of L-histidine, solve the problem of producting L-histidine industrially. This paper studied on the breeding of L-histidine producing mutants starting from filtrating strains, then fermentation of L-histidine was optimized. The main research content and results was as follows:1. First, some measuring method of L-histidine was studied and compared. The conclusion was: the paper chromatography was simple, but only measured L-histidine qualitatively; Pauly colorimetric method was quick and good accuracy, furthermore, not required complex instrument, but it was influenced by many factors; HPLC had better accuracy and sensitivity than the two aforementioned method, but the operation was verbose, long time-consuming and had high cost. The method of paper chromatography being combined with Pauly colorimetric method and HPLC was used to measure L-histidine during the experimentation, according to experimental requirements.2. After determining the method, L-histidine production bacteria were screened from large collected strains. First, paper chromatography was used for qualitative analysis. Then Pauly colorimetric method was used continuously to screen the L-histidine production bacteria which were ensured by paper chromatography. At last, a strain (corynebacterium glutamicum) CG4-2 produced L-histidine 173 mg/L which was highest, this strain was served as original strain.3. The original strain CG4-2 was mutagenized by ultraviolet (UV), lithium chloride (LiCl) and diethyl sulfate (DES). The L-Histidine producing mutant strains which was endued with 6-mercaptopurine resistance, nutritional deficiencies and histidinase-less were obtained. The mutant CG4-2-11-57-40(6-MP~R, transketolase-, histidase-)could accumulate L-Histidine 2.18g/L ,which output was highest. This strain was passed down over six generations for fermentation. The conclusion was: The L-Histidine production was little changed. So the strain had genetic stability characteristic.4. The fermentation of L-histidine was optimized using single factor and orthogonal test in this study. It was considered that the optimal culture medium(g/L) was: glucose 80, ammonium sulfate 35, corn syrup 5, mono-potassium phosphate 1.5, magnesium sulfate 1, calcium carbonate 15; The best fermentation conditions were: the best temperature was 30°C, the initial pH 7.2, the volume of medium 20 mL/250 mL triangular flask. Vaccination was 6.7 percent, flask speed 180 r/min. The ideal strain age was 10 h and the fermentation cycle was 60 h. The production of L-histidine was raised to 4.36 g/L from 2.18 g/L.