Breeding Of L-Histidine Producer And Optimization Of Its Fermentation Conditions

According to the theory of metabolic control fermentation, the paper focuses on the breeding of L-Histidine hyper-producer WH1263, the suitable fermentation condition of shaking flask, the effect of calcium gluconate on L-Histidine fermentation. The main research contents and results are as follows:The methods of breeding L-Histidine producer from Corynebacterium glutamicum were decided based on the relationship with biosynthesis process of nucleotide and fed-back regulatory mechanism of L-Histidine biosynthesis. The L-Histidine producer was derived from the original strain Corynebacterium glutamicum CLW0506 by stepwise mutagenic treatments with ultraviolet (UV), N-methyl-N-nitro-N-nitrosoguanidine (NTG) and diethyl sulfate (DES). The objective mutant strain which absent AMP deaminase, IMP synthetase, IMP dehydrogenase and histidinase was obtained. The L-Histidine producing strain was also endued with other two genetic markers: 6-MPR, 5-FUR. The final mutant WH1263 could accumulate L-Histidine 8.50g/L which was about as 160% as the quantity produced by the parental strain.The WH1263 strain was passed down over ten generations for 72h fermentation the acid production was little changed. That proved the strain with a genetic stability characteristic.Using single factor, orthogonal experiment and response surface methods, Come to the best source of carbon is glucose, nitrogen source is ammonium sulfate, the best medium are (g/L): glucose 99.00, (NH4)2SO4 29.30, K2HPO4·3H2O 1.25, MgSO4·7H2O 0.50, CaCO3 20.00. The best concentrations of Zn2+, Mg2+, Fe2+ and Mn2+ are 2.2mmol/L, 1.8mmol/L, 1.2mmol/L and 0.27mmol/L. The initial pH7.0, vaccination of 6.7 percent, with volume of 15mL/250mL triangular flask, temperature 30°C, flask speed 100/min. The fermentation period is 72h. The mutant strain could accumulate L-Histidine 10.30g/L.By adding calcium gluconate to increase gluconic acid kinase activity, leading to the improvement of hexose monophosphate(HMP) shunt flux and its specific activity greatly. The mutant strain could accumulate L-Histidine 10.50g/L.