Breeding Of L-methionine Producing Strains By Induced Mutations And Study On The Fermentative Conditions Of The Mutant

L-methionine is one of the human indispensable amino acid and it is extensive used to medicine > Food , as well as domestic nutrition industry and so on. The microbe enzymes produced L-Met has some merits, such as economical and environmental protection and so on, so it has industrial application prospectation. The Bacillus strain MV0073 was screened from the west highland of Sichuan province, which has L-5-thiomethylhydantoinase can catalyze D, L-5-thiomethylhydantoin to form L-Met, but the output of L-Met was low. Different means of mutagenesis was performed on MV0073 to breed L-Met overproducing strains, and the fermentative cultral condition of the strain was optimizedUltraviolet ray radiation as the mutagenesis factor, the log phase bacterium fluid of MV0073 was treated with the dose which of the lethal rate was 82. 5% and the positive mutant rate was 13. 7%, and directional isolated overproduction strains by the plate containing ethionine. The L-Met production of mutants had not increased obviously.Experiments verified that Leucin restrains the growth of MV0073, and L-Met can resume the strain growth, so it indicates that the medium containing Leucin can be used to breed L-Met overproducing strains. L-Met high producing strain M0361 was gained through three generations Leu-resistance mutantion. The L-Met production of M0361 was 1.8 times as high as that of MV0073.The M0361 was treated with nitrous acid , then mutants were isolated from the plate containing Leu, but the L-Met production of mutants had not increased obviously.The protoplasts of M0361 was prepared by enzymolysis under the condition that the M0361 cell were incubated with 0. 3 mg/mL Lysozyme at 37C treatment for 2. 5~3h. The protoplasts formation frequency was 86. 2%. The protoplasts was irradiated by UV, and the mutants of L-Met overproduction were isolated from the hypertonic plate containing Fluorouracil, which was the substrate analogue of D, L-5-thiomethylhydantoin. From the substrate analogue resistance breeding,it gained the L-Met overproduction strain M0537.Through the multiple resistance breeding, the mutant M0658 wasobtained. The L-Met production of M0658 was 1. 09g/L, as high as 3 times that of MV0073 and the high level of L- methionine in the culture liquid was sustained during the late stage of culture. Moreover the L-methionine-overproducing capacity still maintained after six generations.The single-factor experiments and the orthogonal experiments were adopted to opitimize the culture condition to improve the L-Met production of M0658. Those experiments ascertained that the opitimized cluture medium consisted of 1% saccharose, 0. 1% NH4NO3 0. 3% K2HPO4, 0. 2%KH2PO4 0.5g/L D, L-5-thiomethylhydantoin, 1% compounds of metal ion, 2% compounds of vitamin, initial pH 8.0-9.0 , and the optimum growth temperature was 35C -37C .Under these conditions, the L-met production of strain M0658 was 1.31 g/L, and it was 1.22 times of the L-met production of M0658 under original conditions.