Breeding Efficient Mutants With Constitutive Hydantoinases Expression By Ion Beam Implantation

Chiral amino acids are elements of semi-synthetic antibiotics, new herbicide, insecticide and bio-active peptides in chemosynthesis. They will have wider application prospects. Hydantoinase process is an important method in the production of chiral amino acids. Hydantoinases are inducible enzymes. The cost and complexity in industrial production of amino acids by hydantoinase process are dramatically increased with addition of inducers. Therefore, the selection of strains with constitutive hydantoinases expression is very significant for application and can provide model strains for research on the regulation of hydantoinases expression. Ion beam implantation mutation is a new technology of breeding strains raised in recent two decades. Due to the higher mutation rate and wider mutational spectrum, it has been widely used in breeding of microbes.Bacillus fordii MH602 isolated from soil by our group was used as a parent strain in this thesis. It is a thermophilic strain producing typical inducible hydantoinase. Ion beam implantation mutation was used to breed mutant strains with efficient constitutive hydantoinases expression.During the mutation process, microbes have to be put under a dry condition without nutrient for a long time. It results in a high death rate of the cells and is disadvantage to the mutation. This thesis firstly resolved this problem. Several oligosaccharides were used as protectors for the exponential-phase cells of MH602 in the mutating process. The best protector was trehalose with the optimal concentration of 2.0%. Negative effect of trehalose on ion beam mutation was not found. According to the survival rate curve, 1.8×1016 ions/cm2 N+ with 10 KeV energy was chosen as a suitable mutation factor. This thesis enriched the protecting methods in the ion beam implantation mutation. These results can help the strains with poor ability resist dry conditions in the ion beam mutation.Benzyl hydantoin and 5-fluorouracil were firstly used as pressures in the...