N ~ + To Inject The Corynebacterium Crenatum The As1.998 Protoplasts Overproducing Strain L-ile,

In this paper, ion beam implanting technology and protoplast mutation technique were applied into original strain improvement of Corynebacterium crenatum AS1.998 for screening high yielding strain of L-Isoleucine. Then the compositions of medium and the conditions in fermentation were studied. The main research contents and results are as follows:1. The conditions of protoplast formation and regeneration of Isoleucine-producing strains Corynebacterium crenatum AS1.998 was studied. The effects of different factors, such as penicillin, lysozyme dosage, temperature of lysozyme hydrolysis, time of lysozyme hydrolysis, rotate speed on protoplast formation. In the end, the conditions of protolast formation and regeneration of Corynebacterium crenatum AS1.998 were determined. Under the optimum conditions, the protoplast formation rate is above 98.53% and the protoplast regeneration rate is about 25.6%～42.2%.2. The method of growth circle bioassay for screening high yielding strain was optimized. The effects of different factors were studied. The accuracy and sensitivity of growth circle bioassay could be improved.3. Paper chromatography-spectrophotometry which was applied into determining the L-Isoleucine in broth was improved. In traditional process, separation and coloration are independent and it usually make the experiment in trouble. In this paper, we made the separation and coloration in the same process. Then we use the method to measure the content of L-Isoleucine in broth .The result showed that the method is highly accurate and precise in the determination of L-Isoleucine , and it is suitable for the screening of strains producing L-Isoleucine..4. The L-Isoleucine producer was derived from the original strain Corynebacterium crenatum AS1.998 protoplast by low energy N~+ ion beam implanting. The Using the growth circle bioassay and the improved paper chromatography-spectrophotometry, in the end, we have isolated a high-yield strain DN-2] which the productivity is 43.66% higher than the original strain's in the rotation-flask experiments.5. The effects of seed medium compositions and fermention medium compositions on the L-Isoleucine production in flask were considered, using single factor analysis method and orthogonality experiments, we get the optimal medium of DN-21. In the optimal medium, the L-isoleucine yield of DN-21 was 8.93g/L.