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This study was conducted to breed a high vitamin B_(12) producer by the fusion of protoplasts between Bacillus natto and Bacillus megaterium. Auxotrophic mutants of Bacillus natto SH-34 (thr-try rif^r) and Bacillus megaterium BK-13 (arg^- ade lys^- str^r) which showed high protease activity and production of vitamin B_(12), respectively, were isolated for the fusion experiment. Protoplasts were induced by incubating the cells with lysis solution containing 500 μg/ml lysozyme, and the ratio of protoplast and regeneration formation were ranged from 99% and 67%, respectively. Fusion frequencies of fusants between Bacillus natto SH-34 and Bacillus megaterium BK-13 were appeared in the ranges of 1.0X 10^(-5) under the treatment of 30% PEG 6000 containing 3% PVP. The fusant, MNF-72 showed the highest product yield of 7.85 μg/g-cell vitamin B_(12) in production medium. For the improvement of productivity, the immobilization of fusants with sodium alginate was carried out. In batch and continuous fermentation systems, the productivity were determined to be 0.58 μg/ml.hr and 0.80 μg/ml.hr vitamin B_(12) under optimum condition, respectivity.
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