Induction of a High Vitamin B12 Productive NattoStrain through Protoplast Fusion

Abstract

To breed a high vitamin B_l2 producible natto strain, protoplast fusion was done betweenBacillus natto, Takahashi No.2 (Arg-mutant), and a vitamin B₁₂ productive strain, Bacillus megaterium IAM 1166 (Try-mutant). Lysozyme of 250μg/ml and 500μg/ml wasadopted for the protoplast preparation of B. natto Takahashi No.2 and B. megaterium IAM1166, respectively, with 10% sucrose at 42°C for 30min. Protoplast fusion was carried outwith 40% polyethyleneglycol 6000, and the regeneration was performed on SucroseGlutamateminimal medium containing 10% sucrose and 3% polyvinylpyrolidon. Preservation of thestringiness and vitamin B₁₂ productivity of the protoplast strains were deeply affected by theconcentrations of lysozyme and sucrose. Three fusants which were capable of producingstringiness and vitamin B₁₂ were isolated on SG medium, and the strong stringiness on asteamed soybean by them were also observed in a similar manner as B. natto.