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To improve Bacillus strains producing biopolymer, conditions for protoplast formation and regeneration were investigated in biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. Bacillus subtilis K-1 mutant (SM-2) and Bacillus coagulans mutants (CM-12) were marked auxotrophic and antibiotics-resistant (SM-2) and an antibiotics-resistant mutants, respectively. To formate protoplasts derived from the mutants, conditions were established as follows. For B. subtilis mutant SM-2, its culture in mid-logarithmic phase was added with penicillin G (1.0 unit/㎖) and further reacted for 1.5 hr. Cells were collected and then treated in lysis fluid (pH 7.0) containing 0.4 M sucrose and lysozyme (25 ㎍/㎖) for 40 min at 37℃. Protoplast formation was very successful (99.6%) and the ratio of cell wall regeneration was 2.4%. For Bacillus coagulans mutant CM-12, its mid-logarithmic phase culture was treated with penicillin G (0.3 unit/㎖) and glycine (0.5%) for lhr. Cells were collected and then resuspended in lysis buffer (pH 7.0) containing 0.6 M lactose and lysozyme (300 ㎍/㎖) for 30 min at 37℃. Protoplast formation was also successful (90.8%) and cell wall regeneration ratio was similar to SM-2 (2.2%). To improve regeneration frequency, regeneration medium was obtained as followed condition,. Cell wall regeneration was improved 2-4 folds with 5.1% for B. subtilis SM-2 and 10.3% for B. coagulans CM-12 when protoplasts mixed with soft top agar(0.4%) was overlaid onto trypticase soy broth medium containing 0.4 M sucrose, 0.7% casamino acid, 1% PVP, 25 mM MgCl₂, 25 mM CaCl₂ and 1.5% agar.
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