Protoplast Formation and Regeneration of Bacillus strains producing biopolymer

Biopolymer 생산성 Bacillus속 균주의 원형질체 형성과 재생

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/ml) 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 $25\;{\mu}g/ml$ for 40 min at $37^{\circ}$. 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/ml) and glycine (0.5%) for 1hr. Cells were collected and then resuspended in lysis buffer (pH 7.0) containing 0.6 M lactose and lysozyme $(300\;{\mu}g/ml)$ for 30 min at $37^{\circ}$. 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 $MgCl_2,\;25\;mM\;CaCl_₂$ and 1.5% agar.

Biopolymer를 생산하는 Bacillus속의 균주개량의 목적으로 biopolymer를 생산하는 균주인 Bacillus subtilis K-1과 유당 이용능이 있는 Bacillus coagulans의 원형질체 형성과 재생에 관하여 조사하였다. 영양요구성과 항생제 내성의 marker가 부여된 두 변이주의 원형질체 형성조건에서 Bacillus subtilis mutant SM-2의 경우, 대수기 중기에 penicillin G(1.0 unit/ml)를 첨가한 다음 1.5시간 반응 후 삼투압 안정제로서 0.4 M sucrose와 $25\;{\mu}g/ml$의 lysozyme이 함유된 lysis fluid(LF, pH 7.0)내에서 $37^{\circ}$, 40분간 반응시켰을 때, 원형질체 형성율은 99.6%, 세포벽 재생율 2.4%였다. Bacillus coagulans mutant CM-12의 경우 대수기 중기에 penicillin G 0.3 unit/ml와 glycine 0.5%를 혼합첨가하고 1시간 반응시킨 후 삼투압 안정제로서 0.6 M lactose와 $300\;{\mu}g/ml$의 lysozyme이 함유된 LF(pH 7.0)내에서 $37^{\circ}$, 30분간 재생시켰을 때, 원형질체 형성율 90.8%, 세포벽 재생율 2.2%였다. 세포벽 재생효율을 높이기 위한 재생배지는 trypticase soy broth(TSB)에 0.4 M sucrose, 0.7% casamino acd, 1% PVP, 25 mM $CaCI_2$, 25 mM $MgCI_2$, 1.5% agar가 함유된 배지에 0.4% soft agar로서 중층 했을 때 Bacillus subtilis SM-2의 재생율은 5.1%, Bacillus coagulans CM-12의 재생율은 10.3%로 $2{\sim}4$배 가량 향상 되었다.