Protoplast Fusion of Alkaline Protease Producing Bacillus subtilis

Alkaline Protease를 생산하는 Bacillus subtilis의 원형질체 융합

To improve alkaline protease producing strain by protoplast fusion, a strain of Bacillus sp. was treated with $100{\mu}g/ml$ NTG (N-methyl-N'-itro-N-nitrosoguanidine) for 45 minutes and mutants of Bacillus subtilts $Arg^-,\;Try^-,\;His^-$ and $Ade^-$ were isolated. The frequency of protoplast formation was about 99%, when teas of exponential phase were treated with $200{\mu}g/ml$ lysozyme at $42^{\circ}C$ for $10{\sim}30$ minutes. In a regeneration medium containing 0.3M sodium succinate, 2.0% polyvinylpyrrolidone, 0.5% casamino acid, 10mM $MgCI_2$ and 20mM $CaCI_2$, regeneration frequency cf the isolated Bacillus subtilis strains was 25.2%. The fusion frequency between mutant was from $2.1{\times}10^{-5}$ to $8.1{\times}10^{-5}$ under optimum condition.

Alkaline protease를 생산하는 Bacillus subtilis를 $100{\mu}g/ml$ NTG 처리하여 $Arg^-,\;Try^-,\;His^-\;Ade^-$ 영양요구성주를 분리하였다. 원형질체 형성은 대수증식기의 균체를 lysozyme $200{\mu}g/ml$로 $42^{\circ}C$에서 $10{\sim}30$분간 처리하였을 때 약 99%였다. 재생배지에 0.3M sodium succinate, polyvinylpyrrolidone 2.0%, casamino acid 0.5%, $10mM\;MgCl_2$ 및 $20mM\;CaCl_2$를 첨가했을 때 재생율은 25.2% 였다. 원형질체 융합은 40%(w/v)의 PEG 6,000 용액을 상온에서 $1{\sim}5$분간 처리시 약 $2{\sim}8{\times}10^{-5}$의 융합빈도를 얻을 수 있었다.