Abstract
To develop cellulase and xylanase-producing strain by protoplast fusion, alkalophilic Bacillus sp. F204 and K17 were treated with NTG(N-methyl-N'-nitro-N-nitrosoguanidine) and isolated anti-biotics resistant strains of S20 (Km$^r$ , Cm$^r$) and G70 (Str$^r$). The frequency of protoplast formation was about 95% when cells of mid-log phase were treated with 200$\mu\textrm{g}$/ml Iysozyme at 37$^{\circ}C$ for 30-45 minutes. Under addition of 0.4-0.5M sodium succinate, 0.5% casamino acid, 1.5% polyvinylpyrrolidone, 25mM MgC1$_2$ and 50mM CaC1$_2$ to the regeneration medium, the regeneration frequency of Bacillus sp. F204 and K17 was 24.9% and 26.2%, respectively. The fusion frequency was 6.6$\times$10$^{-6}$ in the presence of 30% polyethylene glycol 6000 containing 50mM $Ca^{++}$ at 45$^{\circ}C$ for 5 minutes. Cellulase complex and xylanase activities of fusant were compared with parental strains.
호알칼리성 Bacillus sp. F204와 Bacillus sp. K 17간에 원형질체 융합에 의한 cellulase와 xylanase를 동시에 생산하는 균주를 개발하기 위하여 두 균주에 500 $\mu\textrm{g}$/ml NTG를 처리한 후 약제내성변이주 인 S20(Km$^r$, Cm$^r$과 G70(Str$^r$)을 분리하였다. 원형질체 형성율은 균체를 대수증식기 중기까지 배양하여 200$\mu\textrm{g}$/m1 Iysozyme으로 37$^{\circ}C$에서 30-45분간 처리하였을 때 약 95%였다. 재생배지에 0.4-0.5M sodium succinate, 0.5% casamino acid, 1.5% polvinylpyrrolidone, 25mM MgC1$_2$및 50mM CaC1$_2$를 첨가하므로 Bacillus sp. F204와 Bacillus sp. K17의 재생율은 각각 24.9%, 26.2%였다. 50mM $Ca^{++}$을 첨가한 30%의 PEG 6,000용액을 45$^{\circ}C$에서 5분간 융합시킴으로써 6.2$\times$$10^{-6}$ 빈도로 융합주를 얻었다. cellulase, xylanase 및 avicelase를 생산하는 융합주는 모균주와 비교하였다.