Protoplast Formation and Regeneration of Thermophilic Clostridium thermocellum and Clostridium thermohydrosulfuricum

고온성 Clostridium thermocellum과 Clostridium thermohydrosulfuricum의 원형질체 형성 및 재생

The conditions for protoplasts formation and regeneration of thermophilic anaerobic C. thermocellum and C. thermohydrosulfuricum were determined under the anaerobic growth conditions. The cells of C. thermocellum in initial exponential growth phase were identified to be the most suited for protoplast formation. The optimal conditions for protoplast formation were found to be at $37^{\circ}C$ for 2 hours with 0.5 mg/ml of lysozyme in TMG buffer (pH7.5). On the other hand, C. thermohydro-sulfuricum grown in the same medium but excluding glycine was optimally protoplasted at the same conditions but with 0.2 mg/ml of lysozyme. The protoplasts of both strains only subjected to lysozyme treatment of the short time were satisfactorily regenerated after 7-10 days incubation at $60^{\circ}C$ in regeneration medium containing 0.3-0.4 M sorbitol, 0.5% casamino acid, and high concentration of $CaCl_{2}$ and $MgCl_{2}$. The regeneration frequencies of the protoplasts of C. thermocellum and C. thermohydrosulfuricum were found to be very low level of $4.85{\times}10^{-3}$ and $4.23{\times}10^{-2}$, respectively. The nonregenerated L-form cells were also observed inregeneration medium together with regenerated cells.

고온 혐기성 Clostridium thermocellum 및 Clostridium thermohydrosulfuricum의 원형질체 형성 및 재생조건에 대하여 조사하였다. 원형질체 형성은 C. thermocellum의 경우, 0.2% glycine을 첨가한 배지에서 초기대수증식까지 생육한 cell을 0.5mg/ml의 lysozyme을 함유한 100mM TMG buffer(pH 7.5)에서 $37^{\circ}C$, 2시간 처리 했을 때 가장 좋았으며, C. thermohydrosulfuricum은 대수증식기의 cell을 동일 조건에서 0.2mg/ml의 lysozyme으로 처리했을 때 양호하였다. 원형질체 재생은 0.3-0.4M sorbitol. 0.5% casamino acid, 고농도의 $CaCl_2$를 첨가한 재생배지에서 $60^{\circ}C$, 7-10일간 배양했을 때 잘 되었으며, 원형질체 형성시 lysozyme의 처리 시간이 짧을 수록 좋았다. 재생빈도는 C. thermocellum의 경우 $4.85{\times}10^{-3}$, C. thermohydrosulfuricum의 경우 $4.23{\times}10^{-2}$ 이하로 낮은 편이었다. 재생배지에 나타난 colony에는 완전히 재생되지 않은 L-form cell도 함께 존재하였다.