Bacillus thuringiensis의 내독소 단백질의 분리1

Purtification of Parasporal Protein Crystals of Bacillus thuringiensis

해충의 미생물적 방제를 위하여 Bacillus thuringiensis 살충제 개발에 관한 기초자료를 얻고자 3종의 B. thuringiensis var. kurstaki, dendrolimus, aizawai를 이용하여 배지내에서 생장특성을 조사하고 이들의 내독소단백질의 분리를 위해 Renograffin 불연속중층법을 이용한 새로운 분리법을 시도하였다. 1. 각공시균주의 생장곡선을 조사한 결과는 3균주 모두 접종후 3시간 이내에 exponential phase에 이르렀고, 7-8시간 정도에서 stationary phase에 이르렀다. 2. 배지내 pH변화는 exdponential phase때 원래 배지에서 1.4정도 저하하였다가 sporulation의 개시에 따라 회복되기 시작하여 후기에 거의 평형상태를 나타내서 균주간에 차이가 없는 것으로 확인되었다. 3. Renograffin 불연속중층법을 이용한 새로운 B. thuringiensis 내독소단백질 분리법은 50$m\ell$ 원심분리관에 10$m\ell$의 시료를 중층, fixed angel rotor를 이용하여 27,000g에서 1시간 원심했을 경우 99.0% 순도와 5.8%의 회수율을 나타내어 지금까지 보고된 방법들보다 더욱 효율적인 것으로 확인되었다.

The study has been carried out to acquire some basic informations about Bacillus thuringiensis for developing the microbial pesticide. Three strains of Bacillus thuringiensis var. Kurstaki, dendrolimus and aizawai, were used in the experiments as follows. Growth characteristics of each strain were examined and parasporal protein crystals were isolated from the mixtures of spores and protein crystals by the new method of centrifugation in two-layer cushion of Renograffin using a fixed angle rotor. The results are as follows. 1. No difference was shown in growth characterestics among three strains of B. thuringiensis. In growth curves, all strains reached to exponential phase by 2 hr and stationary phase by 7-8 hr after inoculation. 2. The pH of the culture media during exponential growth stage decreased about 1.4 of a pH unit at the beginning of sporulation, but recovered during the early stage of sporulation and then remained nearly constant during the later stage. 3. As 10$m\ell$ sample was applied to two-layer cushion of Renograffin and then centrifuged for 1hr at 27,000g a fixed angle rotor, the purity and recovery ratio was 99.9% and 5.8%, respectively. It has been shown that the new method for the isolation of parasporal protein crystals was more efficient than any from the estabilished methods.