Kinetics of carboxymethyl cellulase (EC 3.2.1.4) production by recombinant cell, $\mbox{\underline{Bacillus}}\quad\mbox{\underline{megaterium}}$ (pCK108), was investigated in a chemically defined medium. The gene expression of plasmid pCK108 was surveyed at different culture conditions.
The ratio of specific growth rate of plasmid-absent to plasmid-carrying cells was determined to 1.31. Phenotypic instability of plasmid in Bacillus megaterium, originated from segregation during cell growth.
The optimal temperature supporting cell growth was determined to be 30℃ at which the plasmid pCK108 could be stably maintained. Cellular plasmid contents, cellulase production rate and the efficiency of gene expression increased significantly with the increase of temperature from 30℃ to 44℃. At temperatures higher than 38℃, however, the plasmid stability decreased sharply to 60% level at the culture time of 30 hours. The optimal pH supporting cell growth and plasmid stability was determined to be 7.0.
The production of the cellulase was not stimulated by sophorose that has been recognized as strong inducer for the production of fungal cellulase. Carboxymethyl cellulose did not also induce the biosynthesis of the cellulase.
The cellulase production with glucose as carbon source, occurred after the cessation of exponential growth, in the mode of non-growth associated. By slow utilizable carbon source such as lactose, however, the enzyme production was also occurred during cell growth. The production of carboxymethyl cellulase from $\mbox{\underline{Bacillus}}\quad\mbox{\underline{megaterium}}$ (pCK108) was regulated by carbon catabolite repression. In batch culture with lactose as sole carbon source (50 g/l), the cellulase was produced to 7.5 U/ml.
The enzyme production in continuous culture was favored when the growth rate was a relatively small fraction of the maximum growth rate under the prevailing culture condition. The gene expression level was higher at lower dilution rate. The enzyme production did not occur at dilution rate higher than $0.22 hr^{-1}$.
The carbon catabolite repression in the synthesis of the carboxymethyl cellulase was recovered by slow feeding of glucose in fed-batch culture which was carried out by programmable feeding system. By constant fed-batch culture, the enzyme was produced to 4.73 U/ml at feed rate of 0.20 ml/min. Yield coefficient for the enzyme production on carbon source was enhanced to 4 times by constant fed-batch culture as compared with batch culture. By exponentially fed-batch culture, the specific growth rate could be maintained at the programmed level.
유전자재조합 균주인 $\mbox{\underline{Bacillus}}\quad \mbox{\underline{megaterium}}$ (pCK108) 을 이용하여 섬유소분해효소를 생산하는 발효과정을 수행하였다. 포도당 1% 를 탄소 및 에너지원으로 하고, 온도 30℃ 및 pH 7.0 에서 생산되는 효소의 양은 1.25 U/ml 이고, 균체당 플라스미드 함량은 0.5 mg/g-cell 이었다. 또한 모균주와 재조합 균주 사이의 성장속도 비율은 약 1.3 이었다.
균체의 성장은 30℃ 에서 가장 좋았으나, 섬유소분해효소의 생산은 플라스미드의 안정성이 급격히 떨어지는 44℃ 에서 도리어 좋았고, 재조합된 유전자의 발현 정도 는 온도가 높을 수록 효율적이었다. 따라서, 장시간의 배양을 위해서는 플라스미드의 안정성이 보장되는 30℃가 유리하였다. 배양액의 pH 는, 온도만큼 큰 영향을 주지는 않지만, pH가 중성인 7.0 을 벗어날 수록 균체성장, 효소생산 그리고 유전자 발현정도가 저조했다.
이 효소의 생성은 탄소대사물에 의해서 억제 받는데, 탄소원이 다량 존재하는 배지상태에서는 그 생성이 억제 되다가 탄소원이 고갈 된 이후부터 생성이 시작되며, 또한 느리게 이용 되는 탄소원이 효소의 생산에 유리하였다. 연속배양의 경우 희석배율 즉 비성장속도에 의해 효소의 생성이 통제 되는데, $0.22 hr^{-1}$ 이상의 희석배율 에서는 그 재조합 유전자의 발현이 거의 일어나지 않았고, $0.143 hr^{-1}$ 이하에서 효소생성이 있었다.
탄소원의 공급을 낮게 통제하고, 균체의 성장속도를 제한시킨 유가식배양에 의해서 효소의 생산이 향상 되었으며, 유전자의 발현도 회분식배양보다 효과적이었다.
