To increase expression efficiency of a $\underline{Cellulomonas}$ $\underline{fimi} β-glucosidase gene in $\underline{Escherichia}$ $\underline{coli}$, the gene previously cloned in our laboratory, was subcloned in expression vectors, pKK223-3, pTTQ 19, pIN IIIAI and pPLc2833 all of which have been known to be strong promoters. Plasmid pKK223-3 and pTTQ 19 contain the hybrid promoter $\underline{tac}$. Plasmid pIN IIIAI contain the 1pp/lacUV5 promoter and translation initiation signal of 1pp gene, and plasmid pLc2833 contains lambda pL promoter followed by multicloning sites. The β-glucosidase gene source were pCF 18 and pCF 16. Recombinant plasmids were obtained by appropriate gene manipulation and the resultant plasmids were named pKCF 16, pTCF 18, pICF 16 and pLCF 16. $\underline{E}$. $\underline{coli}$ cells containing each recombinant plasmids were induced with IPTG and temperature. $\underline{E}$. $\underline{coli}$ JM 103 (pKCF 16) showed very low β-glucosidase activity. In the case of $\underline{E}$. $\underline{coli}$ HB 101 cells transformed with pICF 16 or pTCF 18, the activity of β-glucosidase was increased 11 folds and 24 folds, respectively, compared with that in the $\underline{E}$. $\underline{coli}$ cells carrying pCF 18. The activity of β -glucosidase of $\underline{E}$. $\underline{coli}$ M5248 (pLCF 16) was not higher than that of $\underline{E}$. $\underline{coli}$ JM 83 (pCF 18). However, the production of the enzyme protein itself was high. It was estimated that the protein of β-glucosidase occupied about 5-8% of the total cellular proteins and located in cytoplasmic portion of the cell.
본 실험실에서 분리된, $\underline{Cellulomonas}$ $\underline{fimi} β-glucosidase 유전자를 발현용 벡터인 pKK 223-3, pTTQ 19, pIN III-A1 그리고 pPLc 2833에 서브클론 하였다. 이들은 강력한 프로모터인 tac, lpp/lac UV5 그리고 람다 pL을 각각 가지고 있는 벡터들이다. 섬유소 분해효소가 제대로 서브클론 된 플라스미드들을 각각 pKCF 16, pTCF 18, pICF 16 그리고 pLCF 16 이라고 명명 하였다. 이들을 가지는 대장균 전환주를 가지고 IPTG와 온도변화로 유도되는 효소생산 실험을 수행하였다.
pKCF 16 을 가지는 전환주는 매우 낮은 β-glucosidase 활성을 나타내었다. pICF 16 또는 pTCF 18을 가지는 전환주는 IPTG로 효소합성을 유도하면 효소활성이 서브클론 하기전의 11배와 24배씩 각각 증가하였다.
pLCF 16을 가지는 대장균 전환주는, 온도변화로 효소생산을 유도하면 서브클론 하기전보다 2배 정도 낮은 효소활성을 나타냈으나 생성된 단백질 양은 전체세포 단백질의 5-8 % 정도를 차지하는 것으로 나타났다.
