To make $\underline{Cellulomonas}$ $\underline{fimi}$ $￦beta$-glucosidase gene express in $\underline{Bacillus}$ $\underline{subtilis}$, the gene which was previously cloned in our laboratory, was subcloned in a $\underline{Bacillus}$ expression vector, pPL 708. Plasmid pPL708 contains bacteriophage SpoII promoter followed by multiple cloning sites and CAT gene from $\underline{B}$. $\underline{pumilus}$. The CAT gene contains chloramphenicol control site in the upstream 200 base pair region. A shuttle vector between $\underline{Escherichia}$ $\underline{coli}$ and $\underline{B}$. $\underline{subtilis}$ was constructed using pUC19 and pPL708. A $￦beta$-glucosidase gene-containing pCF16 was treated with appropriate restriction enzymes and Klenow fragment, and ligated to the shuttle vector digested with HpaI. This ligation mixture was used to transform $\underline{E}$. $\underline{coli}$ HB101 cells. From the transformants, recombinant plamids were isolated and designated pPCF84. The $￦beta$-glucosidase gene was relocated under the control region of CAT-86 gene from pPL708. B. subtilis RM125 was transformed with pPCF84 again. The $￦beta$-glucosidase activity was tested both in $\underline{E}$. $\underline{coli}$ and $\underline{B}$. $\underline{subtilis}$. Recombinant $\underline{E}$. $\underline{coli}$ HB101 (pPCF84) was induced with Chloramphenicol when $0.D_{600}$ value reached 0.4. The $￦beta$-glucosidase production was increased up to about 5 times of that of uninduced cells. Expression of ＄￦beta＄-glucosidase gene by chloramphenicol induction did not turn on well in $\underline{E}$. $\underline{coli}$. When the culture of $\underline{B}$. $\underline{subtilis}$ RM125 (pPCF84) was induced with Chloramphenicol, the enzyme activity was increased up to about 17 times higher than that of pCF18, and about 10,000 times than that of gene donor, Cellulomonas fimi. The $￦beta$-glucosidase activity in uninduced $\underline{B}$. $\underline{subtilis}$ (pPCF84) was near to zero, while in uninduced $\underline{E}$. $\underline{coli}$ (pPCF84) enzyme activity was 50% of that of pCF18.
Cellulomonas fimi 의 $￦beta$-Glucosidase 유전자를 Bacillus subtilis 속에서 발현시키기 위해서 고초균의 발현용 벡터인 pPL708 과 대장균의 벡터인 pUC19 을 사용해서 pUP683 을 만들고 pUP683의 CAT 유전자 내의 HpaI제한효소 자리에 $￦beta$-Glucosidase 유전자를 서브클론하여 번역수준에서 유전자의 발현이 조절되도록 하였다. 이렇게 만들어진 Plasmid를 pPCF84 라고 명명하였다. pPCF84 플라즈미드를 가진 고초균 전환주와 대장균 전환주들로부터 Chloramphenicol 로 유도되는 효소생산 실험을 수행하였다.
pPCF84 를 가지는 대장균 전환주는 Chloramphenicol에 의해 발현이 유도되었을때 lac promotor 의 영양하의 $￦beta$-Glucosidase를 가지는 pCF18 보다 2.5배 발현이 증진되었고 Chloramphenicol 에 의해 유도되지 않았을 때에도 pCF18의 약 50%의 효소활성을 나타냈다. 한편, pPCF84를 가지고 있는 고초균 전환주는 Chloramphenicol 에 의해 pCF18 보다 17배까지 발현의 증가가 유도되었고 Chloramphenicol 에 의해 유도되지 않았을 때에는 효소활성이 0에 가까왔다.
