Endo-β-1,4-glucanse gene of $\underline{Clostridium}$ $\underline{thermocellum}$ ATCC 27405 was cloned in $\underline{Escherichia}$ coli JM 83 using pUC 9 plasmid as a cloning vector. Chromosomal DNA of $\underline{C}$. $\underline{thermocellum}$ was isolated and digested with EcoRI. pUC 9 DNA was also digested with EcoRI and dephosphorylated by treating with calf intestinal phosphatase. Both digested DNA preparations were mixed and ligated with T4 DNA ligase. With the ligation mixture $\underline{E}$. $\underline{coli}$ JM 83 was transformed to make a gene library. Approximately 1750 ampicillin resistant $\underline{E}$. $\underline{coli}$ transformants were obtained. The transformants were grown on LB agar plates containing carboxymethylcellulose (CMC) and screened for CMCase-positive colonies using Congo Red dye method. Three colonies formed halos indicating hydrolysis of CMC. From a colony showed the largest halo, recombinant plasmids were isolated, retransferred into E. $\underline{coli}$ JM 83, and through the Congo Red plate-assay insertion of a CMCase directing gene in the recombinant plasmid was confirmed. The new recombinant plasmid contained a 4 kb chromosomal DNA fragment in which a CMCase gene is contained. Restriction sites on the 6.7 Kb pKH 40 were mapped. The crude enzyme produced by E. coli JM 83 (pKH 40) hydrolyzed CMC, pNPC, filter paper and Avicel. The hydrolytic ratios of CMC/pNPC and CMC/filter paper were 200 and 74, respectively. From there, the enzyme encoded by the $\underline{C}$. $\underline{thermocellum}$ gene was identified as endo-β-1,4glucanase. Further characterization of this enzyme differs from that of other $\underline{C}$. $\underline{thermocellum}$ cellulases reported previously. We have cloned a new endo-β-1,4-glucanase gene from $\underline{C}$. $\underline{thermocellum}$.
유전자 운반체로 pUC 9 plasmid를 이용하여 $\underline{Clostridium}$ $\underline{thermocellum}$ 에서 대장균으로 endo-β-1,4-glucanase 유전자를 클로닝 하였다. 모균에서 분리한 염색체와 pUC 9 plasmid 를 EcoRI 제한효소로 절단하여 이들 두가지의 절단된 DNA 를 ligation 한 후 대장균에서 형질 전환 하였다. Congo Red 염료를 이용하여 이들 전환주들 중에서 endo-β-1,4-glucanase 유전자를 가지고 있는 한개의 클론을 선별 하였다. 대장균에서 재조합 plasmid 를 분리하여 약 4 Kb 의 EcoRI 염색체 절편이 존재하는 것을 알 수 있었고 이 plasmid 를 pKH 40 이라 명명하였다. pKH 40에 의해 생성되는 효소는 CMC 뿐만 아니라 pNPC 와 거름종이, Avicel 등을 분해할 수 있는 능력을 가지고 있었고 최적온도는 65℃ 이고 최적산도는 5.0 이었다. 한편, 대장균 내에서 pKH 40 내에 존재하는 외부 DNA 의 유전자 발현은 pUC 9 plasmid 에 존재하는 lacZ promoter 에 의해 영향을 받고 있다. 지금까지 보고된 $\underline{C}$. $\underline{thermoellum}$ 의 섬유소 분해 효소와 비교하여 볼때 pKH 40 에 의해 생성되는 endo-β-1,4-glucanase 는 새로운 효소로 생각이 된다.
