Three different genes; endo-glucanase gene, exo-glucanase gene and ＄￦beta＄- glucosidase gene, are involved in the enzymatic decomposition of cellulose molecules to glucose molecules. The two genes encoding endo-glucanase and ＄￦beta＄- glucosidase have been cloned in our laboratory remaining the gene for exo-glucanase unclosed. In this research, cloning of the exo-glucanase gene was attempted. Chromosomes of $\underline{Clostridium}$ $\underline{thermocellum}$, a cellulolytic organism, were digested with EcoRI and a genomic library was constructed in $\underline{Escherichia}$ $\underline{coli}$ JM 83 using pUC 19 as a vector. A gene located in a 2.9kbp EcoRI fragment was inserted in the vector plasmid and the newly constructed plasmid was named pKM29. $\underline{Escherichia}$ $\underline{coli}$ strains transformed by pKM29 produced enzyme showing strong activity toward p-nitrophenyl-$￦beta$-D-cellobioside(pNPC) and filter paper with little activity toward p-nitrophenyl-$￦beta$-D-glucopyranoside(pNPG). The enzyme hydrolyzed CM-cellulose with negatively Congo Red tests as well as showed exo type mode by viscometric analysis. From these characteristics we have concluded that the enzyme produced by the cloned gene is exo-glucanase. The exo-glucanase produced by $\underline{Escherichia}$ $\underline{coli}$ JM 83 (pKM29) showed optimal at pH 5.5 and was stable at temperature up to 75°C The exo-glucanase activity was enhanced by the presence of endo-glucanase and the synergism between the two enzymes was confirmed.
유전자 운반체로 pUC19 plasmid를 이용하여 Clostridium thermocellum 에서 대장균내로 exoglucanase 유전자를 크로닝 하였다. 모균에서 분리한 염색체와 pUC19 plasmid를 EcoRI 제한효소로 절단하여 이들 두가지의 절단된 DNA를 접합시킨후 대장균에서 형질전환하였다. MUC를 이용하여 이들 전환주들 가운데서 섬유소를 분해하는 하나의 클론을 선별해 내었다. 대장균에서 재조합 plasmid를 분리하여 약 2.9kb의 EcoRI 염색체 절편이 존재하는 것을 알 수 있었고 이 plasmid를 pKM29라고 명명하였다. pKM29에 의해서 생산되는 효소는 Congo Red 실험에서 훈륜을 나타내지 않으면서 CMC를 분해하고, pNPC와 거름종이들을 분해한다. 최종적으로 CMC 분해에 따른 점도의 변화를 관찰하므로써 pKM29에 의해서 생성되는 효소가 exoglucanase라는 것을 확인하였다. 효소 활성의 최적산도는 5.5이었고 75°C까지 안정하게 활성을 유지하였다. 그리고 이 효소는 endoglucanase와 같이 섬유소 분해 반응에 참여할 때 상승작용을 나타내는 것으로 보인다. 지금까지 보고된 C. thermocellum의 섬유소 분해효소와 비교하여 볼 때 pKM29에 의해서 생성되는 exoglucanase는 새로운 효소로 추정이 된다.
