Carboxymethyl cellulose degradating enzyme(CMCase) produced by $\underline{Escherichia coli}$ HB101 harboring plasmid pBS 2115 which contains $\underline{Bacillus}$ cellulase gene has been purified partially and characterized. The purpose of this study was of two fold; to obtain pure CMCase which is a component of the multiple system of microbial cellulases by producing with $\underline{E. coli}$ which had been free of cellulase genes until it was transformed by pBS 2115, and to find any alternative on the expression of the $\underline{Bacillus}$ cellulase gene in $\underline{E. coli}$, if present.
The $\underline{E. coli}$ HB101(pBS 2115) cells at midlogarithmic growth phase were harvested and the CMCase was extracted by applying osmotic shock to the cells. The curde enzyme preparation was purified by DEAE-Sephadex A-25 and hydroxyapatitic chromatography. Through the purification steps, the purity of the enzyme in the osmotic shock extract was increased 300 fold along with the yield reduction from 100 to 22%.
The molecular weight of this enzyme was estimated to be 34,000 in the SDS-polyacrylamide gel electrophoresis. The optimal activity of the enzyme for temperature and pH was at 65℃ and 6.0, respectively. The enzyme was stable at 55℃ for 1 hour. The substrate specificity of this enzyme was broad and hydrolyzed CMC, laminarin, pustulan, polymers of glucose linked by β(1→4), β(1→3), and β(1→6) glycosidic bond. The enzyme was not inhibited by glucose or cellobiose.
The enzyme liquefied CMC with sharp decrease in viscosity at early reaction time followed by a slow decrease, whereas the reducing ends were produced linearly. From these results it was concluded that the CMCase produced by the $\underline{E. coli}$ HB101 (pBS 2115) is endo-β(1→4) glucanase.
B. subtilis의 섬유소 분해 효소를 갖고 있는 E. coli에서 DEAE-Sephadex A-25와 hydroxyapatite column chromatography에 의해 분리하였다.
이 cloning된 효소의 분자량은 SDS-polyacrylamide gel electrophoresis에 약 34,000정도를 나타냈다. 효소의 최적반응 온도는 65℃ 그리고 최적 pH는 6.0이었다. 이 효소의 기질 특이성은 넓어서 CMC, laminarin 그리고 pustulan 즉, β(1→4), β(1→3) 그리고 β(1→6) glycosidic linkage 를 가수 분해하였다. 효소 활성은 50mM glucose와 cellobiose에 의해 저해받지 않았다. 효소의 열 안정성은 55℃에서 1시간 동안 그대로 유지되었다. cloning된 효소의 반응형태는 당화 작용과 액화 작용을 비교함으로서 endo-형태임을 알 수 있었다.
