Purification and characterization of the cloned CMCase of B. subtilis in E. coli = 고초균으로부터 대장균으로 cloning된 섬유소 분해 효소의 분리 및 특성

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Carboxymethyl cellulose degradating enzyme(CMCase) produced by Escherichia coli HB101 harboring plasmid pBS 2115 which contains 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 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 Bacillus cellulase gene in E. coli, if present. The 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 $65\,^\circ\!C$ and 6.0, respectively. The enzyme was stable at $55\,^\circ\!C$ for 1 hour. The substrate specificity of this enzyme was broad and hydrolyzed CMC, laminarin, pustulan, polymers of glucose linked by $\beta(1 \to 4),\; \beta(1 \to 3)$, and $\beta(1 \to 6)$ 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 E. coli HB101 (pBS 2115) is endo-$\beta(1 \to 4)$ glucanase.
Yang, Kyu-Hwan양규환
한국과학기술원 : 생물공학과,
Issue Date
65040/325007 / 000841114

학위논문(석사) - 한국과학기술원 : 생물공학과, 1986.2, [ v, 42 p. ]


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