Putification and characterization of endo-β-1,4-glucanase encoded by bacillus subtilis gene in bacillus megaterium and escherichia coli = Bacillus sybtilis 로 부터 baccillus megaterium 과 escherichia coli로 cloning 된 endo-β-1,4-glucanase 의 분리 및 특성

This study was performed to provide enzymological information which will be needed for the further development in molecular genetics of endo-β-1, 4-glucanase (EC 3.2.1.4) gene, whose product is known to be a member of the cellulase complex. And endo-β-1,4-glucanase gene of $\mbox{\underline{Bacillus}}\quad {\mbox{\underline{subtilis}}$ was previously cloned in $\mbox{\underline{Escherichia}}\quad \mbox{\underline{coli}}$ and subsequently introduced into a cellulase-negative strain of $\mbox{\underline{B.}}\quad \mbox{\underline{megaterium}}$ using a recombinant plasmid pCK98 in our laboratory. Using the new transformant, $\mbox{\underline{B.}}\quad \mbox{\underline{megaterium}}$ (pCK98), the endo-β-1,4-glucanase was produced and used for this study. The enzyme was purified by chromatography including DEAE-Sephadex A-50, Sephadex G-100, and SP-Sephadex C-50. Since the host strain of $\mbox{\underline{B.}}\quad \mbox{\underline{megaterium}}$ was primarily cellulase-negative, only one protein peak on the chromatography showed cellulase activity which is believed to be encoded by the $\mbox{\underline{B.}}\quad \mbox{\underline{subtilis}}$ glucanase gene, and thus could be isolated easily. The purification procedures yielded a 215-fold purification of the enzyme with 15% recovery of the original activity. The purified enzyme was found to be a monomer having molecular weight of 33,000 and contained 5.2% carbohydrate but no metal ion. The isoelectric point of the enzyme was at pH 7.23. The endoglucanase was high in small-nonpolar amino acid residues and low in bulky-aromatic residues. The three consecutive amino acid sequence at the N-terminal of the enzyme was found to be Ala-Gly-Thr. The signal peptide was composed of 29 hydrophobic amino acid residues. The enzyme displayed an absorption maximum at 278 mm with a molar absorption coefficient of $15,000 M^{-1} cm^{-1}$. The maximum hydrolytic activity of the endo-β-1,4-glucanase was observed at pH 5.5 and at 60℃. T...
Advisors
Pack, Moo-Youngresearcher박무영researcher
Publisher
한국과학기술원
Issue Date
1988
Identifier
61172/325007 / 000835120
Language
eng
Description

학위논문(박사) - 한국과학기술원 : 생물공학과, 1988.2, [ xi, 132 p. ]

Keywords

단백질 분리.

URI
http://hdl.handle.net/10203/27268
Link
http://library.kaist.ac.kr/search/detail/view.do?bibCtrlNo=61172&flag=t
Appears in Collection
BS-Theses_Ph.D.(박사논문)
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