Putification and characterization of endo-β-1,4-glucanase encoded by bacillus subtilis gene in bacillus megaterium and escherichia coli

Abstract

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...