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Purification and Characterization of an Endo-$\beta$-1,3-1,4-Glucanase from Escherichia coli(pLL200K)  

김지연 (인제대학교 유전공학연구소)
Publication Information
Korean Journal of Microbiology / v.38, no.4, 2002 , pp. 241-246 More about this Journal
Abstract
A gene coding for endo-$\beta$-1,3-1,4-glucanase of Bacillus circulans was subcloned into Escherichia coli Ml5 using pQE30 as an expression vector. Endo-$\beta$-1,3-1,4-glucanase produced by the recombinant expression plas-mid pLQ43 was intactly purified to a single protein through a nickel-nitrilotriacetic acid (Ni-NTA) metal-affinity chromatography method. The molecular mass of the purified enzyme was estimated to be 28 kDa by SDS-PAGE. The optimum pH and temperature of the enzyme activity were pH 6.8 and $60^{\circ}C$, respectively. This enzyme was fairly stable in the pH ranging 5.5~7.5 and at the temperatures lower than $55^{\circ}C$. The enzyme appeared to be sensitive to most of the metal ions, especially to $Hg^{2+$, and also to methanol, ethanol, isopropanol or 1-butanol at a concentration of 10%(v/v).
Keywords
Bacillus circulans; endo-$\beta$-1,3-1,4-glucanase; Ni-NIA metal-affinity chromatography; purification;
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