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J. Microbiol. Biotechnol. 2003; 13(5): 738-744

Published online October 28, 2003

Copyright © The Korean Society for Microbiology and Biotechnology.

Overexpression, Purification, and Biochemical Characterization of the Thermostable NAD-dependent Alcohol Dehydrogenase from Bacillus stearothermophilus

Shim Eun-Jung , Sang-Hoon Jeon and Kwang-Hoon Kong

Department of Chemistry College of Natural Sciences Chung-Ang University Seoul 156-756 Korea

Abstract

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothennophilus was cloned and overexpressed as a GST fusion protein at a high level in Escherichia coli. The expressed fusion protein was purified simply by glutathione affinity chromatography. GST fusion protein was then cleaved by thrombin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothennophilus. The recombinant enzyme catalyzed the oxidation of a number of alcohols and exhibited high activities towards secondary alcohols. The $K_m\;and\;V_{max}$ values of the recombinant enzyme for ethanol were 5.11 mM and 61.35 U/mg, respectively. Pyridine and imidazole notably inhibited the enzymatic activity. The activity of the recombinant enzyme optimally proceeded at pH 9.0 and $70^{\circ}C$. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately $68^{\circ}C$, and the enzyme was not completely inactivated even at $85^{\circ}C$. The recombinant enzyme showed a high resistance towards denaturing agents (0.05% SDS, 0.1 M urea). Therefore, due to its stability and relatively broad substrate specificity, the recombinant enzyme could be utilized in bio-industrial processes and biosensors.

Keywords: Alcoholdehydrogenase,thermophilicbacterium,Bacillusstearothermophilus,enzymaticcharacterization,over-expressioninE.coli,recombinantenzyme,simplepurification,thermalresistance