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

摘要

The gene ADH encoding NAD-dependent alcohol dehydrogenase from Bacillus stearothermophilus 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 thrambin, while soluble enzyme was further purified by glutathione affinity chromatography. The recombinant enzyme had the same elctrophoretic mobility as the native enzyme from Bacillus stearothermophilus. The recombinanl 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 ℃. The midpoint of the temperature-stability curve for the recombinant enzyme was approximately 68 ℃, and the enzyme was not completely inactivated even at 85 ℃. 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.