Analysis of Protein Expression and Characterization of E.Coli/YqhD Alcohol Dehydrogenase

As global energy demands increase and fossil fuel limitations become more pressing, biofuels such as isobutanol have emerged as promising alternatives due to their high energy density and compatibility with existing fuel infrastructure. This study focuses on optimizing the overexpression and characterization of the Escherichia coli YqhD alcohol dehydrogenase, a key enzyme in the final step of isobutanol biosynthesis. Using varying concentrations of IPTG (50 μM and 100 μM) and induction times (0–3 hours), protein expression was monitored via SDS-PAGE and immunoblotting, revealing that 100 μM IPTG at 3 hours yielded maximal YqhD expression. Affinity chromatography successfully isolated the His6-tagged YqhD protein, with purity confirmed by SDS-PAGE and an experimentally derived molecular weight of approximately 42 kDa. Enzyme concentration was quantified via Bradford assay, yielding a total of 7.097 mg/mL across eluates. Kinetic characterization revealed a Michaelis constant (Km) of 4.93 mM and a Vmax of 0.00393 mM/min, with optimal enzymatic activity observed at pH 8. Additionally, inhibition studies using 4-methylpyrazole demonstrated increased Km and Vmax, suggesting complex inhibition dynamics. These findings establish foundational parameters for scaling YqhD-based isobutanol production and inform future bioengineering strategies aimed at improving biofuel yields through microbial enzyme optimization.