Characterization and Application of Alcohol Dehydrogenase in the Biotransformation of Veratryl Alcohol

Alcohol dehydrogenase (ADH, EC 1.1.1.1) catalyses the biotransfor-mation of alcohols and carbonyl compounds. This study reports the purification and characteriza-tion of ADH from red Rajma seeds (Phaseolus vulgaris) and its catalytic role in veratryl alcohol oxidation. The crude enzyme extract was precipitated with 90% (NH₄)₂SO₄, dialyzed, and purified by DEAE–cellulose chromatography. Purity was confirmed by SDS–PAGE, native PAGE, and MALDI-TOF. pH and temperature optima, substrate specificity, and inhibition by metal ions were studied spectrophotometrically. Biotransformation of veratryl alcohol was confirmed by TLC and HPLC. ADH was purified 12-fold with a specific activity of 8.77 U/mg and a molecular weight of 27 kDa. MALDI-TOF revealed 244 amino acid residues and a pI of 5.02. The enzyme showed optimum activity at pH 10.5 and 40°C. Kinetic analysis revealed ethanol (Vmax 0.112 μmol/min, Km 6.2 mM), allyl alcohol as the best substrate (Vmax 0.192 μmol/min, Km 8.04 mM), and high affinity for NADP⁺ (Km 0.09 mM). Inhibition studies revealed competitive inhibition by Cr³⁺, Fe³⁺, Sr²⁺, Mo⁶⁺, and Hg²⁺; non-competitive inhibition by Ca²⁺, Cu²⁺, and Cd²⁺; and uncompetitive inhi-bition by As³⁺, Sn²⁺, and Ce⁴⁺. Determination of kinetic parameters of ADH for different alcohol substrates helps in understanding the enzyme’s efficiency and specificity. This innovative approach utilizes enzymes as catalysts and offers a more sustainable alternative to traditional chemical synthesis methods. Red Rajma seed is a novel source of ADH with favourable activity, stability, and substrate specificity. Its ability to oxidize veratryl alcohol highlights its potential application as a biocatalyst in industrial biotransformation.