Abstract
A cinnamyl alcohol dehydrogenase (CAD) from the secondary xylem of Leucaena leucocephala has been purified to homogeneity through successive steps of ammonium sulfate fractionation, DEAE cellulose, Sephadex G-75, and Blue Sepharose CL-6B affinity column chromatographies. CAD was purified to 514.2 folds with overall recovery of 13% and specific activity of 812. 5nkat/mg. Native and subunit molecular masses of the purified enzyme were found to be ∼76 and ∼38kDa, respectively, suggesting it to be a homodimer. The enzyme exhibited highest catalytic efficiency (Kcat/Km 3.75μM(-1)s(-1)) with cinnamyl aldehyde among all the substrates investigated. The pH and temperature optima of the purified CAD were pH8.8 and 40°C, respectively. The enzyme activity was enhanced in the presence of 2.0mM Mg(2+), while Zn(2+) at the same concentration exerted an inhibitory effect. The inclusion of 2.0mM EDTA in the assay system activated the enzyme. The enzyme was inhibited with caffeic acid and ferulic acid in a concentration-dependent manner, while no inhibition was observed with salicylic acid. Peptide mass analysis of the purified CAD by MALDI-TOF showed a significant homology to alcohol dehydrogenases of MDR superfamily.
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