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Vanillic acid is an abundant renewable chemical useful for the biocatalytic synthesis of aromatics. The bacterium, Nocardia sp. NRRL 5646 efficiently catalyzes the decarboxylation of vanillic acid to the phenolic product, guaiacol. For purification and characterization of vanillic acid decarboxylase (VAD), a simple, rapid enzyme assay was devised to couple the vanillic acid decarboxylase reaction with horseradish peroxidase/H2O2. Guaiacol formed in the decarboxylation reaction is oxidized by peroxidase to tetraguaiacol, a chromophoric substance, which is measurable at 470?nm. Soluble, VAD activity was inducible in cells grown with vanillic acid. VAD was purified 185-fold by a combination of DEAE cellulose, phenylsepharose and sephacryl S-200 chromatographies. SDS-PAGE gave an estimated molecular mass of 46?±?0.8?kDa while active native VAD was 92?kDa by gel chromatography. VAD required no cofactors, displayed optima at pH 7.0 and 30?°C, and a Km of 0.8?mM for vanillic acid. The N-terminal amino acid sequence was determined to be AEYTLPDLDYDYSALEPHIS. 4-Hydroxybenzoic acid, vanillic acid, and syringic acid were excellent substrates, while VAD showed no activity for isovanillic acid, ferulic acid, p-coumaric acid, 2-hydroxybenzoic acid and 3-hydroxybenzoic acid. Purified VAD required a phenolic functional group para to the carboxylic acid moiety in the best substrates. The vanillic acid decarboxylation reaction is non-oxidative in nature. Conduct of the decarboxylation process in deuterium oxide results in incorporation of one deuterium atom para to the phenolic hydroxyl group of guaiacol. Vanillic acid decarboxylation likely involves enzymatic tautomerization to a vinylogous [beta]-keto acid that spontaneously decarboxylates to guaiacol.
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