Mechanism studies on the oxidation of isoeugenol by Nocardia sp. NRRL 5646

Essential oils contain compounds that are attractive substrates for biotransformation by microorganisms into value added flavors and fragrances. Biotransformations of aromatic aldehydes, ferulic acid, phenolic stilbenes, lignin, eugenol and isoeugenol to vanillin have been widely studied. A few reports have probed the mechanism of biotransformation of eugenol and ferulic acid to vanillin. However, there are no reports investigating the exact mechanism involved in the bioconversion of isoeugenol to vanillin or vanillic acid.; Nocardia species NRRL 5646 catalyzes the biotransformation of isoeugenol to vanillic acid and guaiacol. We proposed three reaction pathways for this biocatalytic process. One involves the oxidation of isoeugenol via coniferyl alcohol and ferulic acid; a second involves initial formation of an epoxide followed by epoxide hydrolysis, alcohol oxidation; and a third pathway is based upon initial hydration of the styrene double bond, alcohol oxidation followed by Baeyer-Villiger oxidation to vanillin.; A variety of chemical, microbial and enzymatic approaches were used to evaluate the bioconversion pathways and to detect putative intermediates. Incubations of ferulate pathway intermediates with both growing and resting Nocardia cells gave no vanillin, vanillic acid or guaiacol as products, thus ruling out this path. Incubation of the putative intermediates of the hydration pathway gave no vanillic acid, ruling out this pathway also. A racemic mixture of trans side-chain epoxides was prepared and further hydrolysis of this epoxide gave the diol. Growing cultures of Nocardia converted the epoxide to vanillic acid and the diol to vanillin suggesting that the epoxide or diol pathways were possible.; Experiments with cytochrome-P-450 inhibitors and lignostilbene-alpha,beta-dioxygenase inhibitors gave inconclusive results that would not enable the identification of these enzymes in the biotransformation process. Labeling experiments with 18O2 and H218O showed that there was incorporation of oxygen from both these sources into vanillic acid.; Experiments with cell fee preparations suggested a novel cofactorless system for this Nocardia mediated biotransformation. The enzyme involved in the conversion of isoeugenol to vanillin was partially purified. Future studies will be aimed at further purification of the oxygenase and also probing the pathway for the cofactorless bioconversion in cell-free extracts.