Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.     

Antioxidant properties of ethyl vanillin in vitro and in vivo

We systematically evaluated the antioxidant activity of ethyl vanillin, a vanillin analog, as compared with the activities of vanillin and other vanillin analogs using multiple assay systems. Ethyl vanillin and vanillin exerted stronger antioxidant effects than did vanillyl alcohol or vanillic acid in the oxygen radical absorbance capacity (ORAC) assay, although the antioxidant activities of vanillyl alcohol and vanillic acid were clearly superior to those of ethyl vanillin and vanillin in the three model radical assays. The antioxidant activity of ethyl vanillin was much stronger than that of vanillin in the oxidative hemolysis inhibition assay, but was the same as that of vanillin in the ORAC assay. Oral administration of ethyl vanillin to mice increased the concentration of ethyl vanillic acid, and effectively raised antioxidant activity in the plasma as compared to the effect of vanillin. These data suggest that the antioxidant activity of ethyl vanillin might be more beneficial than has been thought in daily health practice.     

Antioxidant Properties of Ethyl Vanillin in Vitro and in Vivo

We systematically evaluated the antioxidant activity of ethyl vanillin, a vanillin analog, as compared with the activities of vanillin and other vanillin analogs using multiple assay systems. Ethyl vanillin and vanillin exerted stronger antioxidant effects than did vanillyl alcohol or vanillic acid in the oxygen radical absorbance capacity (ORAC) assay, although the antioxidant activities of vanillyl alcohol and vanillic acid were clearly superior to those of ethyl vanillin and vanillin in the three model radical assays. The antioxidant activity of ethyl vanillin was much stronger than that of vanillin in the oxidative hemolysis inhibition assay, but was the same as that of vanillin in the ORAC assay. Oral administration of ethyl vanillin to mice increased the concentration of ethyl vanillic acid, and effectively raised antioxidant activity in the plasma as compared to the effect of vanillin. These data suggest that the antioxidant activity of ethyl vanillin might be more beneficial than has been thought in daily health practice.     

Metabolism of Ferulic Acid by Paecilomyces variotii and Pestalotia palmarum

Ferulic acid metabolism was studied in cultures of two micromycetes producing different amounts of phenol oxidases. In cultures of the low phenol oxidase producer Paecilomyces variotii, ferulic acid was decarboxylated to 4-vinylguaiacol, which was converted to vanillin and then either oxidized to vanillic acid or reduced to vanillyl alcohol. Vanillic acid underwent simultaneously an oxidative decarboxylation to methoxyhydroquinone and a nonoxidative decarboxylation to guaiacol. Methoxyhydroquinone and guaiacol were demethylated to yield hydroxyquinol and catechol, respectively. Catechol was hydroxylated to pyrogallol. Degradation of ferulic acid by Paecilomyces variotii proceeded mainly via methoxyhydroquinone. The high phenol oxidase producer Pestalotia palmarum catabolized ferulic acid via 4-vinylguaiacol, vanillin, vanillyl alcohol, vanillic acid, and methoxyhydroquinone. However, the main reactions observed with this fungus involved polymerization reactions.     

Peroxidase catalysed oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone

Horseradish peroxidase catalysed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to methoxy-p-benzoquinone. Peroxidase also catalysed the oxidation of vanillyl alcohol to vanillin and vanillic acid; however, neither vanillyl alcohol nor vanillin appeared to give rise to methoxyhydroquinone directly. Correspondingly, peroxidase catalysed the oxidative decarboxylation of syringic acid to 2,6-dimethoxy-p-hydroquinone and subsequent oxidation of the hydroquinone to 2,6-dimethoxy-p-benzoquinone.     

Oxidation of isoeugenol by Nocardia iowensis

Isoeugenol is a starting material for both the synthetic and biotechnological production of vanillin and vanillic acid. Nocardia iowensis DSM 45197 (formerly Nocardia species NRRL 5646) resting cells catalyze the conversion of isoeugenol to vanillic acid, vanillin, vanillyl alcohol and guaiacol. The present study used a variety of chemical, microbial and enzymatic approaches to probe the pathways used by N. iowensis in the oxidation of isoeugenol to these products. Of three possible pathways considered, initial side-chain olefin epoxidation, epoxide hydrolysis to a vicinal diol, and diol cleavage to vanillin and subsequently further oxidation to vanillic acid appears as the most likely route. Isoeugenol was not oxidized to ferulic acid, a well-known microbial transformation precursor for vanillin and vanillic acid. ¹⁸O-Labeled oxygen (one atom) and water (two oxygen atoms) were incorporated into vanillic acid during the whole-cell biotransformation reaction with isoeugenol indicating the likely involvement of oxygenase and hydrolase systems in the bioconversion reaction. Vanillin was converted to singly labeled vanillic acid in the presence of H₂¹⁸O suggesting the presence of an aldehyde oxidase. Cell extracts achieved the conversion of isoeugenol to vanillic acid and vanillin without cofactors. Partial fractionation of two enzyme activities supported the presence of isoeugenol monooxygenase and vanillin oxidase activities in N. iowensis.     

Biocatalytic Synthesis of Vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by ¹H nuclear magnetic resonance and mass spectral analyses.     

Differences in NMR Spectra between Some α- and γ-Glutamyl Dipeptides

A methanol-utilizing phototrophic bacterium, strain M402, was isolated from surface water of an acidic hot spring. The isolated strain was identified as Rhodopseudomonas acidophila from its morphological and physiological characters. Profiles of the utilization of non-aromatic compounds as carbon sources by this strain were in good agreement with those of some strains of R. acidophila reported by Pfennig [J. Bacteriol., 99, 597 (1969)]. However, strain M402 was found to be capable of utilizing vanillic acid, vanillin, vanillyl alcohol, ferulic acid, veratric acid, syringic acid, syringal-dehyde and benzyl alcohol as carbon sources under anaerobic-light conditions. Although Pfennig did not refer to these abilities of his strains, these notable characters of strain M402 seem to be additional new characters of R. acidophila.     

Microbial production of 4-vinylguaiacol from ferulic acid by Bacillus cereus SAS-3006

Abstract

Ferulic acid is an abundant cinnamic acid derivative found in the plant kingdom. It is a commercially available substrate utilized to produce flavor compounds such as 4-vinylguaiacol (4-VG), vanillin, and vanillic acid. The isolate Bacillus cereus SAS-3006 was screened and selected based on its ability to produce 4-VG upon ferulic acid biotransformation. It was identified based on morphological and physiochemical characteristics and its 16S ribosomal DNA sequence (GenBank accession number: KF699134). A maximum amount (79.4 mg/L) of 4-VG accumulation was observed on the 5th day of incubation when the culture was grown on 2.5 mM ferulic acid as sole carbon source. Further conversion of 4-VG to other intermediates such as vanillin, vanillic acid, protocatechuic acid, acetovanillone, and vanillyl alcohol was not observed. In-vitro conversion of ferulic acid to 4-VG was also studied with cell extracts of B. cereus SAS-3006. The present study provides the first evidence for production of 4-VG as the sole product using B. cereus SAS-3006.     

The biotransformation of simple phenolic compounds by Brettanomyces anomalus

Abstract Brettanomyces anomalus is shown here to metabolise p -coumaric, caffeic and ferulic acid to 4-vinyl and 4-ethyl derivatives. We also demonstrate the transformation of vanillin to both vanillyl alcohol and vanillic acid by this yeast. The results presented here show the production of these compounds during the fermentation of this organism and also the effects of these and other simple phenolic compounds on the growth of the organism. The products were analysed and their identities were determined by TLC, HPLC and by mass spectrometry.     

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of ¹⁴CO₂ from O¹⁴CH₃-vanillate. The ¹⁴CO₂ evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH₄NO₃) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.Non-Standard Abbreviations WT wild type Sporotrichum pulverulentum - MHQ methoxyhydroquinone - MQ methoxyquinone - NKM Norkrans medium - DMS dimethylsuccinate - DHP dehydropolymer of coniferyl alcohol     

The influence of lignin degradation products on xylose fermentation by Klebsiella pneumoniae

Summary The inhibitory effects of seven closely related lignin degradation products on xylose fermentation by Klebsiella pneumoniae were studied. Compounds were added in varying concentrations. Less heavily substituted phenolics (at concentrations of, 0.1–0.4 g/l) were more inhibitory to growth and solvent production than vanillyl or syringyl derivatives. All of the cultures recovered from this inhibition after a prolonged incubation period. When the mechanism of the organism's recovery was investigated, GC and LC analysis showed that 43.5% of the vanillin was metabolized to vanillyl alcohol. Several unidentifiable compounds were also detected in trace amounts. K. pneumoniae also metabolized vanilly alcohol (54% of original supplement) and syringaldehyde; however, unlike vanillin, there was no predominant metabolite derived from these compounds. None of the metabolites derived from vanillyl alcohol could be identified while only the corresponding alcohol and trimethoxybenzene were identified among the syringaldehyde derived metabolites.     

The effects of vanilloid analogues structurally related to capsaicin on the transient receptor potential vanilloid 1 channel

Transient receptor potential vanilloid 1 (TRPV1) is known as a receptor of capsaicin, a spicy ingredient of chili peppers. It is also sensitive to a variety of pungent compounds and is involved in nociception. Here, we focused on the structural characteristics of capsaicin, and investigated whether vanillylmanderic acid (VMA), vanillic acid (VAcid), vanillyl alcohol (VAlc), vanillyl butyl ether (VBE), and vanillin, containing a vanillyl skeleton similar to capsaicin, affected the TRPV1 activities. For detection of TRPV1 activity, intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured in HEK 293 cells heterologously expressing mouse TRPV1 (mTRPV1-HEK) and in mouse sensory neurons. Except for vanillin, four vanilloid analogues dose-dependently increased [Ca²⁺]_i in mTRPV1-HEK. The solutions that dissolved VMA, VAcid and vanillin at high concentrations were acidic, whereas those of VAlc and VBE were neutral. Neutralized VAcid evoked [Ca²⁺]_i increases but neutralized VMA did not. Mutation of capsaicin-sensing sites diminished [Ca²⁺]_i responses to VAcid, VAlc and VBE. VAcid, VMA, and vanillin suppressed the activation of TRPV1 induced by capsaicin. VAcid and VMA also inhibited the acid-induced TRPV1 activation. In sensory neurons, VMA diminished TRPV1 activation by capsaicin or acids. The present data indicate that these structural characteristics of chemical compounds on TRPV1 may provide strategies for the development of novel analgesic drugs targeting nociceptive TRPV1.     

Biocatalytic synthesis of vanillin

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylated (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified Nocardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to vanillin. Structures of metabolites were established by (1)H nuclear magnetic resonance and mass spectral analyses.     

Vanillin production from simple phenols by wine-associated lactic acid bacteria

AIMS: The ability of lactic acid bacteria (LAB) to metabolize certain phenolic precursors to vanillin was investigated. METHODS AND RESULTS: Gas chromatography-mass spectrometry (GC-MS) or HPLC was used to evaluate the biosynthesis of vanillin from simple phenolic precursors. LAB were not able to form vanillin from eugenol, isoeugenol or vanillic acid. However Oenococcus oeni or Lactobacillus sp. could convert ferulic acid to vanillin, but in low yield. Only Lactobacillus sp. or Pediococcus sp. strains were able to produce significant quantities of 4-vinylguaiacol from ferulic acid. Moreover, LAB reduced vanillin to the corresponding vanillyl alcohol. CONCLUSIONS: The transformation of phenolic compounds tested by LAB could not explain the concentrations of vanillin observed during LAB growth in contact with wood. SIGNIFICANCE AND IMPACT OF THE STUDY: Important details of the role of LAB in the conversion of phenolic compounds to vanillin have been elucidated. These findings contribute to the understanding of malolactic fermentation in the production of aroma compounds.     

Capsinoid is biosynthesized from phenylalanine and valine in a non-pungent pepper, Capsicum annuum L. cv. CH-19 sweet

The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [(3)H]Valine and [(14)C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. (14)C radioactivity was observed in phenylpropanoid compounds, and in vanillin, vanillylamine, vanillyl alcohol, and vanillic acid. We confirmed that capsinoid is biosynthesized from phenylalanine and valine.     

Harnessing eugenol as a substrate for production of aromatic compounds with recombinant strains of Amycolatopsis sp. HR167

To harness eugenol as cheap substrate for the biotechnological production of aromatic compounds, the vanillyl alcohol oxidase gene (vaoA) from Penicillium simplicissimum CBS 170.90 was cloned in an expression vector suitable for Gram-positive bacteria and expressed in the vanillin-tolerant Gram-positive strain Amycolatopsis sp. HR167. Recombinant strains harboring hybrid plasmid pRLE6SKvaom exhibited a specific vanillyl alcohol oxidase activity of 1.1U/g protein. Moreover, this strain had gained the ability to grow on eugenol as sole carbon source. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, guajacol, and vanillic acid were detected as excreted compounds during growth on eugenol, whereas vanillin could only be detected in trace amounts. Resting cells of Amycolatopsis sp. HR167 (pRLE6SKvaom) produced coniferyl alcohol from eugenol with a maximum conversion rate of about 2.3 mmol/h/l of culture, and a maximum coniferyl alcohol concentration of 4.7 g/1 was obtained after 16 h biotransformation without further optimization. Beside coniferyl alcohol, traces of coniferyl aldehyde and ferulic acid were also detected.     

Metabolism of isoeugenol via isoeugenol-diol by a newly isolated strain of Bacillus subtilis HS8

A bacterium designated as HS8 was newly isolated from soil based on its ability to degrade isoeugenol. The strain was identified as Bacillus subtilis according to its 16S rDNA sequence analysis and biochemical characteristics. The metabolic pathway for the degradation of isoeugenol was examined. Isoeugenol-diol, for the first time, was detected as an intermediate from isoeugenol to vanillin by a bacterial strain. Isoeugenol was converted to vanillin via isoeugenol-diol, and vanillin was then metabolized via vanillic acid to guaiacol by strain HS8. These metabolites, vanillin, vanillic acid, and guaiacol, are all valuable aromatic compounds in flavor production. At the same time, the bipolymerization of isoeugenol was observed, which produced dehydrodiisoeugenol and decreased the vanillin yield. High level of vanillic acid decarboxylase activity was detected in cell-free extract. These findings provided a detailed profile of isoeugenol metabolism by a B. subtilis strain for the first time, which would improve the production of valuable aromatic compounds by biotechnology.     

Biotransformation of ferulic acid to acetovanillone using Rhizopus oryzae

Microbial transformation of ferulic acid to acetovanillone was studied using growing cells of Rhizopus oryzae. Ferulic acid was added to the growing medium (0.5 g L^?1) and incubated for 12 days. The progress of formation of metabolites was monitored by GC and GC-MS after extraction with ethyl acetate. The major metabolite was acetovanillone with minor metabolites formed, such as dihydroferulic acid, coniferyl alcohol and dihydroconiferyl alcohol. Traces of metabolites (≤1–3%), such as vanillin, vanillyl alcohol, vanillic acid and phenyl ethyl alcohol, were also produced. Formation of 4-vinyl guaiacol increased from day 1 (12.4%), reaching a maximum on day 4 (31.7%), and reducing to a minimum on day 12 (3.1%). The formation of acetovanillone increased only from day 2 onward, and reached a maximum (49.2%) on day 12. The optimum concentration of ferulic acid to be added into the medium was found to be only 0.5 g L^?1, as any increase in concentration (0.75 and 1.0 g L^?1) precipitated the precursor, resulting in no further degradation.     

Biotransformation of constituents of essential oils by germinating wheat seed

Wheat seeds, when exposed to essential oils, are able to metabolise certain monoterpenes. The actual amounts of the compounds and their derivatives in the endosperm and embryo of wheat seeds, after exposure to the monoterpenes were determined. Neral and geranial, which are the constituents of citral, are reduced and oxidised to the corresponding alcohols and acids. Similarly citronellal, pulegone and carvacrol are converted partly to the corresponding reduction and oxidation products. The aromatic compound vanillin is partly reduced to vanillyl alcohol or oxidised to vanillic acid. In all cases it seems that part of the compounds applied are degraded, as indicated by the inability to account for all the compounds, which were supplied to the germinated seeds. In most cases the derivatives of the essential oil applied were less toxic than the parent compound. The possible role of non-specific enzymes by which the compounds are oxidised or reduced is discussed.