Abstract
Resting cells of Rhodotorula rubra converted transferulic acid (1) to vanillic acid (2), then to guaiacol (3) and protocatechuic acid (4), under aerobic conditions. In an argon atmosphere, R. rubra transformed ferulic acid to vanillic acid and 4-hydroxy-3-methoxystyrene (5). Metabolites were isolated by solid-phase extraction and characterized by mass spectrometry, 1H, and 13C-nuclear magnetic resonance spectroscopy (NMR). The biotransformation of ferulic acid to vanillic acid by R. rubra cell-free extracts required CoA, ATP, and NAD+. Mass spectrometry and 13C-NMR were used to demonstrate the incorporation of oxygen from H2(18)O during the conversion of ferulic acid to vanillic acid. The results suggest a parallel between this bioconversion reaction and the beta-oxidation of fatty acids. Proton-carbon correlation NMR spectroscopy was used to demonstrate the specific incorporation of deuterium from D2O into guaiacol obtained from vanillic acid. The incorporation of deuterium implicates the involvement of a quinoid vanillic acid tautomer as an intermediate in the decarboxylation reaction.
Highlights
(6).Metaboliteswere isolated by solid-phase extraction and characterized by mass spectrometry, 'H, and 13Cnuclear magnetic resonance spectroscopy (NMR)
The results suggest a parallel between this bioconversion reaction and the &oxidation of fatty acids
Protoncarbon correlation NMR spectroscopy was used to acid, 99%),vanillic acid [2] (4-hydroxy-3-methoxybenzoiaccid, 97%), guaiacol [3](2-methoxyphenol, 98%),protocatechuic acid [4] (3, 4dihydroxybenzoic acid, 97%), and H;'O (95% "0 atom) were purchased from Aldrich Chemical Company (Milwaukee, WI). 4-Hydroxy-3-methoxystyrene ( 5 ) was from Lancaster(Windham, NH) and isolated and characterized in our laboratory [1].The purities of purchased compounds and their structures were confirmed by TLC, HPLC,' and NMR spectral analysis
Summary
(6).Metaboliteswere isolated by solid-phase extraction and characterized by mass spectrometry, 'H, and 13Cnuclear magnetic resonance spectroscopy (NMR). Chemicals-Trans-ferulic acid [1](4-hydroxy-3-methoxycinnamic rubra cell-freeextracts required CoA, ATP, and NAD+. Mass spectrometry and "C-NMR were used to demonstrate the incorporation of oxygen from HZ"O during the conversion of ferulic acid to vanillic acid. We are exploiting the use of contained (w/v) 2% glucose, 0.5% yeast extract, 0.5% soybean meal, enzymes and microbial transformations as means of generat- 0.5% NaC1, and 0.5% K2HP04in distilled water and was adjusted to ing value-addedproducts from ferulic acid [1].An understanding of the biochemical and chemical mechanisms involved in ferulic acid biotransformationsis required asatheoretical basis for the ultimate development of biocatalytic processes pH 7.0 with 6 N HC1 before being autoclaved at 121 "C for 15 min. A 10%inoculum derived from 60-h-old stage I cultures was used to initiate stage I1 cultures, which were incubated for 24 h before addition of solid ferulic acid as for the production of large amounts of ferulic acid-derived substrate to a final concentration of 1mg/ml in medium
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