Vanillin Production Pathways in Alkaline Nitrobenzene Oxidation of Guaiacylglycerol-ß-guaiacyl Ether.

Alkaline nitrobenzene oxidation (AN oxidation) is a significant method for chemical analysis of lignin. Despite its importance in lignin chemistry, the detailed chemical reactions involved in AN oxidation are not yet fully understood. Surprisingly, there is almost no experimentally supported information available regarding the reaction pathways in the AN oxidation of guaiacyl glycerol-ß-guaiacyl ether (GG), a common model compound in lignin chemistry. This study reports the results of our investigation into the formation pathway of vanillin (4-hydroxy-3-methoxybenzaldehyde) in the AN oxidation of GG. Our series of experiments proposed a vanillin formation pathway involving an enol ether with a C2 side chain, 2-methoxy-4-[2-(2-methoxyphenoxy)-ethenyl]-phenol C2EE, as an intermediate, in which C2EE is produced by the non-oxidative degradation of GG by alkali. Another enol ether with a C3 side-chain, Z-4-[3-hydroxy-2-(2-methoxyphenoxy)-1-propen-1-yl]-2-methoxyphenol (C3EE), and the condensation products formed under alkaline conditions were found to be insignificant as vanillin sources. On the other hand, the comparison of the vanillin yields from GG and isolated C2EE (80.7 and 86.5 mol %, respectively) in their AN oxidation to the C2EE yield from GG in the absence of nitrobenzene (69.9 mol %) also suggested that the vanillin formation from GG involved unknown pathways in which C2EE is not an intermediate.

Degradation of the phenolic ß-ether lignin model dimer and dyes by dye-decolorizing peroxidase from Bacillus amyloliquefaciens.

OBJECTIVE: The dye-decolorizing peroxidase from Bacillus amyloliquefaciens, BaDyP, was identified to be an efficient catalyst for the degradation of phenolic ß-ether lignin model dimer guaiacylglycerol-ß-guaiacyl ether (GGE) and dyes. RESULTS: Efeb gene encoding BaDyP from B. amyloliquefaciens MN-13 consisted of 1257 bp and the open reading frame encoded 418 amino acids. The efeb gene was expressed in Escherichia coli BL21 and a recombinant BaDyP of 50 kDa was achieved. The BaDyP exhibited activity in oxidizing GGE and decolorizing azo and triphenylmethane dyes. At pH 4.5 and 30 °C the BaDyP not only completely degraded GGE by the cleavage of ß-O-4 ether bond and Cα-Cß bond, and Cα oxidation without any oxidative mediator, but also decolorized four synthetic dyes, including congo red, bromine cresol green, eriochrome black T and crystal violet. This was achieved with decolorization rates of 65.7%, 70.62%, 80.06% and 62.09%, respectively, after 72 h of incubation. CONCLUSIONS: BaDyP was identified as a bacteria peroxidase with great potential for the degradation of lignin and bioremediation of dye-contamination.

Bacillus amyloliquefaciens CotA degradation of the lignin model compound guaiacylglycerol-ß-guaiacyl ether.

The cotA gene from Bacillus amyloliquefaciens MN-13 was cloned and expressed in Escherichia coli Transetta. Nucleotide sequence analysis showed an open reading frame of 1542 bp encoding a polypeptide comprised of 513 amino acids. The degradation of lignin model compounds by recombinant CotA was investigated by HPLC-MS with guaiacylglycerol-ß-guaiacyl ether as the substrate. The compounds including guaiacol, 3-(4-hydro-3-methoxyphenyl)-3-oxo-propanol and 4-hydro-3-methoxy acetophenone detected by HPLC-MS verified the rupture of ß-O-4 bond and oxidation Cα bond of guaiacylglycerol-ß-guaiacyl ether by CotA. 4-vinylguaiacol and 1-(4-hydroxy-3-methoxyl phenyl)-1-(2-methoxyl) phenoxyl ethylene were first time found in the degradation products of guaiacylglycerol-ß-guaiacyl ether. The appearance of 4-vinylguaiacol and 4-hydro-3-methoxy acetophenone confirmed the cleavage of Cß-Cγ bond. 1-(4-hydroxy-3-methoxyl phenyl)-2-(2-methoxyl) phenoxyl ethylene was coupled by the radical reaction of 4-vinylguaiacol with guaiacol. Otherwise, no corresponding degradation product was found to give a proof of cleavage of Cα-Cß bond in guaiacylglycerol-ß-guaiacyl ether by CotA.

Phylogenetic and kinetic characterization of a suite of dehydrogenases from a newly isolated bacterium, strain SG61-1L, that catalyze the turnover of guaiacylglycerol-ß-guaiacyl ether stereoisomers.

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-ß-guaiacyl ether (GGE), which contains a key ß-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.

Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol beta-aryl ether linkages.

Cleavage of the arylglycerol beta-aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial beta-etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the beta-aryl ether linkage cleavage of high-molecular lignin in the soil fungi. We screened and isolated 2BW-1 cells by using a highly sensitive fluorescence assay system. The beta-aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW-1, and is secreted into the extracellular fraction. The beta-aryl ether cleavage enzyme converts the guaiacylglycerol beta-O-guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the C alpha alcohol structure and p-hydroxyl group and specifically attacks the beta-aryl ether linkage of high-molecular mass lignins with addition of two water molecules at the C alpha and C beta positions.

Degradation of labelled lignins and veratrylglycerol-beta-guaiacyl ether by Acinetobacter sp.

Acinetobacter sp. evolved 14CO2 from 14C-(ring)DHP lignin and 14C-teakwood lignin. Veratrylglycerol-beta-guaiacyl ether, a lignin model compound with beta-o-4 linkage was cleaved by Acinetobacter sp. Veratrylglycerol-beta-guaiacyl ether into 2(o-methoxyphenoxy) ethanol and veratrylalcohol 2(o-methoxyphenoxy) ethanol was degraded to guaiacol and then to catechol whereas veratrylalcohol was converted to veratraldehyde, veratric acid, vanillic acid, protocatechuic acid and catechol. Both catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase were detected in veratrylglycerol-beta-guaiacyl ether grown cultures.

Anaerobic degradation of veratrylglycerol-beta-guaiacyl ether and guaiacoxyacetic acid by mixed rumen bacteria.

Veratrylglycerol-beta-guaiacyl ether (0.2 g/liter), a lignin model compound, was found to be degraded by mixed rumen bacteria in a yeast extract medium under strictly anaerobic conditions to the extent of 19% within 24 h. Guaiacoxyacetic acid, 2-(o-methoxyphenoxy)ethanol, vanillic acid, and vanillin were detected as degradation products of veratrylglycerol-beta-guaiacyl ether by thin-layer chromatography, gas chromatography, and gas chromatography-mass spectrometry. Guaiacoxyacetic acid (0.25 g/liter), when added into the medium as a substrate, was entirely degraded within 36 h, resulting in the formation of phenoxyacetic acid, guaiacol, and phenol. These results suggest that the beta-arylether bond, an important intermonomer linkage in lignin, can be cleaved completely by these rumen anaerobes.

[Investigations of guaifenesine metabolism with gas chromatography-mass spectrometry]. / Unersuchungen zum Metabolismus von Guaifenesin mit Hilfe der Gaschromatographie-Massenspektrometrie.

Recently the authors about toxicological investigations after uptake of guaiacol glyceryl ether containing drugs. There had been detected two intensive pink spots on the TLC from urine samples after detection with Marquis-reagent. It is reported about the identification of Catechol glyceryl ether and Hydroxyguaifenesine via Gas Chromatography Mass Spectrometry. Until today only the oxidation of guaiacol glyceryl ether to beta-(2-methoxyphenoxy)lactic acid is described. The demethylation of G. is performed by O-Demethylase localized in liver microsomes. This enzyme seems to be the main enzyme for the metabolism of G. It is pointed out to a pharmacologically interesting relationship between the chemical structure of guaiacol glyceryl ether and codeine.