Effects of a biologically relevant antioxidant on the dehydrogenative polymerization of coniferyl alcohol

Dehydrogenation polymers (DHPs or synthetic lignins) were synthesized from coniferyl alcohol by enzymatic oxidation in the presence of ascorbic acid to study the potential effects of an antioxidant upon their structure. Specific interunit substructures (beta-O-4', beta-beta', and beta-5') were quantified by 13C NMR, which showed how ascorbic acid altered their amounts compared with control syntheses without this antioxidant, especially by increasing the amount of beta-O-4' substructures. The effect of ascorbic acid increased with its concentration. Surprisingly, no influence on the sizes of the synthetic lignins, as determined by size exclusion chromatography, was observed. The chemistry of this antioxidant effect during dehydrogenative polymerization and the potential biological significance (cell wall lignification) of these observations are discussed.     

In vitro analysis of the monolignol coupling mechanism using dehydrogenative polymerization in the presence of peroxidases and controlled feeding ratios of coniferyl and sinapyl alcohol

In this study, dehydrogenative polymers (DHP) were synthesized in vitro through dehydrogenative polymerization using different ratios of coniferyl alcohol (CA) and sinapyl alcohol (SA) (10:0, 8:2, 6:4, 2:8, 0:10), in order to investigate the monolignol coupling mechanism in the presence of horseradish peroxidase (HRP), Coprinus cinereus peroxidase (CiP) or soybean peroxidase (SBP) with H₂O₂, respectively. The turnover capacities of HRP, CiP and SBP were also measured for coniferyl alcohol (CA) and sinapyl alcohol (SA), and CiP and SBP were found to have the highest turnover capacity for CA and SA, respectively. The yields of HRP-catalyzed DHP (DHP-H) and CiP-catalyzed DHP (DHP-C) were estimated between ca. 7% and 72% based on the original weights of CA/SA in these synthetic conditions. However, a much lower yield of SBP-catalyzed DHP (DHP-S) was produced compared to that of DHP-H and DHP-C. In general, the DHP yields gradually increased as the ratio of CA/SA increased. The average molecular weight of DHP-H also increased with increasing CA/SA ratios, while those of DHP-C and DHP-S were not influenced by the ratios of monolignols. The frequency of β-O-4 linkages in the DHPs decreased with increasing CA/SA ratios, indicating that the formation of β-O-4 linkages during DHP synthesis was influenced by peroxidase type.     

Enzymatic production of cyclodextrins

Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.     

Aggregation during coniferyl alcohol polymerization in pectin solution: a biomimetic approach of the first steps of lignification

Coniferyl alcohol was polymerized in pectin solution in order to mimic the lignification that is the final step of biosynthesis of plant cell wall. Dehydrogenated polymers (DHP = coniferyl alcohol polymers = synthetic lignin) interact with pectin to form hydrophobic clusters as monitored by pyrene fluorescence spectroscopy. The structure of these clusters was studied during the polymerization of synthetic lignin by static and quasielastic light scattering and small angle neutron scattering experiments. We show that synthetic lignin and pectin contribute to the same clusters, but the inner structure of these clusters is very heterogeneous and displays three phases. One observes a segregation between well separated pectin and lignin rich phases at length scales below approximately 30 nm. As a corollary of this segregation, clusters embody a large amount of solvent. On average, the density of the polymer rich phase (lignin plus pectin) inside clusters increases while its specific surface area decreases throughout the polymerization process. These results are discussed with respect to in vivo lignification of the plant cell wall.     

Bacterial degradation of dehydropolymers of coniferyl alcohol

A bacterial isolate identified as Xanthomonas sp. proved to be ligninolytic due to its ability to degrade ¹⁴C-labeled dehydropolymers of coniferyl alcohol (DHP) and [14C]lignocellulose complexes from corn plants (Zea mays). Several parameters of ligninolysis were evaluated and it was shown that resting cells degrade DHP as sole carbon source. Enhancement of DHP degradation in the presence of ferulic acid or water-soluble fractions of DHP or of dioxane lignin from wheat was demonstrated. It is shown that a dissociation of DHP takes place during incubation in the absence of the bacteria which is reflected in a shift of DHP to lower molecular weight fractions. Bacterial degradation of [14C] DHP results in the release of ¹⁴CO₂ and in the incorporation of the ¹⁴C-label into the biomass of the bacteria, as shown by chemical and biological methods.Abbreviations Bq Becquerel, measure for radioactivity according to SI nomenclature - DHP dehydropolymers of coniferyl alcohol - DMF dimethylformamide - DMSO dimethyl sulfoxide - HPLC high performance liquid chromatography - TCA trichloroacetic acid - THF tetrahydrofuran     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia

[This corrects the article on p. 1445 in vol. 46.].     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Biodegradation of hydrocarbons in the presence of cyclodextrins

Aliphatic hydrocarbons are one of the main components of oil contamination. Bioremediation is considered to be a cost-effective treatment option among the conventional treatment methods with bioavailability being the limitation. Chemical surfactants could be used to increase the bioavailability of the hydrocarbons but they showed marked toxicity and environmental pollution. Cyclodextrins are cyclic oligosaccharides which can alter the solubility of the hydrocarbons by incorporating suitably sized hydrophobic molecules into their hydrophobic cavities. This paper focuses on studying the degradation of hydrocarbons by Pseudomonas like species named as Vid1 isolated previously from bilge oil contaminated waters in the presence of cyclodextrins. Among the three cyclodextrins (α, β and γ) tested at different concentrations, 2.5 mM of β-cyclodextrin showed higher amount of biodegradation when n-hexadecane was used as a model hydrocarbon compound. The percentage of residual hexadecane remaining in the 2.5 mM β-cyclodextrin supplied medium at 120 h was found to be 15% in comparison with the biotic control which was 43%. In the next experimental setup, degradation of mixture of hydrocarbons (tetradecane, hexadecane and octadecane) by Vid1 (Pseudomonas like species) was studied at a concentration of 2.5 mM β-cyclodextrin. The residual percentage of tetradecane, hexadecane and octadecane at 120 h was found to be 32, 43 and 61% in comparison with the biotic control 50, 58 and 67%, respectively. Our studies show that among a mixture of hydrocarbons (tetradecane, hexadecane and octadecane) in the presence of β-cyclodextrin, the highest concentration of hydrocarbon degradation was found in tetradecane, hexadecane and octadecane, respectively.     

Stereospecificity of cinnamyl alcohol dehydrogenase and synthesis of stereospecifically labelled coniferyl alcohol

Using horse liver alcohol dehydrogenase, stereospecifically tritiated (R)- and (S)-(γ-³H)-coniferyl alcohol was synthesized. Using both of these substrates it was demonstrated that cinnamyl alcohol dehydrogenase from lignifying Forsythia tissue specifically removes the pro-R-hydrogen atom of coniferyl alcohol in the oxidation to the aldehyde. This also means that in the reverse reaction the A-hydrogen of NADPH is transferred to the Re-site of coniferyl aldehyde.     

Anaerobic degradation of coniferyl alcohol by methanogenic consortia.

Coniferyl alcohol was shown to be completely biodegradable to carbon dioxide and methane under strictly anaerobic culture conditions. The mineralization of 300 mg of the substrate per liter was observed in acclimated ferulic acid-degrading methanogenic consortia, as well as in anaerobic enrichments on coniferyl alcohol seeded with sewage sludge. Ferulic and phenylpropionic acids were detected in the cultures degrading coniferyl alcohol as the sole carbon and energy source, suggesting that this compound is oxidized to ferulic acid, which is then degraded as previously described.     

Enzymatic synthesis of oligosaccharides from branched cyclodextrins

6'-alpha-Maltosyl-maltotriose and 6'-alpha-D-glucosyl-maltotriose were prepared from Novamyl degradation of 6-O-alpha-maltosyl-alpha-cyclodextrin and 6-O-alpha-D-glucosyl-alpha-cyclodextrin, respectively. NMR spectroscopy was used to elucidate their structural identities, in a combination of COSY experiments. Further, a mechanism for the degradation was proposed based on the Novamyl active site geometry.     

Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.     

Hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins.

The oxidation of xenobiotics by the hydroperoxidase activity of lipoxygenase in the presence of cyclodextrins was studied. These produced an inhibitory effect on xenobiotics oxidation, based on their degree of hydrophobicity and the charge (isoproterenol < 4-methyl-catechol (4MC) < 4-tert-butylcatechol (TBC) < 4-tert-octylcatechol (TOC)). This inhibitory effect was due to the complexation of xenobiotics in the hydrophobic cavity of cyclodextrins. The complexation constant Kc was calculated by nonlinear regression of the inhibition curves obtained in the presence of cyclodextrins, and the values obtained were 400, 16,250, and 35,127 M-1 for 4MC, TBC, and TOC, respectively. The validity of these values was checked at different points of the Michaelis-Menten saturation curve, and a sigmoidal inhibition curve was obtained at the saturating concentration of the o-diphenol, TBC, with no change in the Kc value. This demonstrates the validity of the equations used to calculate Kc for the complete range of the Michaelis-Menten equation.     

Fractal analysis of STM images of photochemical polymer of coniferyl alcohol

Fractal analysis was applied to images of photochemical lignin polymer obtained using scanning tunneling microscope. We studied the polymer obtained in vitro by ionic mechanism through UV radiation--induced polymerization. The analysis showed the regularity of the lignin-like polymer at different levels of organization. At the 95% confidence level, there was no significant difference in the fractal dimension between images representing different organizational levels of photochemical lignin. That means that lignin produced in in vitro conditions by photochemical mechanism of synthesis, has a fractal structural organization. The obtained values of the fractal dimension are in good agreement with the theoretically predicted value for the polyaddition and polycondensation mechanism of polymerization, known as the bulk model.     

Spectral comparisons of coniferyl and cinnamyl alcohol epoxide derivatives with a purported cis-epoxyconiferyl alcohol isolate

The reported isolation of cis-epoxyconiferyl alcohol must be incorrect, based upon comparison of the reported Nuclear Magnetic Resonance (NMR) spectral data for the isolate with those for synthesized coniferyl and cinnamyl alcohol epoxide derivatives. Attempts to prepare cis- and trans-coniferyl alcohols were unsuccessful, although their acetate derivatives could be synthesized. The NMR spectral data for a synthetic sample of pinoresinol were in excellent agreement with those for the purported isolate.     

Partial purification and characterization of a coniferyl alcohol dehydrogenase fromRhodococcus erythropolis

A nicotinamide adenine dinucleotide (NAD)-dependent coniferyl alcohol dehydrogenase was enriched 1,200-fold from crude extracts ofRhodococcus erythropolis. The purification procedure involved ion exchange chromotography, gel filtration on Biogel A 1,5 and Sephadex G-200, and hydroxyapatite treatment. The enzyme had a molecular weight of approximately 200,000 and displayed maximal activity at pH 9.0. The apparentK _m values for NAD and coniferyl alcohol were, respectively, 0.22 and 0.645 mM. Nicotinamide adenine dinucleotide phosphate (NADP) could only partially replace NAD. The enzyme was active with vanillyl alcohol and aromatic alcohols bearing the ,-unsaturated side chain of coniferyl alcohols. These aromatic alcohols included the dilignols dehydrodiconiferyl alcohol and guaiacylglycerol--coniferyl ether.     

Microbial synthesis of coniferyl alcohol by the fungus Byssochlamys fulva V107.

Coniferyl alcohol (123 mM = 21.9 g/l) was synthesized from eugenol with a yield of 94.6% in a 36 h fed-batch bioconversion using resting cells of the fungus Byssochlamys fulva V107.     

Distribution of lignin and its coniferyl alcohol and coniferyl aldehyde groups in Picea abies and Pinus sylvestris as observed by Raman imaging

Wood cell wall consists of several structural components, such as cellulose, hemicelluloses and lignin, whose concentrations vary throughout the cell wall. It is a composite where semicrystalline cellulose fibrils, acting as reinforcement, are bound together by amorphous hemicelluloses and lignin matrix. Understanding the distribution of these components and their functions within the cell wall can provide useful information on the biosynthesis of trees. Raman imaging enables us to study chemistry of cell wall without altering the structure by staining the sample or fractionating it. Raman imaging has been used to analyze distributions of lignin and cellulose, as well as the functional groups of lignin in wood. In our study, we observed the distribution of cellulose and lignin, as well as the amount of coniferyl alcohol and aldehyde groups compared to the total amount of lignin in pine (Pinus sylvestris) and spruce (Picea abies) wood samples. No significant differences could be seen in lignin and cellulose distribution between these samples, while clear distinction was observed in the distribution of coniferyl alcohols and coniferyl aldehyde in them. These results could provide valuable insight on how two similar wood species control biosynthesis of lignin differently during the differentiation of cell wall.     

Ferulate–coniferyl alcohol cross-coupled products formed by radical coupling reactions

Radical coupling reactions between ethyl ferulate (Et-FA), a simple model for feruloyl polysaccharides in planta, and coniferyl alcohol (CA), a monolignol, were studied in order to better understand the polymer cross-coupling interactions among polysaccharides and monolignols or lignin, mediated by ferulate (FA), in plant cell walls. Cross-coupled FA/CA dimers produced in an aqueous buffer (pH 5.0) containing peroxidase/hydrogen peroxide were isolated and characterized by NMR. The total coupling products were characterized by 2D ¹³C–¹H correlation (HSQC) NMR spectroscopy and GC–MS. Results from this study showed that ferulate readily cross-couples with coniferyl alcohol through free radical coupling mechanisms producing a series of cross-coupled FA/CA dimers with β-O-4-, β-5-/8-5-, and 8-β-linkages; the syntheses and isolation of β-5- and 8-5-cross-coupled dimers are reported here. The transformation from 8-β-coupled FA/CA hydroxyl esters into lactones through intramolecular transesterification is demonstrated for the first time and mechanisms behind these transformations are discussed. The finding of both β-5- and 8-5-cross-coupled dimers in this study suggests that analogs of both may be present in plant cell walls. Finally it is suggested that ferulates in plants indeed react with monolignols through free radical mechanisms producing a more diverse array of cross-coupled dimers than previously reported.