Studies of xylan interactions and cross-linking to synthetic lignins formed by bulk and end-wise polymerization: a model study of lignin carbohydrate complex formation

The mechanism of lignin carbohydrate complex formation by addition of polysaccharides on quinone methide (QM) generated during lignin polymerisation was investigated using a model approach. Dehydrogenation polymers (DHPs, lignin model compounds) were synthesized from coniferyl alcohol in the presence of a glucuronoarabinoxylan (GAX) extracted from oat spelts, by Zutropfverfahren (ZT) and Zulaufverfahren (ZL) methods. The methods ZT and ZL differed in their distribution of QM over the reaction period but generated roughly the same QM amount. Steric exclusion chromatography of the ZT and ZL reaction products showed that only the ZT reaction produced high molar mass compounds. Covalent linkages in the ZT reaction involving ether bonds between GAX moiety and α carbon of the lignin monomer were confirmed by ¹³C NMR and xylanase-based fractionation. The underlying phenomena were further investigated by examining the interactions between GAX and DHP in sorption experiments. GAX and DHPs were shown to interact to form hydrophobic aggregates. In the ZT process, slow addition permitted polymer reorganisation which led to dehydration around the lignin-like growing chains thereby limiting the addition of water on the quinone methide formed during polymerisation and thus favoured lignin–carbohydrate complex (LCC) formation.     

Effect of reaction media concentration on the solubility and the chemical structure of lignin model compounds

In plant cell walls, lignin polymerization occurs in concentrated polysaccharide gel. The effect of high polysaccharide concentrations on the structure of lignin-like polymers (DHPs=dehydrogenation polymers), were investigated by running lignification-like polymerization under three reaction conditions in which the concentrations of all reactants (xylan/coniferyl alcohol (CA)/oxidising system) were gradually increased. Control experiments were also run in similar conditions but without polysaccharides. DHPs showed increased solubility with increased concentrations of reactants in the presence of xylans but were mostly insoluble in buffer control experiments. The structures of DHPs were characterized by thioacidolysis and size exclusion chromatography (SEC). Results indicated that the frequency of beta-alkyl aryl ether bonds and DHP molecular weight increased with increasing concentration of the reaction mixture in the presence of xylans whereas those of control DHPs decreased slightly under the same conditions. This emphasizes the role of the pre-existing polysaccharide gel and high concentrations existing in the cell wall during construction of the lignin polymer.     

Peroxidase activity can dictate the in vitro lignin dehydrogenative polymer structure

The objective of this study was to assess the influence of the peroxidase/coniferyl alcohol (CA) ratio on the dehydrogenation polymer (DHP) synthesis. The soluble and unsoluble fractions of horseradish peroxidase (HRP)-catalyzed CA dehydrogenation mixtures were recovered in various proportions, depending on the polymerization mode (Zutropf ZT/Zulauf ZL) and HRP/CA ratio (1.6-1100purpurogallin U mmol(-1)). The ZL mode yielded 0-57%/initial CA of unsoluble condensed DHPs (thioacidolysis yields <200micromolg(-1)) with a proportion of uncondensed CA end groups increasing with the HRP/CA ratio (7.2-55.5%/total uncondensed CA). Systematically lower polymer yields (0-49%/initial CA) were obtained for the ZT mode. In that mode, a negative correlation was established between the beta-O-4 content (thioacidolysis yields: 222-660micromolg(-1)) and the HRP/CA ratio. In both modes, decreasing the HRP/CA ratio below 18Ummol(-1) favoured an end-wise polymerization process evidenced by the occurrence of tri-, tetra- and pentamers involving at least one beta-O-4 bond. At low ratio, the unsoluble ZT DHP was found to better approximate natural lignins than DHPs previously synthesized with traditional methods. Besides its possible implication in lignin biosynthesis, peroxidase activity is a crucial parameter accounting for the structural variations of in vitro DHPs.     

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.     

On the role of the monolignol γ-carbon functionality in lignin biopolymerization

In order to investigate the importance of the monomeric γ-carbon chemistry in lignin biopolymerization and structure, synthetic lignins (dehydrogenation polymers; DHP) were made from monomers with different degrees of oxidation at the γ-carbon, i.e., carboxylic acid, aldehyde and alcohol. All monomers formed a polymeric material through enzymatic oxidation. The polymers displayed similar sizes by size exclusion chromatography analyses, but also exhibited some physical and chemical differences. The DHP made of coniferaldehyde had poorer solubility properties than the other DHPs, and through contact angle of water measurement on spin-coated surfaces of the polymeric materials, the DHPs made of coniferaldehyde and carboxylic ferulic acid exhibited higher hydrophobicity than the coniferyl alcohol DHP. A structural characterization with ¹³C NMR revealed major differences between the coniferyl alcohol-based polymer and the coniferaldehyde/ferulic acid polymers, such as the predominance of aliphatic double bonds and the lack of certain benzylic structures in the latter cases. The biological role of the reduction at the γ-carbon during monolignol biosynthesis with regard to lignin polymerization is discussed.     

Extracellular laccases and peroxidases from sycamore maple (Acer pseudoplatanus) cell-suspension cultures

We have investigated the abilities of extracellular enzymes from dark-grown cell-suspension cultures of sycamore maple (Acer pseudoplatanus L.) to oxidize monolignols, the precursors for lignin biosynthesis in plants, as well as a variety of other lignin-related compounds. Laccase and peroxidase both exist as a multiplicity of isoenzymes in filtrates of spent culture medium, but their abilities to produce water-insoluble, dehydrogenation polymers (DHPs) from the monolignols (in the presence of hydrogen peroxide for the peroxidase reaction) appear identical whether or not the enzymes are purified from the concentrated filtrates or left in a crude mixture. The patterns of bonds formed in these DHPs are identical to those found in DHPs synthesized using horseradish peroxidase or fungal laccase, and many of these bonds are found in the natural lignins extracted from different plant sources. On the other hand, sycamore maple laccase is very much less active on phenolic substrates containing multiple aromatic rings than is sycamore maple peroxidase. We suggst that whereas laccase may function during the early stages of lignification to polymerize monolignols into oligo-lignols, cell-wall peroxidases may function when H₂O₂ is produced during the later stages of xylem cell development or in response to environmental stresses.Abbreviations DHP dehydrogenation polymer - IEF isoelectric focuring - NMR nuclear magnetic resonance - PAGE polyacrylamide gel electrophoresis The authors wish to thank Dr. Masahiro Samejima (University of Tokyo) for provision of lignin model compounds and Dr. Göran Gellerstadt (Royal Institute of Technology, Sweden) for helpful suggestions regarding stilbene formation and light spectroscopy. Monolignols were prepared by Mr. Nate Weymouth with help from Dr. Herb Morrison (USDA/ARS, Richard B. Russell Research Center, Athens, GA). Thanks also to Ms. Izabella Poppe of the Complex Carbohydrate Research Center (CCRC) for assistance with carbohydrate analyses, and Mr. Vincent Sorrentino for help with the growth of cell-suspension cultures.     

Polymerization of coniferyl alcohol by Mn3+‐mediated (enzymatic) oxidation: Effects of H2O2 concentration,aqueous organic solvents,and pH

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)‐malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn²⁺ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5 ≤ pH ≤ 6.0 and with 0.75 ≤ H₂O₂:CA ratio ≤ 1. The results obtained from the Mn³⁺‐malonate‐mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis‐GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H₂O₂/CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of β–β linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81–90, 2018     

Structural differences between the lignin-carbohydrate complexes present in wood and in chemical pulps

Lignin-carbohydrate complexes (LCCs) were prepared in quantitative yield from spruce wood and from the corresponding kraft and oxygen-delignified pulps and were separated into different fractions on the basis of their carbohydrate composition. To obtain an understanding of the differences in lignin structure and reactivity within the various LCC fractions, thioacidolysis in combination with gas chromatography was used to quantify the content of beta-O-4 structures in the lignin. Periodate oxidation followed by determination of methanol was used to quantify the phenolic hydroxyl groups. Furthermore, size exclusion chromatography (SEC) of the thioacidolysis fractions was used to monitor any differences between the original molecular size distribution and that after the delignification processes. Characteristic differences between the various LCC fractions were observed, clearly indicating that two different forms of lignin are present in the wood fiber wall. These forms are linked to glucomannan and xylan, respectively. On pulping, the different LCCs have different reactivities. The xylan-linked lignin is to a large extent degraded, whereas the glucomannan-linked lignin undergoes a partial condensation to form more high molecular mass material. The latter seems to be rather unchanged during a subsequent oxygen-delignification stage. On the basis of these findings, a modified arrangement of the fiber wall polymers is suggested.     

Effects of dihydropyridines on calcium release from the isolated membrane complex consisting of the transverse tubule and sarcoplasmic reticulum.

We have investigated a) the effects of the dihydropyridines (DHPs) nifedipine and nimodipine on depolarization-induced (T-tubule-mediated) Ca2+ release in the vesicles consisting of the complex of the T-tubule and SR, and b) the binding of [3H]nimodipine to these vesicles. These DHPs inhibited the slow but not the fast phase of depolarization-induced release, both of which are mediated via the T-tubule. The DHPs have no effect on caffeine-induced release in which T-tubules are not involved. There are two classes of DHP binding sites: one, with high affinity and small capacity, and another, exhibiting low affinity and a much larger capacity. The inhibition paralleled the low affinity binding of DHP with no correlation with the high affinity binding. These results suggest that the low affinity DHP binding sites located probably in the DHP receptor, rather than the high affinity DHP binding site, are responsible for the inhibition of e-c coupling.     

<Emphasis Type="Italic">Phoma herbarum</Emphasis>, a soil fungus able to grow on natural lignin and synthetic lignin (DHP) as sole carbon source and cause lignin degradation

The fungus Phoma herbarum isolated from soil showed growth on highly pure lignin extracted from spruce wood and on synthetic lignin (DHP). The lignin remaining after cultivation was shown to have a lower molecular weight. The reduction in the numbers of ether linkages of the extracted lignins was also observed by derivatization followed by reductive cleavage (DFRC) in combination with ³¹P NMR studies. The fungal strain showed an ability to degrade synthetic lignin by extracellular catalysts. GC–MS was applied to study the evolution of low molar mass adducts, e.g., monolignols and it was shown that a reduced coniferyl alcohol product was produced from DHP in a cell-free environment. The work has demonstrated the ability of soil microbes to grow on lignin as sole carbon source. The potential impact is in the production of low molar mass renewable phenols for material application.     

Enhancing the enzymatic hydrolysis of lignocellulosic biomass by increasing the carboxylic acid content of the associated lignin

To assess the effects that the physical and chemical properties of lignin might have on the enzymatic hydrolysis of pretreated lignocellulosic substrates, protease treated lignin (PTL) and cellulolytic enzyme lignin (CEL) fractions, isolated from steam and organosolv pretreated corn stover, poplar, and lodgepole pine, were prepared and characterized. The adsorption of cellulases to the isolated lignin preparations corresponded to a Langmuir adsorption isotherm. It was apparent that, rather than the physical properties of the isolated lignin, the carboxylic acid functionality of the isolated lignin, as determined by FTIR and NMR spectroscopy, had much more of an influence when lignin was added to typical hydrolysis of pure cellulose (Avicel). An increase in the carboxylic content of the lignin preparation resulted in an increased hydrolysis yield. These results suggested that the carboxylic acids within the lignin partially alleviate non-productive binding of cellulases to lignin. To try to confirm this possible mechanism, dehydrogenative polymers (DHP) of monolignols were synthesized from coniferyl alcohol (CA) and ferulic acid (FA), and these model compounds were added to a typical enzymatic hydrolysis of Avicel. The DHP from FA, which was enriched in carboxylic acid groups compared with the DHP from CA, adsorbed a lower mount of cellulases and did not decrease hydrolysis yields when compared to the DHP from CA, which decreased the hydrolysis of Avicel by 8.4%. Thus, increasing the carboxylic acid content of the lignin seemed to significantly decrease the non-productive binding of cellulases and consequently increased the enzymatic hydrolysis of the cellulose.     

Nonmodal gating of cardiac calcium channels as revealed by dihydropyridines

The hypothesis that dihydropyridine (DHP)-sensitive calcium channels have three distinct modes of gating has been examined. The major prediction is that the relative frequencies among modes depend on DHP concentration while the kinetics within a mode do not. We tested this by studying whole-cell and single-channel calcium currents in neonatal rat and adult guinea pig cardiac myocytes in different concentrations of several DHPs. In the absence of DHPs calcium currents declined with time but the kinetics, which are the focus of this study, were unchanged. Open-time frequency distributions had insignificant numbers of prolonged openings and were well fit by single tau's. Agonist DHP stereoisomers produced concentration-dependent changes in whole-cell tail current tau's. The frequency distribution of single calcium channel current open times became biexponential and the tau's were concentration dependent. The average number of openings per trace of channels with customary open times increased with increases in DHP concentration. Latencies to first opening for the customary openings and for prolonged openings were shorter in the presence of DHPs. A second larger conductance is another important feature of DHP-bound single calcium channels. Thus DHPs not only caused prolonged openings; they produced numerous changes in the kinetics of customary openings and increased channel conductance. It follows that these effects of DHPs do not support the hypothesis of modal gating of calcium channels. The mode model is not the only model excluded by the results; models in which DHPs are allowed to act only or mainly on open states are excluded, as are models in which the effects are restricted to inactivated states. We suggest a different type of model in which cooperative binding of DHPs at two sites produces the essential changes in kinetics and conductance.     

Synthesis, characterisation and water sorption properties of pectin-dehydrogenation polymer (lignin model compound) complex

Coniferyl alcohol was polymerised in the presence of pectin and a pectin-DHP complex was isolated. Characterisation of the complex has demonstrated that DHP (20% wt) was covalently linked by ester bonds to the pectin. The water sorption behaviour of the pectin-DHP complex was determined at several relative vapour pressures and compared with those obtained on pectin, DHP and a blend of both polymers in the same proportion as in the complex. The complex exhibited a lower hydrophilicity resulting from three associated phenomena: grafting, pectin-DHP interactions and the formation of a chemical network.     

Structure and optical properties of plant cell wall bio-inspired materials: cellulose-lignin multilayer nanocomposites

Interfacial affinity between lignin model compound (dehydrogenation polymer [DHP]) and cellulose nanocristals (CN) was studied before building a nanocomposite cellulose/lignin in multilayer form by spin-coating method. The adsorption isotherm of DHP was measured by ellipsometry at the liquid/CN film interface and showed that the surface concentration of adsorbed DHP increases with the bulk concentration in solution. The DHP appeared as globular structures on cellulosic film, as observed by AFM. Spreading a dense lignin layer on CN film gave rise to the disappearance of the InfraRed resonance bands related to the DHP aromatics. The film obtained from alternate layers of cellulose/DHP was transparent in visible light and had weak absorption in UV wavelengths. Optical properties measured in the visible wavelength range by ellipsometry and spectrophotometry indicated that beyond six bilayers (cellulose/DHP), the composite exhibits antireflexion properties.     

Characterization of arabinoxylan-dehydrogenation polymer (synthetic lignin polymer) nanoparticles

Coniferyl alcohol (G monomer) and a mixture of coniferyl alcohol/sinapyl alcohol (GS monomers, 1/1 ratio) were polymerized to dehydrogenation polymers (DHPs) in presence of two structurally related heteroxylans (HX) differing only in their phenolic substitution patterns. One (HX-40) was enriched in ferulate (FA) while the other (HX-90) was almost devoid of FA. The morphology of the resulting nanoparticles was studied by transmission electron microscopy whereas formation of particles was followed by size exclusion chromatography with online multiangle laser light scattering. HX-40-DHP-G- and HX-40-DHP-GS-derived particles display complex morphological patterns whereas HX-90-DHP-G and HX-90-DHP-GS present rather spherical shapes. The determination of particle sizes and molar masses showed that HX-90 samples formed denser particles than HX-40 ones. These differences are discussed in relation to the ferulate substitution level.     

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.     

Dihydropyridine Action on Voltage-dependent Potassium Channels Expressed in Xenopus Oocytes

Dihydropyridines (DHPs) are well known for their effects on L-type voltage-dependent Ca²⁺ channels. However, these drugs also affect other voltage-dependent ion channels, including Shaker K⁺ channels. We examined the effects of DHPs on the Shaker K⁺ channels expressed in Xenopus oocytes. Intracellular applications of DHPs quickly and reversibly induced apparent inactivation in the Shaker K⁺ mutant channels with disrupted N- and C-type inactivation. We found that DHPs interact with the open state of the channel as evidenced by the decreased mean open time. The DHPs effects are voltage-dependent, becoming more effective with hyperpolarization. A model which involves binding of two DHP molecules to the channel is consistent with the results obtained in our experiments.     

beta-fluoro-coniferyl alcohol does not inhibit lignin biosynthesis in suspension cultures of Picea abies (L.) Karst

A fluorinated analogue of coniferyl alcohol has been reported to be a specific inhibitor of oxidases involved in the biosynthesis of lignin. The Z isomer of beta-fluoro-coniferyl alcohol was synthesized and used for the preparation of dehydrogenation polymers (DHPs) and was also tested on lignin producing suspension cultures of spruce (Picea abies (L.) Karst.). The growth of the cells or the production of lignin by the suspension cultures was not significantly affected by the addition of fluoroconiferyl alcohol. This analogue did not form polymers quite as easily as did coniferyl alcohol in oxidation with hydrogen peroxide and horseradish peroxidase. In both cases the beta-fluoroconiferyl alcohol became incorporated in the polymeric product. We were unable to detect any specific inhibition of peroxidase activity, which is at variance with earlier reports of pronounced inhibition of lignin biosynthesis in poplar plantlets by fluoroconiferin, a potential inhibitor of oxidases involved in lignin biosynthesis.     

The effect of mebudipine and dibudipine, two new Ca2+ channel blockers, in comparison with nifedipine on Ca2+ spikes of F1 neuronal soma membrane in Helix aspersa

Mebudipine and dibudipine are two new dihydropyridine (DHP) Ca2+ channel blockers that have been synthesized by Mahmoudian et al. (1997). In previous studies, they showed considerable relaxant effect on vascular and ileal smooth muscles. These two compounds also reduced the contraction force of rat left atrium (20, 22). In the present study, we attempted to compare the inhibitory actions of these new DHPs and nifedipine on the high threshold Ca2+ spikes of F1 neuronal soma membrane in the subesophageal ganglia of Helix aspersa, using current-clamp method. At a concentration of 1 microM, two new DHP compounds (mebudipine and dibudipine) were tested for their L-type Ca2+ channel blocker activity. Both compounds reversibly reduced the peak amplitude of action potential and after hyperpolarization potential and markedly decreased the duration of Ca2+ spikes. The most potent of these DHPs was mebudipine. Neither the two new DHPs nor nifedipine changed the resting membrane potential in a statistically significant way.     

Vascular Occlusions in the Stem of Ailanthus excelsa Roxb.

The stem of Ailanthus excelsa Roxb. (Simaroubaceae) may developvascular occlusions and gum-resin cavities in the xylem as aresponse to injury and infection. Fungal hyphae are observedin the vessel lumen and in the adjacent parenchyma cells. Theocclusions appeared in various forms and showed varying chemicalcomposition. The chemical nature of the occluding material differedfrom that of the exudate formed in the cavities. The majorityof occlusions contained lipids, protein, polysaccharides, phenolics,lignin and probably pectin, whereas, the exudate contained lipids,protein and four soluble sugars. All the components of the occlusionexcept lignin and pectin appear to have been formed in the parenchymacells adjacent to the vessel and migrated to vessel lumen throughthe vessel wall pits. The different constituents migrate intothe vessel lumen independently where they mix to form occlusions. Ailanthus excelsa Roxb., vascular occlusions, lignin, fluorescence, exudation