Bernard Kurek

Hydrolysis of wheat bran and straw by an endoxylanase: production and structural characterization of cinnamoyl-oligosaccharides

Hydrolysis of wheat bran and wheat straw by a 20.7 kDa thermostable endoxylanase released 35 and 18% of the cell-wall xylan content, respectively. Separation of the cinnamoyl-oligosaccharides (accounting for 6%) from the bulk of total oligosaccharides was achieved by specific anion-exchange chromatography. The cinnamoyl-oligosaccharides were further purified by preparative paper chromatography (PPC) and their molecular weight was determined by MALDI-TOF mass spectrometry. The partially purified hydrolysis end-products contained from 4 to 16 and from 4 to 12 pentose residues for wheat bran and straw, respectively, and only one cinnamic acid per molecule. The primary structure of the new feruloyl arabinoxylopentasaccharide from wheat bran hydrolysis, which has been determined using 2D NMR spectroscopy, is O-beta-D-xylopyranosyl-(1-->4)-O-[5-O- (feruloyl)-alpha-L-arabinofuranosyl-(1-->3)]-O-beta-D-xylopyranosy l-(1-->4) -O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose.

The unmasking of lignin structures in wheat straw by alkali

This study reports on the structural modifications of wheat straw cell wall promoted by potassium carbonate and sodium hydroxide that lead to the unmasking of some lignin structures. The first impact of the treatments was the extraction of a particular fraction of lignin enriched in C-C linked structures compared to the mean composition in reference wheat straw. Concomitantly, an apparent increase in the amount of lignin monomers released by the cleavage of alkyl-aryl ether bonds was observed in alkali-extracted samples. By summing the amount of ether linked monomers analyzed by thioacidolysis in the solubilized lignin to that found in the extracted wheat straw, an excess of up to 37% is apparent, relative to the corresponding amount in the reference wheat straw. Other modifications of the cell wall were also found. Indeed, a fraction of uronic acids was lost during the treatments and a new fractionation pattern of the lignin-carbohydrate complexes was evidenced. It can thus be concluded that a significant proportion of lignin within the cell wall was unmasked after (i) the selective removal of a particular lignin fraction, (ii) a partial saponification of the esterified fraction of lignin with uronic acids and (iii) a modification of the interactions between the cell wall constituents.

Influence of the physical state of lignin on its degradability by the lignin peroxidase of Phanerochaete chrysosporium

Abstract The rate of oxidation of spruce milled wood lignin by Phanerochaete chrysosporium was investigated with precipitated lignin as well as lignin finely dispersed in water (colloidal lignin). H2O2 consumption rates in the course of lignin oxidation in the presence of purified lignin peroxidase were much higher with colloidal lignin compared with precipitated lignin. Characterization of reaction products and molecular-size distribution confirms that reaction rate of lignin peroxidase with 14C-lignin is strongly dependent on the physical state of lignin. The lignin peroxidase catalyse mainly repolymerization of colloidal lignin. However, in the presence of veratryl alcohol, both polymerization and depolymerization were observed.

Physiological regulation of glyoxal oxidase from Phanerochaete chrysosporium by peroxidase systems

Glyoxal oxidase (GLOX) is an H2O2-producing enzyme secreted by ligninolytic cultures of Phanerochaete chrysosporium. The oxidase is reversibly inactivated during purification, but can be reactived when coupled to lignin peroxidase (LiP) with veratryl alcohol as the peroxidase substrate. To characterize the modulation of this extracellular oxidase activity, we studied effects of pH, peroxide concentration, peroxidase source (fungal vs plant), and peroxidase substrate with recombinant GLOX (rGLOX). Our results show that a peroxidase system is not required for rGLOX activity. However, the activity is transient and the enzyme is partly and reversibly inactivated by the produced peroxide. rGLOX activity is more sustained at pH 6 than pH 4.5, and therefore the activation at pH 4.5 by a coupled peroxidase system is more clearly demonstrable. Results with peroxidase substrates of widely varying redox potentials strongly suggest that oxidized intermediates produced by coupled peroxidases are the GLOX activators. Both LiP and horseradish peroxidase (HRP) may be used to fully activate rGLOX using methoxybenzenes as peroxidase substrates. Notably, rGLOX is activated when lignin itself is used in coupled reactions with LiP. In contrast, guaiacol and catechols are both inactivating and lignin degradation products are expected to have similar effects. Taken together, our results suggest that ligninolysis by peroxidase could be regulated by GLOX activity and influenced by the presence of veratryl alcohol, lignin, and lignin degradation products. Such coordinated metabolism would influence the kinetics of free radical generation by the LiPs and, therefore, the overall efficiency of lignin depolymerization.

Oxidative Degradation of Alkali Wheat Straw Lignin by Fungal Lignin Peroxidase, Manganese Peroxidase and Laccase: A Comparative Study

Lignin peroxidase (LiP), manganese peroxidase (MnP) from Phanerochaete chrysosporium and laccase from Pleurotus eryngii were separately used to degrade alkali wheat straw lignin (AL). In order to characterize the catalytic action of the different enzymes, the chemical structure and the hydrodynamic properties of treated lignin were analyzed by thioacidolysis-gas chromatography and molecular size exclusion chromatography. The results confirmed that only LiP was able to degrade guaiacyl (G) and syringyl (S) structures in non-phenolic methylated lignins. However, provided that some phenolic terminal structures are present, MnP and laccase were able to degrade the non-phenolic part of the polymer linked by β-O-4 alkyl aryl ether bonds. This suggested that the oxidative reactions catalyzed in alkali straw lignin could progress through bond cleavages generating phenoxy radicals. The molecular size distribution of both thioacidolysis products and the oxidized polymer showed that AL underwent condensation side-reactions regardless of the enzymic treatment, but only LiP oxidation led to the increase in the hydrodynamic volume of the recovered lignin. This indicated that modification by enzymes of bonding patterns in lignin is not always associated with alterations in the spatial network of the polymer.

Viscoelastic properties of woody hemp core

Abstract The aim of this study was to determine the effect of removing extractives from the woody core of hemp (chènevotte) on the chain mobility of hemicelluloses and lignins, which can react during technological transformation such as de-fibering and/or composite materials production. Extractives are molecules with low molecular weight, which are present in the cell wall matrix and can be readily removed by solvents. In the present paper, the nature and amounts of extractives, removed under different conditions and with solvents of different polarities, were determined. The mobility and structural relaxations of lignins and hemicelluloses were stu-died in situ by dynamic mechanical analysis and dielectric analysis under controlled moisture content. Extractions at low temperature led to rigidification of lignins and plasticizing of hemicelluloses, probably due to local changes by the selective removal of molecules interacting with the polymers. Probably, the accessibility of hemicelluloses to plasticizing water was increased at controlled humidity. In contrast, hot extractions including water induced rigidification of the hemi-celluloses and plasticizing of lignins. This could be related to a combination of molecule extractions and chemical modi-fications of both polymers. This interpretation is supported by the variation of activation energy for relaxation of hemi-celluloses. It can be concluded that each type of extraction has a clear specific effect on the relaxation properties of the amorphous cell wall polymers.

Abiotic and enzymatic degradation of wheat straw cell wall: a biochemical and ultrastructural investigation.

The action of an abiotic lignin oxidant and a diffusible xylanase on wheat straw was studied and characterized at the levels of the molecular structures by chemical analysis and of the cell wall ultrastructure by transmission electron microscopy. While distinct chemical changes in the target polymers were observed when each system was used separately, a combination of the two types of catalysts did not significantly increase either lignin oxidation or hemicellulose hydrolysis. Microscopic observations however revealed that the supramolecular organization of the cell wall polymers was significantly altered. This suggests that the abiotic Mn-oxalate complex and the xylanase cooperate in modifying the cell wall architecture, without noticeably enhancing the degradation of the constitutive polymers.

Oxidation of spruce lignin by fungal lignin peroxidase and horseradish peroxidase: Comparison of their actions on molecular structure of the polymer in colloidal solution

Abstract Oxidation of spruce milled-wood lignins in colloidal state catalyzed by horseradish peroxidase (HRP) and lignin peroxidase (LiP) in the presence of H 2 O 2 was compared in nonbuffered dimethyl-formamide/water. Lignins were characterized for their hydrodynamic properties by size-exclusion chromatography and structural bonding pattern by analysis after thioacidolysis. In contrast to LiP, HRP did not induce modification of lignin hydrodynamic properties. The lignin content in β-O-4-linked guaiacyl monomers and dimeric structures, however, decreased after oxidation by the two enzymes, indicating large structural changes in the polymer. Preferential degradation by both LiP and HRP of β-5 and β-1 lignin dimeric units was also observed. This suggests a greater susceptibility to enzyme oxidation, as compared to the 5-5′ and 4-O-5 lignin substructures. In conclusion, the action of HRP on the lignin polymer is similar in many respects to that of fungal LiP, but is distinctly different in its inability to cause a net destructuring of the macromolecular three-dimensional network.

Energy saving with fungal enzymatic treatment of industrial poplar alkaline peroxide pulps

An alkaline peroxide industrial pulp from poplar was treated with a manganese peroxidase (MnP) from the hypersecretory strain of Phanerochaete chrysosporium I-1512 after a second stage of refining. The enzymatic treatment caused an improvement in the pulp quality by inducing an enzymatic refining onto the fibers. Transmission electron microscopy showed that the enzymatic refining was characterized by internal and external fibrillation of the fibers. Chemical modifications of lignin caused by MnP treatment facilitated the post-refining stage before papersheet manufacture. All together the enzymatic treatment resulted in energy savings of 25% during beating.

Structural features of lignin determining its biodegradation by oxidative enzymes and related systems

Peroxidases and laccases are key enzymes in the lignin biodegradation process. They oxidize phenolic and non-phenolic lignin model compounds into their phenoxy and cation radicals, respectively. Further non-enzymatic evolution lead then to various C-C and ether bond cleavages. Nevertheless, almost no information on the structural alterations undergone in vitro or in situ by lignin after enzymatic catalysis is available. We report here on the molecular structure of lignin oxidized by various (per)oxidasic systems. The oxidizability of phenolic and non-phenolic structures of the guaiacyl and syringyl type in the lignin network will be discussed as well as the modification of the macromolecular properties of the polymer oxidized in situ or in isolated state.