Deciphering the enzymatic grafting of vanillin onto lignosulfonate for the production of versatile aldehydes-bearing biomaterials.

Modification of lignin plays a crucial role in extending its applications. While chemical functionalization has been extensively applied, exploring the enzyme-catalyzed approach for grafting phenolic molecules presents a promising avenue. Herein, we investigate the controlled laccase-mediated grafting of vanillin onto lignosulfonates (LS) as a sustainable approach to introduce aldehydes into LS, paving the way for further (bio)chemical functionalizations (e.g., reductive amination and Knoevenagel-Doebner condensations). The resulting vanillin-grafted LS is comprehensively characterized (HPLC, SEC, Pyrolysis-GC/MS, FTIR). The study reveals four key steps in the grafting process: (i) vanillin acts as a mediator, generating the phenoxyl radical that initiates LS oxidation, (ii) the oxidation leads to depolymerization of LS, resulting in a decrease in molecular weight, (iii) rearrangement in the vanillin-grafted LS, evidenced by the replacement of labile bonds by stronger 5-5 bonds that resist to pyrolysis, and (iv) if the reaction is prolonged after complete consumption of vanillin, condensation of the vanillin-grafted LS occurs, leading to a significant increase in molecular weight. This study provides valuable insights on the behavior of vanillin and LS throughout the process and allows to identify the optimal reaction conditions, thereby enhancing the production of vanillin-grafted LS for its subsequent functionalization.

Use of Pyrolysis-Gas Chromatography/Mass Spectrometry as a Tool to Study the Natural Variation in Biopolymers in Different Tissues of Economically Important European Softwood Species.

Intraspecific macromolecule variation in stemwood, knotwood, and branchwood was studied using analytical pyrolysis with the intention of introducing a rapid working method to assess the variance in lignin content using analytical pyrolysis and highlight variability markers. The study was performed on Picea abies, Abies alba, and Pseudotsuga menziesii. Lignin determined via analytical pyrolysis-GC/MS (Py-lignin) can be used to identify variations in lignin content, compared to using classical Klason lignin values as a reference method for lignin determination, which requires a correction factor. Principal component analysis (PCA) was performed to identify biopolymer pyrolysis product markers for different species, tissues, or heights that could help highlight structural differences. Douglas fir was differentiated from spruce and silver fir in the levoglucosan amount. Guaiacol was more present in spruce wood, and creosol was more present in Douglas fir. Knotwood was structurally close to stemwood in spruce and silver fir, but there was a clear transition between stemwood and branchwood tissue in Douglas fir. Knotwood was differentiated by higher furan compounds. Branchwood was clearly separate from stemwood and knotwood and presented the same markers as compression wood in the form of phenylpropanoid lignins (H-lignin) as well as isoeugenol and vinyl guaiacol, the two most produced lignin pyrolysis products.

Knotwood and Branchwood Polyphenolic Extractives of Silver Fir, Spruce and Douglas Fir and Their Antioxidant, Antifungal and Antibacterial Properties.

The extractive contents of three softwood species largely used in the wood industry, namely Abies alba (Silver fir), Picea abies (spruce) and Pseudotsuga menziesii (Douglas fir), have been determined quantitatively for knots and at different points chosen along their branches, before analysis using high-performance liquid chromatography coupled with Mass Spectrometry (HPLC-MS). The results indicated that branchwood samples located in close proximity to the stem present high contents of extractives similar to those recorded for the knots. HPLC analysis showed quite similar chemical compositions, indicating that first cm of the branches could be considered as an additional source of knotwood. The antibacterial, antifungal and antioxidant activities of knot's extractives have been investigated with the dual objective of better understanding the role of high levels of secondary metabolites present in the knot and evaluating their potential for biorefinery applications. The antioxidant activity study showed that crude extracts of Douglas fir knotwood presented higher radical scavenging activity levels than the extracts of Silver fir and spruce, which presented more or less the same activities. Silver fir and spruce knotwood extracts presented higher antibacterial activity levels than the Douglas fir knotwood extracts did, while Douglas fir knotwood extracts presented more fungal growth inhibition than the spruce and fir knotwood extracts did. The structure-activity relationships indicate that radical scavenging and antifungal activities are associated with a higher relative quantity of flavonoids in the crude extracts, while higher relative quantities of lignans are associated with antibacterial activity.

Effect of Tannins Addition on Thermal Stability of Furfurylated Wood.

This article presents the effect of the addition of condensed tannins, used as a reticulation agent, on the polymerization of furfuryl alcohol during wood furfurylation, as well as the effect of these condensed tannins on the thermal stability of modified wood. Three kinds of dicarboxylic acids (adipic acid, succinic acid, and tartaric acid), as well as glyoxal, used as model of a wood reticulation agent, were used to catalyze the polymerization of furfuryl alcohol or tannin-furfuryl alcohol solutions. Impregnation of furfuryl alcohol or tannin-furfuryl alcohol solution into the wood, followed by curing at 103 °C for a specific duration, was performed for the wood modification. The thermal stability of the obtained tannin-furfuryl alcohol polymers and their corresponding modified woods was investigated. The leaching resistance and dimensional stability of the modified woods were also evaluated. Results indicated that the partial substitution of furfuryl alcohol by the tannins improved the polymerization reactivity in conditions where furfuryl alcohol alone did not lead to the formation of a solid polymeric material. The thermal stability and leaching resistance of the furfurylated wood in the presence of tannins were improved. Dimensional stability was also improved for furfurylated samples, but the effect of tannin addition was not so obvious, depending on the acidic catalyst used.

Highly Branched Tannin-Tris(2-aminoethyl)amine-Urea Wood Adhesives.

Condensed tannin copolymerized with hyperbranched tris(2-aminoethyl)amine-urea formed by amine-amido deamination yields a particleboard thermosetting adhesive without any aldehydes satisfying the requirements of relevant standards for the particleboard internal bond strength. The tannin-triamine-urea cures well at 180 °C, a relatively low temperature for today's particleboard hot pressing. As aldehydes were not used, the formaldehyde emission was found to be zero, not even in traces due to the heating of wood. The effect is ascribed to the presence of many reactive sites, such as amide, amino, and phenolic groups belonging to the three reagents used. The tannin appears to function as an additional cross-linking agent, almost a nucleating agent, for the triamine-urea hyperbranched oligomers. Chemical analysis by MALDI ToF and 13C NMR has shown that the predominant cross-linking reaction is that of the substitution of the tannin phenolic hydroxyls by the amino groups of the triamine. The reaction of tannin with the still-free amide groups of urea is rather rare, but it may occur with the rarer tannin flavonoid units in which the heterocyclic ring is opened. Due to the temperature gradient between the surfaces and the board core in the particleboard during hot pressing, the type and the relative balance of covalent and ionic bonds in the resin structure may differ in the surfaces and the board core.

Elicitation of Antimicrobial Active Compounds by Streptomyces-Fungus Co-Cultures.

The bacteria of the genus Streptomyces and Basidiomycete fungi harbor many biosynthetic gene clusters (BGCs) that are at the origin of many bioactive molecules with medical or industrial interests. Nevertheless, most BGCs do not express in standard lab growth conditions, preventing the full metabolic potential of these organisms from being exploited. Because it generates biotic cues encountered during natural growth conditions, co-culture is a means to elicit such cryptic compounds. In this study, we explored 72 different Streptomyces-fungus interaction zones (SFIZs) generated during the co-culture of eight Streptomyces and nine fungi. Two SFIZs were selected because they showed an elicitation of anti-bacterial activity compared to mono-cultures. The study of these SFIZs showed that co-culture had a strong impact on the metabolic expression of each partner and enabled the expression of specific compounds. These results show that mimicking the biotic interactions present in this ecological niche is a promising avenue of research to explore the metabolic capacities of Streptomyces and fungi.

Study of the effectiveness of a presoaking process for reducing the additives migration from babies toys.

This research aims to study a process of steeping in the n-heptane, used for reducing the migration of additives contained initially in toys for babies plasticized with di-octylphtalate (DOP) based on poly (vinyl chloride) (PVC) and stabilized with epoxidized sunflower oil (ESO). Two formulations were carried out at different levels of DOP plasticizers (15% and 45%). The migration tests were conducted in the synthetic saliva in the absence and in the presence of α-amylase with or without agitation at 37° C for 1, 3, and 6 h. The migration phenomenon was studied on the basis of preliminary studies based on the mass variation of the two formulations and where the physico-chemical technical analysis: Fourier-transform infrared spectroscopy (FTIR) and gas chromatography coupled with gas chromatography-mass spectrometry (GC-MS) were performed. This work shows that the presoak method can be used successfully to reduce the migration phenomenon of the additives and to decrease the interactions between the PVC samples and the saliva stimulant. This treatment has allowed a notable decrease of the overall migration of all the additives from saliva. It is noted that the high pH value (7.17) was obtained with the F45% formulation under agitation and in the presence of α-amylase, a mass loss of the order of 0.9004 and a minimum DOP concentration of 0.024 ppm. The analysis by GC-MS provided the DOP chromatograms of the control and the specimens, which have undergone migration tests and treatments. In addition, the amount of DOP, migrated in the case of the F15% and F45%, controls the formulations and was greater than those of the presoaked formulations, which have indicated the efficiency of the applied process. This study shows that migration has taken place, and that the soaking treatment has reduced the migration of all the additives present in the PVC samples.

Glutathione Transferases: Surrogate Targets for Discovering Biologically Active Compounds.

Glutathione transferases comprise a large class of multifunctional enzymes, some involved in detoxification pathways. Since these enzymes are able to interact with potentially toxic molecules, they could be used as targets to screen for compounds with biological activity. To test this hypothesis, glutathione transferases (GSTs) from the white-rot fungus Trametes versicolor have been used to screen for antifungal molecules from a library of tropical wood extracts. The interactions between a set of six GSTs from the omega class and 116 extracts from 21 tropical species were quantified using a high-throughput thermal shift assay. A correlation between these interactions and the antifungal properties of the tested extracts was demonstrated. This approach has been extended to the fractionation of an Andira coriacea extract and led to the detection of maackiain and lapachol in this wood. Altogether, the present results supported the hypothesis that such detoxification enzymes could be used to detect biologically active molecules.

Yield and compositions of bark phenolic extractives from three commercially significant softwoods show intra- and inter-specific variation.

Tree bark is rich in commercially valuable secondary metabolites such as polyphenolic compounds like flavonoids and tannins. The yield and composition of bark extractives from Abies alba varies longitudinally within the stem. A. alba bark above the crown had the greatest extractive content, but the concentration of polyphenolic compounds was greatest below the crown. Here, we use a nonlinear model describing how bark extractive yields of A. alba, Picea abies and Pseudotsuga menziesii change with height, where differences among species are accounted for using different model coefficients. For all species there is longitudinal variability in extract yield. For P. abies and P. menziesii, the form of that variation differs depending on whether the bark was located at the same position in the stem as branches. Although the relationship form of total extractive yield differs between branched and un-branched samples, the relationship forms for individual compound yields does not change depending on branch presence. Despite trees from thinned stands having longer crowns and faster growth rates, indicative of greater photosynthetic activity, there was no evidence that thinning had affected either the yield or composition of extractives in these species. In P. abies, the proportions of flavonoids was higher in bark from the top of the tree, whereas epi-gallocatechin gallate was found in high proportions at the stem base. In P. menziesii bark extracts, taxifolin was the dominant compound, present in significantly higher proportions in bark towards the base of the stem.

In-situ extraction of depolymerization products by membrane filtration against lignin condensation.

Catalytic depolymerization of lignin is a challenging process due to competitive repolymerization reactions. In this paper, the oxidative depolymerization of lignin was catalyzed by a commercial laccase both in a batch experiment and in a membrane bioreactor using the same catalytic conditions. The membrane bioreactor was previously optimized to reach high permeation flux (25 L.h-1.m-2) during lignin diafiltration. While the lignin was exclusively condensed in the batch experiment leading to high molecular weight macromolecules (from 9 to 16 kDa), its depolymerization was effective in the bioreactor producing fragments of less than 1 kDa thanks to the in-situ extraction of the reaction products. This paper demonstrates that the reactor configuration is playing an essential role in triggering or preventing lignin condensation. It also reports the first proof-of-concept demonstrating that in-situ membrane extraction of the reactive fragments of lignin from the bulk medium can be useful against detrimental repolymerization reactions.

Mining the Biosynthetic Potential for Specialized Metabolism of a Streptomyces Soil Community.

The diversity and distribution of specialized metabolite gene clusters within a community of bacteria living in the same soil habitat are poorly documented. Here we analyzed the genomes of 8 Streptomyces isolated at micro-scale from a forest soil that belong to the same species or to different species. The results reveal high levels of diversity, with a total of 261 biosynthesis gene clusters (BGCs) encoding metabolites such as terpenes, polyketides (PKs), non-ribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs) with potential bioactivities. A significant part of these BGCs (n = 53) were unique to only one strain when only 5 were common to all strains. The metabolites belong to very diverse chemical families and revealed that a large diversity of metabolites can potentially be produced in the community. Although that analysis of the global metabolome using GC-MS revealed that most of the metabolites were shared between the strains, they exhibited a specific metabolic pattern. We also observed that the presence of these accessory pathways might result from frequent loss and gain of genes (horizontal transfer), showing that the potential of metabolite production is a dynamic phenomenon in the community. Sampling Streptomyces at the community level constitutes a good frame to discover new biosynthetic pathways and it appears as a promising reservoir for the discovery of new bioactive compounds.

A reverse chemical ecology approach to explore wood natural durability.

The natural durability of wood species, defined as their inherent resistance to wood-destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white-rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long-term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood-decaying fungi and also wood natural durability.

Lignin Carbohydrate Complexes structure preserved throughout downstream processes for their valorization after recovery from industrial process water.

Firstly studied and pointed out because of their major role in wood recalcitrance to delignification, LCCs are gradually raising interest about their own valorization thanks to their various interesting properties. Considering their future industrial production, this work is focused on evaluating the impact of different downstream processes (DSP) on their structure including moist heat sterilization, simple effect evaporation and spray-drying. LCCs were recovered from the mill water of a thermomechanical plant by successive membrane filtration steps. Samples were taken after the different DSP and compared to a freeze-dried control. Global and structural characterizations given by elemental analysis, acid hydrolysis, pyrolysis coupled to gas-chromatography, SEC, 13C and 2D-HSQC NMR showed that the LCCs structure is well preserved during all of these DSP. This paper demonstrates the feasibility of producing high quality industrial LCC grade for commercialization in integrated biorefineries.

Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium.

Extensive evidence showed that the efficiency of fungal wood degradation is closely dependent on their ability to cope with the myriad of putative toxic compounds called extractives released during this process. By analysing global gene expression of Phanerochaete chrysosporium after short oak extractive treatment (1, 3 and 6 h), we show that the early molecular response of the fungus concerns first mitochondrial stress rescue followed by the oxidation and finally conjugation of the compounds. During these early responses, the lignolytic degradative system is not induced, rather some small secreted proteins could play an important role in cell protection or signaling. By focusing on the functional characterization of an hitherto uncharacterized glutathione transferase, we show that this enzyme interacts with wood molecules suggesting that it could be involved in the detoxification of some of them, or act as a scavenger to prevent their cytosolic toxicity and favour their transport.

Molecular recognition of wood polyphenols by phase II detoxification enzymes of the white rot Trametes versicolor.

Wood decay fungi have complex detoxification systems that enable them to cope with secondary metabolites produced by plants. Although the number of genes encoding for glutathione transferases is especially expanded in lignolytic fungi, little is known about their target molecules. In this study, by combining biochemical, enzymatic and structural approaches, interactions between polyphenols and six glutathione transferases from the white-rot fungus Trametes versicolor have been demonstrated. Two isoforms, named TvGSTO3S and TvGSTO6S have been deeply studied at the structural level. Each isoform shows two distinct ligand-binding sites, a narrow L-site at the dimer interface and a peculiar deep hydrophobic H-site. In TvGSTO3S, the latter appears optimized for aromatic ligand binding such as hydroxybenzophenones. Affinity crystallography revealed that this H-site retains the flavonoid dihydrowogonin from a partially purified wild-cherry extract. Besides, TvGSTO6S binds two molecules of the flavonoid naringenin in the L-site. These data suggest that TvGSTO isoforms could interact with plant polyphenols released during wood degradation.

Derivatives of the Lignan 7'-Hydroxymatairesinol with Antioxidant Properties and Enhanced Lipophilicity.

The lignan 7'-hydroxymatairesinol (1), extracted from the knotwoods of fir (Abies alba), spruce (Picea abies), and Douglas fir (Pseudotsuga menziesii), exhibited unexpected reactivity when esterification reactions were attempted on the hydroxy group at position C-7'. To circumvent the rapid intramolecular cyclization procedure, leading quantitatively to the lignan conidendrin (7), a simple strategy for 7'-esterification of 1 under mild conditions (three steps, up to 80% overall yield) was developed. Compared to hydroxymatairesinol (1) (log K'w = 1.49), the derivatives (2-5) had increased lipophilicity with log K'w > 3.1, as determined by a UHPLC method. Compounds 1-5 exhibited potent antioxidant properties in the same range as the standards ascorbic acid and α-tocopherol (IC50 = 20-25 µM) and higher than that of BHT using a DPPH radical-scavenging assay.

Hepatoprotective and antidiabetic effects of Pistacia lentiscus leaf and fruit extracts.

Pistacia lentiscus (Anacardiaceae) is commonly used in folk medicine to treat various diseases. The aim of the present study was to evaluate the hepatoprotective and antioxidant activities of extracts of P. lentiscus leaves (PL) and fruits (PF) against experimentally induced liver damage. Furthermore, characterization of extracts was attempted by a spectroscopic methodology (Fourier transform infrared spectroscopy) and high-performance liquid chromatography with diode array detection analysis. A hepatoprotective potential against paracetamol [165 mg/kg body weight (b.w.)] toxicity was noticed in mice pretreated with the same dose of PL or PF extract (125 mg/kg b.w.) or a combination of both (PL/PF 63/63 mg/kg b.w.), as revealed by an analysis of biochemical parameters (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase activities and total bilirubin). These results were confirmed by histological examination of the liver, which revealed significant protection against paracetamol-induced hepatic necrosis. Furthermore, PF extract exhibited a promising antidiabetic activity in streptozotocin-induced diabetic rats, similar to the reference drug glibenclamide (0.91 g/L), a result confirmed by in vitro inhibition of α-amylase. We demonstrated that the leaf crude extract showed the best effect in all tested methods, compared to its fruit counterpart, probably due to the presence of higher amounts of phenolic compounds, as determined by phytochemical and Fourier transform infrared spectroscopy analyses. Moreover, high-performance liquid chromatography with diode array detection led to the identification of six compounds for each part of the plant. Gallic acid, a characteristic compound of Pistacia species, was most abundant in leaves and fruits, while luteolin was detected for the first time in fruits. Obtained activities of P. lentiscus extracts may well be due, at least in part, to the presence of the above compounds.

The GSTome Reflects the Chemical Environment of White-Rot Fungi.

White-rot fungi possess the unique ability to degrade and mineralize all the different components of wood. In other respects, wood durability, among other factors, is due to the presence of extractives that are potential antimicrobial molecules. To cope with these molecules, wood decay fungi have developed a complex detoxification network including glutathione transferases (GST). The interactions between GSTs from two white-rot fungi, Trametes versicolor and Phanerochaete chrysosporium, and an environmental library of wood extracts have been studied. The results demonstrate that the specificity of these interactions is closely related to the chemical composition of the extracts in accordance with the tree species and their localization inside the wood (sapwood vs heartwood vs knotwood). These data suggest that the fungal GSTome could reflect the chemical environment encountered by these fungi during wood degradation and could be a way to study their adaptation to their way of life.

Glutathionyl-hydroquinone reductases from poplar are plastidial proteins that deglutathionylate both reduced and oxidized glutathionylated quinones.

Glutathionyl-hydroquinone reductases (GHRs) catalyze the deglutathionylation of quinones via a catalytic cysteine. The two GHR genes in the Populus trichocarpa genome, Pt-GHR1 and Pt-GHR2, are primarily expressed in reproductive organs. Both proteins are localized in plastids. More specifically, Pt-GHR2 localizes in nucleoids. At the structural level, Pt-GHR1 adopts a typical GHR fold, with a dimerization interface comparable to that of the bacterial and fungal GHR counterparts. Pt-GHR1 catalyzes the deglutathionylation of both reduced and oxidized glutathionylated quinones, but the enzyme is more catalytically efficient with the reduced forms.

Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.