Publications by authors named "José C del Rio"

68 Publications

Radical Coupling Reactions of Hydroxystilbene Glucosides and Coniferyl Alcohol: A Density Functional Theory Study.

Front Plant Sci 2021 2;12:642848. Epub 2021 Mar 2.

Department of Energy Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin, Madison, WI, United States.

The monolignols, -coumaryl, coniferyl, and sinapyl alcohol, arise from the general phenylpropanoid biosynthetic pathway. Increasingly, however, authentic lignin monomers derived from outside this process are being identified and found to be fully incorporated into the lignin polymer. Among them, hydroxystilbene glucosides, which are produced through a hybrid process that combines the phenylpropanoid and acetate/malonate pathways, have been experimentally detected in the bark lignin of Norway spruce (). Several interunit linkages have been identified and proposed to occur through homo-coupling of the hydroxystilbene glucosides and their cross-coupling with coniferyl alcohol. In the current work, the thermodynamics of these coupling modes and subsequent rearomatization reactions have been evaluated by the application of density functional theory (DFT) calculations. The objective of this paper is to determine favorable coupling and cross-coupling modes to help explain the experimental observations and attempt to predict other favorable pathways that might be further elucidated via polymerization aided by synthetic models and detailed structural studies.
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http://dx.doi.org/10.3389/fpls.2021.642848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7960926PMC
March 2021

Structural Characteristics of the Guaiacyl-Rich Lignins From Rice ( L.) Husks and Straw.

Front Plant Sci 2021 19;12:640475. Epub 2021 Feb 19.

Department of Plant Biotechnology, Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Seville, Spain.

Rice ( L.) is a major cereal crop used for human nutrition worldwide. Harvesting and processing of rice generates huge amounts of lignocellulosic by-products such as rice husks and straw, which present important lignin contents that can be used to produce chemicals and materials. In this work, the structural characteristics of the lignins from rice husks and straw have been studied in detail. For this, whole cell walls of rice husks and straw and their isolated lignin preparations were thoroughly analyzed by an array of analytical techniques, including pyrolysis coupled to gas chromatography-mass spectrometry (Py-GC/MS), nuclear magnetic resonance (NMR), and derivatization followed by reductive cleavage (DFRC). The analyses revealed that both lignins, particularly the lignin from rice husks, were highly enriched in guaiacyl (G) units, and depleted in -hydroxyphenyl (H) and syringyl (S) units, with H:G:S compositions of 7:81:12 (for rice husks) and 5:71:24 (for rice straw). These compositions were reflected in the relative abundances of the different interunit linkages. Hence, the lignin from rice husks were depleted in β--4' alkyl-aryl ether units (representing 65% of all inter-unit linkages), but presented important amounts of β-5' (phenylcoumarans, 23%) and other condensed units. On the other hand, the lignin from rice straw presented higher levels of β--4' alkyl-aryl ethers (78%) but lower levels of phenylcoumarans (β-5', 12%) and other condensed linkages, consistent with a lignin with a slightly higher S/G ratio. In addition, both lignins were partially acylated at the γ-OH of the side-chain (ca. 10-12% acylation degree) with -coumarates, which overwhelmingly occurred over S-units. Finally, important amounts of the flavone tricin were also found incorporated into these lignins, being particularly abundant in the lignin of rice straw.
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http://dx.doi.org/10.3389/fpls.2021.640475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7932998PMC
February 2021

High Epoxidation Yields of Vegetable Oil Hydrolyzates and Methyl Esters by Selected Fungal Peroxygenases.

Front Bioeng Biotechnol 2020 5;8:605854. Epub 2021 Jan 5.

Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Seville, Spain.

Epoxides of vegetable oils and free and methylated fatty acids are of interest for several industrial applications. In the present work, refined rapeseed, sunflower, soybean, and linseed oils, with very different profiles of mono- and poly-unsaturated fatty acids, were saponified and transesterified, and the products treated with wild unspecific peroxygenases (UPOs, EC 1.11.2.1) from the ascomycete (UPO) and the basidiomycete (UPO), as well as with recombinant UPO of the ascomycete (rUPO), as an alternative to chemical epoxidation that is non-selective and requires strongly acidic conditions. The three enzymes were able of converting the free fatty acids and the methyl esters from the oils into epoxide derivatives, although significant differences in the oxygenation selectivities were observed between them. While UPO selectively produced "pure" epoxides (monoepoxides and/or diepoxides), UPO formed also hydroxylated derivatives of these epoxides, especially in the case of the oil hydrolyzates. Hydroxylated derivatives of non-epoxidized unsaturated fatty acids were practically absent in all cases, due to the preference of the three UPOs selected for this study to form the epoxides. Moreover, rUPO, in addition to forming monoepoxides and diepoxides of oleic and linoleic acid (and their methyl esters), respectively, like the other two UPOs, was capable of yielding the triepoxides of α-linolenic acid and its methyl ester. These enzymes appear as promising biocatalysts for the environmentally friendly production of reactive fatty-acid epoxides given their self-sufficient monooxygenase activity with selectivity toward epoxidation, and the ability to epoxidize, not only isolated pure fatty acids, but also complex mixtures from oil hydrolysis or transesterification containing different combinations of unsaturated (and saturated) fatty acids.
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http://dx.doi.org/10.3389/fbioe.2020.605854DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7813931PMC
January 2021

Differentiation of Tracheary Elements in Sugarcane Suspension Cells Involves Changes in Secondary Wall Deposition and Extensive Transcriptional Reprogramming.

Front Plant Sci 2020 18;11:617020. Epub 2020 Dec 18.

Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.

Plant lignocellulosic biomass, mostly composed of polysaccharide-rich secondary cell walls (SCWs), provides fermentable sugars that may be used to produce biofuels and biomaterials. However, the complex chemical composition and physical structure of SCWs hinder efficient processing of plant biomass. Understanding the molecular mechanisms underlying SCW deposition is, thus, essential to optimize bioenergy feedstocks. Here, we establish a xylogenic culture as a model system to study SCW deposition in sugarcane; the first of its kind in a C4 grass species. We used auxin and brassinolide to differentiate sugarcane suspension cells into tracheary elements, which showed metaxylem-like reticulate or pitted SCW patterning. The differentiation led to increased lignin levels, mainly caused by S-lignin units, and a rise in -coumarate, leading to increased -coumarate:ferulate ratios. RNAseq analysis revealed massive transcriptional reprogramming during differentiation, with upregulation of genes associated with cell wall biogenesis and phenylpropanoid metabolism and downregulation of genes related to cell division and primary metabolism. To better understand the differentiation process, we constructed regulatory networks of transcription factors and SCW-related genes based on co-expression analyses. Accordingly, we found multiple regulatory modules that may underpin SCW deposition in sugarcane. Our results provide important insights and resources to identify biotechnological strategies for sugarcane biomass optimization.
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http://dx.doi.org/10.3389/fpls.2020.617020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814504PMC
December 2020

Cell wall remodeling under salt stress: Insights into changes in polysaccharides, feruloylation, lignification, and phenolic metabolism in maize.

Plant Cell Environ 2020 09 13;43(9):2172-2191. Epub 2020 Jul 13.

Department of Biochemistry, State University of Maringá, Maringá, Brazil.

Although cell wall polymers play important roles in the tolerance of plants to abiotic stress, the effects of salinity on cell wall composition and metabolism in grasses remain largely unexplored. Here, we conducted an in-depth study of changes in cell wall composition and phenolic metabolism induced upon salinity in maize seedlings and plants. Cell wall characterization revealed that salt stress modulated the deposition of cellulose, matrix polysaccharides and lignin in seedling roots, plant roots and stems. The extraction and analysis of arabinoxylans by size-exclusion chromatography, 2D-NMR spectroscopy and carbohydrate gel electrophoresis showed a reduction of arabinoxylan content in salt-stressed roots. Saponification and mild acid hydrolysis revealed that salinity also reduced the feruloylation of arabinoxylans in roots of seedlings and plants. Determination of lignin content and composition by nitrobenzene oxidation and 2D-NMR confirmed the increased incorporation of syringyl units in lignin of maize roots. Salt stress also induced the expression of genes and the activity of enzymes enrolled in phenylpropanoid biosynthesis. The UHPLC-MS-based metabolite profiling confirmed the modulation of phenolic profiling by salinity and the accumulation of ferulate and its derivatives 3- and 4-O-feruloyl quinate. In conclusion, we present a model for explaining cell wall remodeling in response to salinity.
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http://dx.doi.org/10.1111/pce.13805DOI Listing
September 2020

Lignin from Tree Barks: Chemical Structure and Valorization.

ChemSusChem 2020 Sep 2;13(17):4537-4547. Epub 2020 Jun 2.

Department of Plant Biotechnology, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Av. Reina Mercedes, 10, 41012-, Seville, Spain.

Lignins from different tree barks, including Norway spruce (Picea abies), eucalyptus (Eucalyptus globulus), mimosa (Acacia dealbata) and blackwood acacia (A. melanoxylon), are thoroughly characterized. The lignin from E. globulus bark is found to be enriched in syringyl (S) units, with lower amounts of guaiacyl (G) and p-hydroxyphenyl (H) units (H/G/S ratio of 1:26:73), which produces a lignin that is highly enriched in β-ether linkages (83 %), whereas those from the two Acacia barks have similar compositions (H/G/S ratio of ≈5:50:45), with a predominance of β-ethers (73-75 %) and lower amounts of condensed carbon-carbon linkages; the lignin from A. dealbata bark also includes some resorcinol-related compounds, that appear to be incorporated or intimately associated to the polymer. The lignin from P. abies bark is enriched in G units, with lower amounts of H units (H/G ratio of 14:86); this lignin is thus depleted in β-O-4' alkyl-aryl ether linkages (44 %) and enriched in condensed linkages. Interestingly, this lignin contains large amounts of hydroxystilbene glucosides that seem to be integrally incorporated into the lignin structure. This study indicates that lignins from tree barks can be seen as an interesting source of valuable phenolic compounds. Moreover, this study is useful for tailoring conversion technologies for bark deconstruction and valorization.
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http://dx.doi.org/10.1002/cssc.202000431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540371PMC
September 2020

Hydroxystilbene Glucosides Are Incorporated into Norway Spruce Bark Lignin.

Plant Physiol 2019 07 25;180(3):1310-1321. Epub 2019 Apr 25.

Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, Avda. Reina Mercedes, 10, 41012-Seville, Spain

Recent investigations have revealed that, in addition to monolignols, some phenolic compounds derived from the flavonoid and hydroxystilbene biosynthetic pathways can also function as true lignin monomers in some plants. In this study, we found that the hydroxystilbene glucosides isorhapontin (isorhapontigenin--glucoside) and, at lower levels, astringin (piceatannol--glucoside) and piceid (resveratrol--glucoside) are incorporated into the lignin polymer in Norway spruce () bark. The corresponding aglycones isorhapontigenin, piceatannol, and resveratrol, along with glucose, were released by derivatization followed by reductive cleavage, a chemical degradative method that cleaves β-ether bonds in lignin, indicating that the hydroxystilbene glucosides are (partially) incorporated into the lignin structure through β-ether bonds. Two-dimensional NMR analysis confirmed the occurrence of hydroxystilbene glucosides in this lignin, and provided additional information regarding their modes of incorporation into the polymer. The hydroxystilbene glucosides, particularly isorhapontin and astringin, can therefore be considered genuine lignin monomers that participate in coupling and cross-coupling reactions during lignification in Norway spruce bark.
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http://dx.doi.org/10.1104/pp.19.00344DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6752895PMC
July 2019

Valorization of Tomato Processing by-Products: Fatty Acid Extraction and Production of Bio-Based Materials.

Materials (Basel) 2018 Nov 7;11(11). Epub 2018 Nov 7.

Smart Materials, Istituto Italiano di Tecnologia, 16163 Genova, Italy.

A method consisting of the alkaline hydrolysis of tomato pomace by-products has been optimized to obtain a mixture of unsaturated and polyhydroxylated fatty acids as well as a non-hydrolysable secondary residue. Reaction rates and the activation energy of the hydrolysis were calculated to reduce costs associated with chemicals and energy consumption. Lipid and non-hydrolysable fractions were chemically (infrared (IR) spectroscopy, gas chromatography/mass spectrometry (GC-MS)) and thermally (differential scanning calorimetry (DSC), thermogravimetric analysis (TGA)) characterized. In addition, the fatty acid mixture was used to produce cutin-based polyesters. Freestanding films were prepared by non-catalyzed melt-polycondensation and characterized by Attenuated Total Reflected-Fourier Transform Infrared (ATR-FTIR) spectroscopy, solid-state nuclear magnetic resonance (NMR), DSC, TGA, Water Contact Angles (WCA), and tensile tests. These bio-based polymers were hydrophobic, insoluble, infusible, and thermally stable, their physical properties being tunable by controlling the presence of unsaturated fatty acids and oxygen in the reaction. The participation of an oxidative crosslinking side reaction is proposed to be responsible for such modifications.
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http://dx.doi.org/10.3390/ma11112211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266337PMC
November 2018

Lavender- and lavandin-distilled straws: an untapped feedstock with great potential for the production of high-added value compounds and fungal enzymes.

Biotechnol Biofuels 2018 2;11:217. Epub 2018 Aug 2.

1UMR1163 BBF Biodiversité et Biotechnologie Fongiques, INRA, Aix Marseille Univ, 13288 Marseille Cedex 09, France.

Background: Lavender () and lavandin (a sterile hybrid of  × ) essential oils are among those most commonly used in the world for various industrial purposes, including perfumes, pharmaceuticals and cosmetics. The solid residues from aromatic plant distillation such as lavender- and lavandin-distilled straws are generally considered as wastes, and consequently either left in the fields or burnt. However, lavender- and lavandin-distilled straws are a potentially renewable plant biomass as they are cheap, non-food materials that can be used as raw feedstocks for green chemistry industry. The objective of this work was to assess different pathways of valorization of these straws as bio-based platform chemicals and fungal enzymes of interest in biorefinery.

Results: Sugar and lignin composition analyses and saccharification potential of the straw fractions revealed that these industrial by-products could be suitable for second-generation bioethanol prospective. The solvent extraction processes, developed specifically for these straws, released terpene derivatives (e.g. τ-cadinol, β-caryophyllene), lactones (e.g. coumarin, herniarin) and phenolic compounds of industrial interest, including rosmarinic acid which contributed to the high antioxidant activity of the straw extracts. Lavender and lavandin straws were also suitable inducers for the secretion of a wide panel of lignocellulose-acting enzymes (cellulases, hemicellulases and oxido-reductases) from the white-rot model fungus Interestingly, high amounts of laccase and several lytic polysaccharide monooxygenases were identified in the lavender and lavandin straw secretomes using proteomics.

Conclusions: The present study demonstrated that the distilled straws of lavender and lavandin are lignocellulosic-rich materials that can be used as raw feedstocks for producing high-added value compounds (antioxidants, aroma) and fungal oxidative enzymes, which represent opportunities to improve the decomposition of recalcitrant lignocellulose into biofuel. Hence, the structure and the physico-chemical properties of these straws clearly open new perspectives for use in biotechnological processes involving especially filamentous fungi. These approaches represent sustainable strategies to foster the development of a local circular bioeconomy.
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http://dx.doi.org/10.1186/s13068-018-1218-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071384PMC
August 2018

Fate of -hydroxycinnamates and structural characteristics of residual hemicelluloses and lignin during alkaline-sulfite chemithermomechanical pretreatment of sugarcane bagasse.

Biotechnol Biofuels 2018 5;11:153. Epub 2018 Jun 5.

1Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP 12602-810 Brazil.

Background: Preparing multiple products from lignocellulosic biomass feedstock enhances the profit and sustainability of future biorefineries. Grasses are suitable feedstocks for biorefineries as they permit a variety of possible by-products due to their particular chemical characteristics and morphology. Elucidating the fate of -hydroxycinnamates (ferulates-FAs and -coumarates-CAs) and major structural components during bioprocessing helps to discriminate the sources of recalcitrance in grasses and paves the way for the recovery of -hydroxycinnamates, which have multiple applications. To address these subjects, we assessed sugarcane bagasse biorefining under alkaline-sulfite chemithermomechanical (AS-CTM) pretreatment and enzymatic saccharification.

Results: The mass balances of the major bagasse components were combined with 2D-NMR structural evaluation of process solids to advance our understanding of sugarcane bagasse changes during biorefining. AS-CTM pretreatment provided a high yield and thoroughly digestible substrates. The pretreated material was depleted in acetyl groups, but retained 62 and 79% of the original lignin and xylan, respectively. Forty percent of the total FAs and CAs were also retained in pretreated material. After pretreatment and enzymatic hydrolysis, the residual solids contained mostly lignin and ester-linked CAs, with minor amounts of FAs and non-digested polysaccharides. Saponification of the residual solids, at a higher alkali load, cleaved all the ester linkages in the CAs; nevertheless, a significant fraction of the CAs remained attached to the saponified solids, probably to lignin, through 4- ether-linkages.

Conclusion: AS-CTM pretreatment provided soundly digestible substrates, which retain substantial amounts of xylans and lignin. Acetyl groups were depleted, but 40% of the total FAs and CAs remained in pretreated material. Ester-linked CAs detected in pretreated material also resisted to the enzymatic hydrolysis step. Only a more severe saponification reaction cleaved ester linkages of CAs from residual solids; nevertheless, CAs remained attached to the core lignin through 4- ether-linkages, suggesting the occurrence of an alkali-stable fraction of CAs in sugarcane bagasse.
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http://dx.doi.org/10.1186/s13068-018-1155-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987574PMC
June 2018

Structural Characterization of Lignin from Maize ( Zea mays L.) Fibers: Evidence for Diferuloylputrescine Incorporated into the Lignin Polymer in Maize Kernels.

J Agric Food Chem 2018 May 24;66(17):4402-4413. Epub 2018 Apr 24.

Department of Energy Great Lakes Bioenergy Research Center , Wisconsin Energy Institute, University of Wisconsin-Madison , Madison , Wisconsin 53726 , United States.

The structure of the phenolic polymer in maize grain fibers, with 5.5% Klason lignin content, has been studied. For this, the milled wood lignin (MWL) and dioxane lignin (DL) preparations were isolated and analyzed. The data indicated that the lignin in maize fibers was syringyl rich, mostly involved in β-aryl ether, resinol, and phenylcoumaran substructures. 2D NMR and derivatization followed by reductive cleavage (DFRC) also revealed the occurrence of associated ferulates together with trace amounts of p-coumarates acylating the γ-OH of lignin side chains, predominantly on S-lignin units. More interesting was the occurrence of diferuloylputrescine, a ferulic acid amide, which was identified by 2D NMR and comparison with a synthesized standard, that was apparently incorporated into this lignin. A phenylcoumaran structure involving a diferuloylputrescine coupled through 8-5' linkages to another diferuloylputrescine (or to a ferulate or a guaiacyl lignin unit) was found, providing compelling evidence for its participation in radical coupling reactions. The occurrence of diferuloylputrescine in cell walls of maize kernels and other cereal grains appears to have been missed in previous works, perhaps due to the alkaline hydrolysis commonly used for composition studies.
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http://dx.doi.org/10.1021/acs.jafc.8b00880DOI Listing
May 2018

Variability in Lignin Composition and Structure in Cell Walls of Different Parts of Macaúba (Acrocomia aculeata) Palm Fruit.

J Agric Food Chem 2018 Jan 22;66(1):138-153. Epub 2017 Dec 22.

Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC , Av. Reina Mercedes, 10, 41012 Seville, Spain.

The lignins from different anatomical parts of macaúba (Acrocomia aculeata) palm fruit, namely stalks, epicarp, and endocarp, were studied. The lignin from stalks was enriched in S-lignin units (S/G 1.2) and β-ether linkages (84% of the total) and was partially acylated at the γ-OH of the lignin side-chains (26% lignin acylation), predominantly with p-hydroxybenzoates and acetates. The epicarp lignin was highly enriched in G-lignin units (S/G 0.2) and consequently depleted in β-ethers (65%) and enriched in condensed structures such as phenylcoumarans (24%) and dibenzodioxocins (3%). The endocarp lignin was strikingly different from the rest and presented large amounts of piceatannol units incorporated into the polymer. This resulted in a lignin polymer depleted in β-ethers but enriched in condensed structures and linked piceatannol moieties. The incorporation of piceatannol into the lignin polymer seems to have a role in seed protection.
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http://dx.doi.org/10.1021/acs.jafc.7b04638DOI Listing
January 2018

Xylan extraction from pretreated sugarcane bagasse using alkaline and enzymatic approaches.

Biotechnol Biofuels 2017 7;10:296. Epub 2017 Dec 7.

Departamento de Biotecnologia, Escola de Engenharia de Lorena, Universidade de São Paulo, Lorena, SP 12602-810 Brazil.

Background: New biorefinery concepts are necessary to drive industrial use of lignocellulose biomass components. Xylan recovery before enzymatic hydrolysis of the glucan component is a way to add value to the hemicellulose fraction, which can be used in papermaking, pharmaceutical, and food industries. Hemicellulose removal can also facilitate subsequent cellulolytic glucan hydrolysis.

Results: Sugarcane bagasse was pretreated with an alkaline-sulfite chemithermomechanical process to facilitate subsequent extraction of xylan by enzymatic or alkaline procedures. Alkaline extraction methods yielded 53% (w/w) xylan recovery. The enzymatic approach provided a limited yield of 22% (w/w) but produced the xylan with the lowest contamination with lignin and glucan components. All extracted xylans presented arabinosyl side groups and absence of acetylation. 2D-NMR data suggested the presence of -methyl-glucuronic acid and -coumarates only in enzymatically extracted xylan. Xylans isolated using the enzymatic approach resulted in products with molecular weights (Mw) lower than 6 kDa. Higher Mw values were detected in the alkali-isolated xylans. Alkaline extraction of xylan provided a glucan-enriched solid readily hydrolysable with low cellulase loads, generating hydrolysates with a high glucose/xylose ratio.

Conclusions: Hemicellulose removal before enzymatic hydrolysis of the cellulosic fraction proved to be an efficient manner to add value to sugarcane bagasse biorefining. Xylans with varied yield, purity, and structure can be obtained according to the extraction method. Enzymatic extraction procedures produce high-purity xylans at low yield, whereas alkaline extraction methods provided higher xylan yields with more lignin and glucan contamination. When xylan extraction is performed with alkaline methods, the residual glucan-enriched solid seems suitable for glucose production employing low cellulase loadings.
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http://dx.doi.org/10.1186/s13068-017-0981-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719793PMC
December 2017

Fatty Acid Chain Shortening by a Fungal Peroxygenase.

Chemistry 2017 Dec 20;23(67):16985-16989. Epub 2017 Nov 20.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, 41012, Seville, Spain.

A recently discovered peroxygenase from the fungus Marasmius rotula (MroUPO) is able to catalyze the progressive one-carbon shortening of medium and long-chain mono- and dicarboxylic acids by itself alone, in the presence of H O . The mechanism, analyzed using H O , starts with an α-oxidation catalyzed by MroUPO generating an α-hydroxy acid, which is further oxidized by the enzyme to a reactive α-keto intermediate whose decarboxylation yields the one-carbon shorter fatty acid. Compared with the previously characterized peroxygenase of Agrocybe aegerita, a wider heme access channel, enabling fatty acid positioning with the carboxylic end near the heme cofactor (as seen in one of the crystal structures available) could be at the origin of the unique ability of MroUPO shortening carboxylic acid chains.
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http://dx.doi.org/10.1002/chem.201704773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5725704PMC
December 2017

Oxidoreductases on their way to industrial biotransformations.

Biotechnol Adv 2017 Nov 15;35(6):815-831. Epub 2017 Jun 15.

Instituto de Catálisis y Petroleoquímica, CSIC, Madrid, Spain.

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with HO as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating HO that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other HO generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.
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http://dx.doi.org/10.1016/j.biotechadv.2017.06.003DOI Listing
November 2017

Lignin Films from Spruce, Eucalyptus, and Wheat Straw Studied with Electroacoustic and Optical Sensors: Effect of Composition and Electrostatic Screening on Enzyme Binding.

Biomacromolecules 2017 04 23;18(4):1322-1332. Epub 2017 Mar 23.

Departments of Forest Biomaterials and Chemical and Biomolecular Engineering, North Carolina State University , Raleigh, North Carolina 27695, United States.

Lignins were isolated from spruce, wheat straw, and eucalyptus by using the milled wood lignin (MWL) method. Functional groups and compositional analyses were assessed via 2D NMR and P NMR to realize their effect on enzyme binding. Films of the lignins were fabricated and ellipsometry, atomic force microscopy, and water contact angle measurements were used for their characterization and to reveal the changes upon enzyme adsorption. Moreover, lignin thin films were deposited on quartz crystal microgravimetry (QCM) and surface plasmon (SPR) resonance sensors and used to gain further insights into the lignin-cellulase interactions. For this purpose, a commercial multicomponent enzyme system and a monocomponent Trichoderma reesei exoglucanase (CBH-I) were considered. Strong enzyme adsorption was observed on the various lignins but compared to the multicomponent cellulases, CBH-I displayed lower surface affinity and higher binding reversibility. This resolved prevalent questions related to the affinity of this enzyme with lignin. Remarkably, a strong correlation between enzyme binding and the syringyl/guaiacyl (S/G) ratio was found for the lignins, which presented a similar hydroxyl group content (P NMR): higher protein affinity was determined on isolated spruce lignin (99% G units), while the lowest adsorption occurred on isolated eucalyptus lignin (70% S units). The effect of electrostatic interactions in enzyme adsorption was investigated by SPR, which clearly indicated that the screening of charges allowed more extensive protein adsorption. Overall, this work furthers our understanding of lignin-cellulase interactions relevant to biomass that has been subjected to no or little pretreatment and highlights the widely contrasting effects of the nature of lignin, which gives guidance to improve lignocellulosic saccharification and related processes.
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http://dx.doi.org/10.1021/acs.biomac.7b00071DOI Listing
April 2017

Modification of Monolignol Biosynthetic Pathway in Jute: Different Gene, Different Consequence.

Sci Rep 2017 01 4;7:39984. Epub 2017 Jan 4.

Molecular Biology Laboratory, Department of Biochemistry and Molecular Biology, University of Dhaka, Dhaka-1000, Bangladesh.

Lignin, a cross-linked macromolecule of hydrophobic aromatic structure, provides additional rigidity to a plant cell wall. Although it is an integral part of the plant cell, presence of lignin considerably reduces the quality of the fiber of fiber-yielding plants. Decreasing lignin in such plants holds significant commercial and environmental potential. This study aimed at reducing the lignin content in jute-a fiber crop, by introducing hpRNA-based vectors for downregulation of two monolignoid biosynthetic genes- cinnamate 4-hydroxylase (C4H) and caffeic acid O-methyltransferase (COMT). Transgenic generations, analyzed through Southern, RT-PCR and northern assays showed downregulation of the selected genes. Transgenic lines exhibited reduced level of gene expression with ~ 16-25% reduction in acid insoluble lignin for the whole stem and ~13-14% reduction in fiber lignin content compared to the control lines. Among the two transgenic plant types one exhibited an increase in cellulose content and concomitant improvement of glucose release. Composition of the lignin building blocks was found to alter and this alteration resulted in a pattern, different from other plants where the same genes were manipulated. It is expected that successful COMT-hpRNA and C4H-hpRNA transgenesis in jute will have far-reaching commercial implications leading to product diversification and value addition.
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http://dx.doi.org/10.1038/srep39984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5209690PMC
January 2017

Lignin Composition and Structure Differs between Xylem, Phloem and Phellem in L.

Front Plant Sci 2016 27;7:1612. Epub 2016 Oct 27.

Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade de Lisboa Lisboa, Portugal.

The composition and structure of lignin in different tissues-phellem (cork), phloem and xylem (wood)-of was studied. Whole cell walls and their respective isolated milled lignins were analyzed by pyrolysis coupled with gas chromatography/mass spectrometry (Py-GC/MS), two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) and derivatization followed by reductive cleavage (DFRC). Different tissues presented varied -hydroxyphenyl:guaiacyl:syringyl (H:G:S) lignin compositions. Whereas lignin from cork has a G-rich lignin (H:G:S molar ratio 2:85:13), lignin from phloem presents more S-units (H:G:S molar ratio of 1:58:41) and lignin from xylem is slightly enriched in S-lignin (H:G:S molar ratio 1:45:55). These differences were reflected in the relative abundances of the different interunit linkages. Alkyl-aryl ethers (β--4') were predominant, increasing from 68% in cork, to 71% in phloem and 77% in xylem, as consequence of the enrichment in S-lignin units. Cork lignin was enriched in condensed structures such as phenylcoumarans (β-5', 20%), dibenzodioxocins (5-5', 5%), as corresponds to a lignin enriched in G-units. In comparison, lignin from phloem and xylem presented lower levels of condensed linkages. The lignin from cork was highly acetylated at the γ-OH of the side-chain (48% lignin acetylation), predominantly over G-units; while the lignins from phloem and xylem were barely acetylated and this occurred mainly over S-units. These results are a first time overview of the lignin structure in xylem, phloem (generated by cambium), and in cork (generated by phellogen), in agreement with literature that reports that lignin biosynthesis is flexible and cell specific.
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http://dx.doi.org/10.3389/fpls.2016.01612DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081372PMC
October 2016

Selective ligninolysis of wheat straw and wood chips by the white-rot fungus Lentinula edodes and its influence on in vitro rumen degradability.

J Anim Sci Biotechnol 2016 22;7:55. Epub 2016 Sep 22.

Animal Nutrition Group, Wageningen University, De Elst 1, 6708 WD Wageningen, The Netherlands.

Background: The present work investigated the influence of lignin content and composition in the fungal treatment of lignocellulosic biomass in order to improve rumen degradability. Wheat straw and wood chips, differing in lignin composition, were treated with Lentinula edodes for 0, 2, 4, 8 and 12 wk and the changes occurring during fungal degradation were analyzed using pyrolysis-gas chromatography-mass spectrometry and detergent fiber analysis.

Results: L. edodes preferentially degraded lignin, with only limited cellulose degradation, in wheat straw and wood chips, leaving a substrate enriched in cellulose. Syringyl (S)-lignin units were preferentially degraded than guaiacyl (G)-lignin units, resulting in a decreased S/G ratio. A decreasing S/G ratio (wheat straw: r = -0.72, wood chips: r = -0.75) and selective lignin degradation (wheat straw: r = -0.69, wood chips: r = -0.88) were correlated with in vitro gas production (IVGP), a good indicator for rumen degradability.

Conclusions: L. edodes treatment increased the IVGP of wheat straw and wood chips. Effects on IVGP were similar for wheat straw and wood chips indicating that lignin content and 3D-structure of cell walls influence in vitro rumen degradability more than lignin composition.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5034620PMC
http://dx.doi.org/10.1186/s40104-016-0110-zDOI Listing
October 2016

From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase.

Angew Chem Int Ed Engl 2016 09 30;55(40):12248-51. Epub 2016 Aug 30.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, 41012, Seville, Spain.

A new heme-thiolate peroxidase catalyzes the hydroxylation of n-alkanes at the terminal position-a challenging reaction in organic chemistry-with H2 O2 as the only cosubstrate. Besides the primary product, 1-dodecanol, the conversion of dodecane yielded dodecanoic, 12-hydroxydodecanoic, and 1,12-dodecanedioic acids, as identified by GC-MS. Dodecanal could be detected only in trace amounts, and 1,12-dodecanediol was not observed, thus suggesting that dodecanoic acid is the branch point between mono- and diterminal hydroxylation. Simultaneously, oxygenation was observed at other hydrocarbon chain positions (preferentially C2 and C11). Similar results were observed in reactions of tetradecane. The pattern of products formed, together with data on the incorporation of (18) O from the cosubstrate H2 (18) O2 , demonstrate that the enzyme acts as a peroxygenase that is able to catalyze a cascade of mono- and diterminal oxidation reactions of long-chain n-alkanes to give carboxylic acids.
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http://dx.doi.org/10.1002/anie.201605430DOI Listing
September 2016

Effects of Fe deficiency on the protein profiles and lignin composition of stem tissues from Medicago truncatula in absence or presence of calcium carbonate.

J Proteomics 2016 05 30;140:1-12. Epub 2016 Mar 30.

USDA-ARS Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, 1100 Bates Street, Houston, TX 77030, USA. Electronic address:

Unlabelled: Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago truncatula. Two-dimensional electrophoresis separation of stem protein extracts resolved 276 consistent spots in the whole experiment. Iron deficiency in absence or presence of CaCO3 caused significant changes in relative abundance in 10 and 31 spots, respectively, and 80% of them were identified by mass spectrometry. Overall results indicate that Fe deficiency by itself has a mild effect on the stem proteome, whereas Fe deficiency in the presence of CaCO3 has a stronger impact and causes changes in a larger number of proteins, including increases in stress and protein metabolism related proteins not observed in the absence of CaCO3. Both treatments resulted in increases in cell wall related proteins, which were more intense in the presence of CaCO3. The increases induced by Fe-deficiency in the lignin per protein ratio and changes in the lignin monomer composition, assessed by pyrolysis-gas chromatography-mass spectrometry and microscopy, respectively, further support the existence of cell wall alterations.

Biological Significance: In spite of being essential for the delivery of nutrients to the shoots, our knowledge of stem responses to nutrient deficiencies is very limited. The present work applies 2-DE techniques to unravel the response of this understudied tissue to Fe deficiency. Proteomics data, complemented with mineral, lignin and microscopy analyses, indicate that stems respond to Fe deficiency by increasing stress and defense related proteins, probably in response of mineral and osmotic unbalances, and eliciting significant changes in cell wall composition. The changes observed are likely to ultimately affect solute transport and distribution to the leaves.
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http://dx.doi.org/10.1016/j.jprot.2016.03.017DOI Listing
May 2016

Lignin-carbohydrate complexes from sisal (Agave sisalana) and abaca (Musa textilis): chemical composition and structural modifications during the isolation process.

Planta 2016 May 5;243(5):1143-58. Epub 2016 Feb 5.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, P.O. Box 1052, 41080, Seville, Spain.

Main Conclusion: Two types of lignins occurred in different lignin-carbohydrate fractions, a lignin enriched in syringyl units, less condensed, preferentially associated with xylans, and a lignin with more guaiacyl units, more condensed, associated with glucans. Lignin-carbohydrate complexes (LCC) were isolated from the fibers of sisal (Agave sisalana) and abaca (Musa textilis) according to a plant biomass fractionation procedure recently developed and which was termed as "universally" applicable to any type of lignocellulosic material. Two LCC fractions, namely glucan-lignin (GL) and xylan-lignin (XL), were isolated and differed in the content and composition of carbohydrates and lignin. In both cases, GL fractions were enriched in glucans and comparatively depleted in lignin, whereas XL fractions were depleted in glucans, but enriched in xylans and lignin. Analysis by two-dimensional Nuclear Magnetic Resonance (2D-NMR) and Derivatization Followed by Reductive Cleavage (DFRC) indicated that the XL fractions were enriched in syringyl (S)-lignin units and β-O-4' alkyl-aryl ether linkages, whereas GL fractions have more guaiacyl (G)-lignin units and less β-O-4' alkyl-aryl ether linkages per lignin unit. The data suggest that the structural characteristics of the lignin polymers are not homogeneously distributed within the same plant and that two different lignin polymers with different composition and structure might be present. The analyses also suggested that acetates from hemicelluloses and the acyl groups (acetates and p-coumarates) attached to the γ-OH of the lignin side chains were extensively hydrolyzed and removed during the LCC fractionation process. Therefore, caution must be paid when using this fractionation approach for the structural characterization of plants with acylated hemicelluloses and lignins. Finally, several chemical linkages (phenylglycosides and benzyl ethers) could be observed to occur between lignin and xylans in these plants.
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http://dx.doi.org/10.1007/s00425-016-2470-1DOI Listing
May 2016

Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.

Appl Environ Microbiol 2015 Jun 10;81(12):4130-42. Epub 2015 Apr 10.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Seville, Spain

The goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106-118, 2013, http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.
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http://dx.doi.org/10.1128/AEM.00660-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524154PMC
June 2015

Isolation and structural characterization of the milled wood lignin, dioxane lignin, and cellulolytic lignin preparations from brewer's spent grain.

J Agric Food Chem 2015 Jan 7;63(2):603-13. Epub 2015 Jan 7.

Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC, P.O. Box 1052, E-41080 Seville, Spain.

The structure of the lignin from brewer's spent grain (BSG) has been studied in detail. Three different lignin preparations, the so-called "milled-wood" lignin (MWL), dioxane lignin (DL), and cellulolytic lignin (CEL), were isolated from BSG and then thoroughly characterized by pyrolysis GC/MS, 2D-NMR, and derivatization followed by reductive cleavage (DFRC). The data indicated that BSG lignin presents a predominance of guaiacyl units (syringyl/guaiacyl ratio of 0.4-0.5) with significant amounts of associated p-coumarates and ferulates. The flavone tricin was also present in the lignin from BSG, as also occurred in other grasses. 2D-NMR (HSQC) revealed that the main substructures present are β-O-4' alkyl-aryl ethers (77-79%) followed by β-5' phenylcoumarans (11-13%) and lower amounts of β-β' resinols (5-6%) and 5-5' dibenzodioxocins (3-5%). The results from 2D-NMR (HMBC) and DFRC indicated that p-coumarates are acylating the γ-carbon of lignin side chains and are mostly involved in condensed structures. DFRC analyses also indicated a minor degree of γ-acylation with acetate groups, which takes place preferentially on S lignin (6% of S units are acetylated) over G lignin (only 1% of G units are acetylated).
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http://dx.doi.org/10.1021/jf505808cDOI Listing
January 2015

Analysis of lignin-carbohydrate and lignin-lignin linkages after hydrolase treatment of xylan-lignin, glucomannan-lignin and glucan-lignin complexes from spruce wood.

Planta 2014 May 15;239(5):1079-90. Epub 2014 Feb 15.

Department of Fibre and Polymer Technology, Royal Institute of Technology, KTH, Teknikringen 56-58, 10044, Stockholm, Sweden.

Xylan-lignin (XL), glucomannan-lignin (GML) and glucan-lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin-carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4' ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4' substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.
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http://dx.doi.org/10.1007/s00425-014-2037-yDOI Listing
May 2014

Comprehensive study of valuable lipophilic phytochemicals in wheat bran.

J Agric Food Chem 2014 Feb 4;62(7):1664-73. Epub 2014 Feb 4.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC , P.O. Box 1052, E- 41080 Seville, Spain.

Wheat bran, the major side-stream generated in the milling of wheat grains in the production of white flour, contains significant quantities of carbohydrate and proteins. While not interfering with flour utilization, the bran could be considered as an important feedstock within a biorefinery concept. Wheat bran also contains some amounts of lipids that can be used as a source of valuable phytochemicals. Gas chromatography and mass spectrometry analysis of the lipid composition of destarched wheat bran demonstrated that the predominant lipids found in wheat bran were free fatty acids (ca. 40% of total lipids), followed by acylglycerols (40%). Additionally, important amounts of alkylresorcinols (13% of total lipids) and steroid compounds (hydrocarbons, ketones, free sterols, sterol glycosides, sterol esters, and sterol ferulates) (7% of total lipids) were also present among the lipids of wheat bran. The use of wheat bran as a valuable source of phytochemicals of interest in the context of a wheat bran biorefinery is discussed.
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http://dx.doi.org/10.1021/jf404772bDOI Listing
February 2014

Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock.

Biotechnol Biofuels 2014 Jan 8;7(1). Epub 2014 Jan 8.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes, 10, E-41012 Seville, Spain.

Background: Biofuel production from lignocellulosic material is hampered by biomass recalcitrance towards enzymatic hydrolysis due to the compact architecture of the plant cell wall and the presence of lignin. The purpose of this work is to study the ability of an industrially available laccase-mediator system to modify and remove lignin during pretreatment of wood (Eucalyptus globulus) feedstock, thus improving saccharification, and to analyze the chemical modifications produced in the whole material and especially in the recalcitrant lignin moiety.

Results: Up to 50% lignin removal from ground eucalypt wood was attained by pretreatment with recombinant Myceliophthora thermophila laccase and methyl syringate as mediator, followed by alkaline peroxide extraction in a multistage sequence. The lignin removal directly correlated with increases (approximately 40%) in glucose and xylose yields after enzymatic hydrolysis. The pretreatment using laccase alone (without mediator) removed up to 20% of lignin from eucalypt wood. Pyrolysis-gas chromatography/mass spectrometry of the pretreated wood revealed modifications of the lignin polymer, as shown by lignin markers with shortened side chains and increased syringyl-to-guaiacyl ratio. Additional information on the chemical modifications produced was obtained by two-dimensional nuclear magnetic resonance of the whole wood swollen in dimethylsulfoxide-d6. The spectra obtained revealed the removal of guaiacyl and syringyl lignin units, although with a preferential removal of the former, and the lower number of aliphatic side-chains per phenylpropane unit (involved in main β-O-4' and β-β' inter-unit linkages), in agreement with the pyrolysis-gas chromatography/mass spectrometry results, without a substantial change in the wood polysaccharide signals. However, the most noticeable modification observed in the spectra was the formation of Cα-oxidized syringyl lignin units during the enzymatic treatment. Further insight into the modifications of lignin structure, affecting other inter-unit linkages and oxidized structures, was attained by nuclear magnetic resonance of the lignins isolated from the eucalypt feedstock after the enzymatic pretreatments.

Conclusions: This work shows the potential of an oxidative enzymatic pretreatment to delignify and improve cellulase saccharification of a hardwood feedstock (eucalypt wood) when applied directly on the ground lignocellulosic material, and reveals the main chemical changes in the pretreated material, and its recalcitrant lignin moiety, behind the above results.
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http://dx.doi.org/10.1186/1754-6834-7-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3917704PMC
January 2014

Understanding pulp delignification by laccase-mediator systems through isolation and characterization of lignin-carbohydrate complexes.

Biomacromolecules 2013 Sep 14;14(9):3073-80. Epub 2013 Aug 14.

Department of Fibre and Polymer Technology, Royal Institute of Technology, KTH, Teknikringen 56-58, SE-10044 Stockholm, Sweden.

The effects and mechanism of pulp delignification by laccases in the presence of redox mediators have been investigated on unbleached eucalyptus kraft pulp treated with laccases from Pycnoporus cinnabarinus (PcL) and Myceliophthora thermophila (MtL) and 1-hydroxybenzotriazole (HBT) and methyl syringate (MeS) as mediators, respectively. Determination of the corrected κ number in eucalyptus pulps after the enzymatic treatments revealed that the PcL-HBT system exhibited a more remarkable delignification effect than the MtL-MeS system. To obtain further insight, lignin-carbohydrate complexes were fractionated and subsequently characterized by nuclear magnetic resonance, thioacidolysis (followed by gas chromatography and size exclusion chromatography), and pyrolysis-gas chromatography-mass spectrometry (pyrolysis-GC-MS) analyses before and after the enzymatic treatments and their controls. We can conclude that the laccase-mediator treatments altered the lignin structures in such a way that more lignin was recovered in the xylan-lignin fractions, as shown by Klason lignin estimation, with smaller amounts of both syringyl (S) and guaiacyl (G) uncondensed units, as shown by thioacidolysis and gas chromatography, especially after the PcL-HBT treatment. The laccase-mediator treatment produced oxidation at Cα and cleavage of Cα and Cβ bonds in pulp lignin, as shown by pyrolysis-GC-MS. The general mechanism of residual lignin degradation in the pulp by laccase-mediator treatments is discussed in light of the results obtained.
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http://dx.doi.org/10.1021/bm4006936DOI Listing
September 2013

Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from Coprinopsis cinerea.

Biotechnol Bioeng 2013 Sep 22;110(9):2323-32. Epub 2013 Apr 22.

Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Reina Mercedes 10, Seville, Spain.

The goal of this study is the selective oxyfunctionalization of aliphatic compounds under mild and environmentally friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advantage from a new peroxidase type that catalyzes monooxygenase reactions with H2 O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxygenase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identified by mass spectrometry. Regioselective hydroxylation of saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (only at the ω-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also ω-1 or ω-2 hydroxylated, but di- and tri-glycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the ω-1 or ω-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or di-hydroxylated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production.
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http://dx.doi.org/10.1002/bit.24904DOI Listing
September 2013

Structural characterization of lignin isolated from coconut (Cocos nucifera) coir fibers.

J Agric Food Chem 2013 Mar 26;61(10):2434-45. Epub 2013 Feb 26.

Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS), CSIC , PO Box 1052, E-41080 Seville, Spain.

The structure of the isolated milled "wood" lignin from coconut coir has been characterized using different analytical methods, including Py-GC/MS, 2D NMR, DFRC, and thioacidolysis. The analyses demonstrated that it is a p-hydroxyphenyl-guaiacyl-syringyl (H-G-S) lignin, with a predominance of G units (S/G ratio 0.23) and considerable amounts of associated p-hydroxybenzoates. Two-dimensional NMR indicated that the main substructures present in this lignin include β-O-4' alkyl aryl ethers followed by phenylcoumarans and resinols. Two-dimensional NMR spectra also indicated that coir lignin is partially acylated at the γ-carbon of the side chain with p-hydroxybenzoates and acetates. DFRC analysis showed that acetates preferentially acylate the γ-OH in S rather than in G units. Despite coir lignin's being highly enriched in G-units, thioacidolysis indicated that β-β' resinol structures are mostly derived from sinapyl alcohol. Finally, we find evidence that the flavone tricin is incorporated into the coconut coir lignin, as has been recently noted for various grasses.
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http://dx.doi.org/10.1021/jf304686xDOI Listing
March 2013