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he hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.

Las enzimas que degradan la lignina son de interés biotecnológico para las biorrefinerías de la lignocelulosa [1]. En concreto, las lignina peroxidasas (LiPs) han recibido una atención especial por su capacidad para oxidar directamente este polímero aromático [2]. Hasta ahora, estas enzimas solo se habían identificado en basidiomicetos del orden Polyporales, donde se incluyen la mayoría de las especies degradadoras de madera.Tras la secuenciación del genoma de Agrocybe pediades, caracterizado por crecer sobre hojarasca, identificamos la primera enzima ligninolítica de esta familia (ApeLiP) en un hongo del orden Agaricales [3]. La secuencia de ApeLiP se clonó, expresó en cuerpos de inclusión en Eschericchia coli y replegó in vitro, obteniéndose así la enzima activa. Una vez purificada demostramos su capacidad para oxidar compuestos modelo de lignina fenólicos y no fenólicos. Esta actividad se asoció a un triptófano catalítico (Trp166) expuesto al solvente (identificado en la estructura de ApeLiP obtenida mediante difracción de rayos-X) tras verificar la pérdida de la capacidad oxidativa en la variante Trp166Ala. Diferentes estudios, incluyendo la determinación de su potencial redox y estabilidad a pH, determinaron que las propiedades de ApeLiP son compatibles con su capacidad para oxidar y modificar estructuralmente la lignina, tal como se demostró utilizando lignina real mediante espectroscopía de flujo detenido y 2D-NMR. Este trabajo demuestra que no solo los Polyporales degradadores de madera, sino también los hongos Agaricales formadores de setas, tienen enzimas de enorme relevancia tanto para el reciclado del carbono en la naturaleza como para la modificación biotecnológica de la lignina.

Phenolic metabolism is rewired during internode development and in response to cinnamyl alcohol dehydrogenase perturbation in sorghum, with different sets of genes and accumulating compounds. The molecular mechanisms associated with secondary cell wall (SCW) deposition in sorghum remain largely uncharacterized. Here, we employed untargeted metabolomics and large-scale transcriptomics to correlate changes in SCW deposition with variation in global gene expression profiles and metabolite abundance along an elongating internode of sorghum, with a major focus on lignin and phenolic metabolism. To gain deeper insight into the metabolic and transcriptional changes associated with pathway perturbations, a bmr6 mutant [with reduced cinnamyl alcohol dehydrogenase (CAD) activity] was analyzed. In the wild type, internode development was accompanied by an increase in the content of oligolignols, p-hydroxybenzaldehyde, hydroxycinnamate esters, and flavonoid glucosides, including tricin derivatives. We further identified modules of genes whose expression pattern correlated with SCW deposition and the accumulation of these target metabolites. Reduced CAD activity resulted in the accumulation of hexosylated forms of hydroxycinnamates (and their derivatives), hydroxycinnamaldehydes, and benzenoids. The expression of genes belonging to one specific module in our co-expression analysis correlated with the differential accumulation of these compounds and contributed to explaining this metabolic phenotype. Metabolomics and transcriptomics data further suggested that CAD perturbation activates distinct detoxification routes in sorghum internodes. Our systems biology approach provides a landscape of the metabolic and transcriptional changes associated with internode development and with reduced CAD activity in sorghum.

Papyrus, produced from the white pith of Cyperus papyrus L., has been used for millennia as the major writing support by ancient cultures, but there was no continuous papyrus production until modern times. Therefore, papyrus production had to be rediscovered. Modern Egyptian papyrus producers claim that their sheets possess 'the same physical and chemical properties as ancient papyri'. To study if this is accurate, commercially available papyrus sheets were compared to ancient papyri and papyri produced according to Pliny's historic description. Material characterization was performed with a focus on the potentially color-bearing lignin. Two-dimensional nuclear magnetic resonance spectroscopy, derivatization followed by reductive cleavage, and pyrolysis-gas chromatography/mass spectrometry were complemented with microscopy and tests for surface pH and sodium content. The lignin data in the native pith and commercial sheets were compared to 10 ancient samples from the Papyrus Museum Vienna. The analytical data clearly show the involvement of a strong alkaline treatment followed by chlorine bleaching for commercial papyri, as expressed by higher pH values, altered lignin structures, and chlorinated lignin compounds. The inclusion of an alkaline step in ancient papyrus manufacture is discussed but dismissed, since the alkali-treatment causes a huge decrease in lignin content, that was not found for the analysed ancient specimen. We assume that this additional treatment was introduced to obtain yellowish papyrus sheets meeting optical expectations of modern spectators. Linguistic and art historic evidence indicates that such a step would not have made sense in antiquity, since it was desired to produce white papyri.

Unspecific peroxygenases (UPOs) are extracellular fungal enzymes of biotechnological interest as self-sufficient (and more stable) counterparts of cytochrome P450 monooxygenases, the latter being present in most living cells. Expression hosts and structural information are crucial for exploiting UPO diversity (over eight thousand UPO-type genes were identified in sequenced genomes) in target reactions of industrial interest. However, while many thousands of entries in the Protein Data Bank include molecular coordinates of P450 enzymes, only 19 entries correspond to UPO enzymes, and UPO structures from only two species (Agrocybe aegerita and Hypoxylon sp.) have been published to date. In the present study, two UPOs from the basidiomycete Marasmius rotula (rMroUPO) and the ascomycete Collariella virescens (rCviUPO) were crystallized after sequence optimization and Escherichia coli expression as active soluble enzymes. Crystals of rMroUPO and rCviUPO were obtained at sufficiently high resolution (1.45 and 1.95 angstrom, respectively) and the corresponding structures were solved by molecular replacement. The crystal structures of the two enzymes (and two mutated variants) showed dimeric proteins. Complementary biophysical and molecular biology studies unveiled the diverse structural bases of the dimeric nature of the two enzymes. Intermolecular disulfide bridge and parallel association between two alpha-helices, among other interactions, were identified at the dimer interfaces. Interestingly, one of the rCviUPO variants incorporated the ability to produce fatty acid diepoxides-reactive compounds with valuable cross-linking capabilities-due to removal of the enzyme C-terminal tail located near the entrance of the heme access channel. In conclusion, different dimeric arrangements could be described in (short) UPO crystal structures.

Three Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile strains (C130915_07T, C150915_16 and C150915_17) were isolated from lymph nodes of Algerian cows. On the basis of 16S rRNA gene and whole genome similarities, the isolates were almost identical and clearly grouped in the genus Pseudochrobactrum . This allocation was confirmed by the analysis of fatty acids (C19:cyclo, C18 : 1, C18 : 0, C16 : 1 and C16 : 0) and of polar lipids (major components: phosphatidylethanolamine, ornithine-lipids, phosphatidylglycerol, cardiolipin and phosphatidylcholine, plus moderate amounts of phosphatidylmonomethylethanolamine, phosphatidyldimethylethanolamine and other aminolipids). Genomic, physiological and biochemical data differentiated these isolates from previously described Pseudochrobactrum species in DNA relatedness, carbon assimilation pattern and growth temperature range. Thus, these organisms represent a novel species of the genus Pseudochrobactrum , for which the name Pseudochrobactrum algeriensis sp. nov. is proposed (type strain C130915_07T=CECT30232T=LMG 32378T).

Rice (Oryza sativa L.) straw is a highly abundant, widely available, and low cost agricultural waste that can be used as a source to extract valuable phytochemicals of industrial interest. Hence, in the present work, the chemical composition of the lipophilic compounds present in rice straw was thoroughly characterized by gas chromatography and mass spectrometry using medium-length high-temperature capillary columns, which allowed the identification of a wide range of lipophilic compounds, from low molecular weight fatty acids to high molecular weight sterols esters, sterol glucosides, or triglycerides in the same chromatogram. The most abundant lipophilic compounds in rice straw were fatty acids, which accounted for up to 6,400 mg/kg (41.0% of all identified compounds), followed by free sterols (1,600 mg/kg; 10.2%), sterol glucosides (1,380 mg/kg; 8.8%), fatty alcohols (1,150 mg/kg; 7.4%), and triglycerides (1,140 mg/kg; 7.3%), along with lower amounts of high molecular weight wax esters (900 mg/kg; 5.8%), steroid ketones (900 mg/kg; 5.8%), monoglycerides (600 mg/kg; 3.8%), alkanes (400 mg/kg; 2.6%), diglycerides (380 mg/kg; 2.4%), sterol esters (380 mg/kg; 2.4%), tocopherols (340 mg/kg; 2.2%), and steroid hydrocarbons (60 mg/kg; 0.4%). This information is of great use for the valorization of rice straw to obtain valuable lipophilic compounds of interest for the nutraceutical, pharmaceutical, cosmetic, and chemical industries. Moreover, this knowledge is also useful for other industrial uses of rice straw, as in pulp and papermaking, since some lipophilic compounds are at the origin of the so-called pitch deposits during pulping. Copyright © 2022 Rosado, Marques, Rencoret, Gutiérrez and del Río.

Lignin is largely produced by the oxidative radical coupling of the three monolignols, p-coumaryl, coniferyl, and sinapyl alcohols, which form the p-hydroxyphenyl, guaiacyl, and syringyl lignin units in the lignin polymer. However, it is increasingly appreciated that other “unconventional” phenolic compounds can also behave as lignin monomers participating in oxidative radical reactions during the lignification process and becoming integrated into the lignin polymer in some plants. Various phenolic compounds have been found incorporated into the lignin, including phenolic compounds derived from the monolignol biosynthetic pathway (e.g., monolignol ester conjugates, caffeyl and 5-hydroxyconiferyl alcohols, hydroxycinnamaldehydes, among others). More fascinating still was the discovery in some plants of a series of phenolic compounds arising from other polyphenolic biosynthetic pathways, including flavonoids (tricin, dihydrotricin, naringenin, naringenin chalcone), hydroxystilbenes (resveratrol, piceatannol, and isorhapontigenin, and their O-glucosylated counterparts piceid, astringin, and isorhapontin), and ferulic acid amides (feruloyltyramine, and diferuloylputrescine), that are integrated into the lignin polymer. All of these discoveries indicate that the lignin structure is far more complex than previously thought, that the definition of lignin should not be restricted to a polymer derived solely from the three traditional monolignols, and that other unconventional lignin monomers must be considered, thus expanding the established definition of lignin. The overriding factor remains, however, that lignification, the process of polymerization to the lignin polymer, allows this compositional flexibility because it is a simple chemical process. © 2022 Elsevier Ltd

Agro-industrial residues were used as a successful, cheap and renewable substrate, capable of increasing activity of ligninolytic enzymes for further use in melanin degradation assays to develop inputs for the treatment of skin hyperpigmentation. The analysis of melanin degradation was performed in a quantitative and qualitative way. The enzymatic crude broth extract, rich in laccases, produced by Marasmiellus palmivorus VE111 in medium formulated with lignocellulosic residues of Araucaria angustifolia subjected to hydrolysis and alcoholic fermentation, degraded almost 33% of the melanin content and enzymatic crude broth extract produced by Pycnoporus sanguineus PR_32 degraded 13%, in just 8 h, using vanillin as a mediator. Proton 2D NMR spectra show melanin changes after laccase treatment. This work has provided an insight for the development of ecofriendly, sustainable and innovative cosmetics to deal with skin hyperpigmentation. © 2022 Elsevier B.V.

Terminal alkenes are among the most attractive starting materials for the synthesis of epoxides, which are essential and versatile intermediate building blocks for the pharmaceutical, fla-voring, and polymer industries. Previous research on alkene epoxidation has focused on the use of several oxidizing agents and/or different enzymes, including cytochrome P450 monooxygenases, as well as microbial whole-cell catalysts that have several drawbacks. Alternatively, we explored the ability of unspecific peroxygenases (UPOs) to selectively epoxidize terminal alkenes. UPOs are attractive biocatalysts because they are robust extracellular enzymes and only require H2O2 as cosub-strate. Here, we show how several UPOs, such as those from Cyclocybe (Agrocybe) aegerita (AaeUPO), Marasmius rotula (MroUPO), Coprinopsis cinerea (rCciUPO), Humicola insolens (rHinUPO), and Daldinia caldariorum (rDcaUPO), are able to catalyze the epoxidation of long-chain terminal alkenes (from C12:1 to C20:1) after an initial optimization of several reaction parameters (cosolvent, cosub-strate, and pH). In addition to terminal epoxides, alkenols and other hydroxylated derivatives of the alkenes were formed. Although all UPOs were able to convert and epoxidize the alkenes, nota-ble differences were observed between them, with rCciUPO being responsible for the highest sub-strate turnover and MroUPO being the most selective with respect to terminal epoxidation. The potential of peroxygenases for epoxidizing long-chain terminal alkenes represents an interesting and green alternative to the existing synthesis technologies. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

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 Chaetomium globosum (CglUPO) and the basidiomycete Marasmius rotula (MroUPO), as well as with recombinant UPO of the ascomycete Humicola insolens (rHinUPO), 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 CglUPO selectively produced "pure" epoxides (monoepoxides and/or diepoxides), MroUPO 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, rHinUPO, 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 alpha-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.

Lignin is a phenylpropanoid polymer derived essentially from the oxidative radical coupling of three p-hydroxycinnamyl alcohols, the so-called monolignols: p-coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all originating from the shikimate-derived monolignol biosynthetic pathway, have been found to behave as lignin monomers in many plants, including the monolignol ester conjugates (with acetate, p-coumarate, ferulate, and p-hydroxybenzoate) or the incompletely methylated monomers caffeyl and 5-hydroxyconiferyl alcohols, among others. However, recent studies have revealed that some phenolic compounds derived from other biosynthetic pathways also act as true lignin monomers, participating in radical coupling reactions during lignification in several plants. This is the case for the flavone tricin, established as a monomer in the lignins from grasses and other monocots, and the newly discovered hydroxystilbenes (resveratrol, isorhapontigenin, and piceatannol) that are implicated as monomers in the lignification of palm fruit endocarps, or the stilbene glucosides that have been found incorporated into the lignin of Norway spruce bark. The discovery of ‘nonconventional’ phenolic precursors arising from other biosynthetic pathways than the canonical monolignol biosynthetic pathway expands the traditional definition of lignin and reveals that any phenolic compound that is delivered to the cell wall may be oxidized and incorporated into the lignin polymer during lignification, subject exclusively to its chemical compatibility.

Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography−mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the cannel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the ability of the MroUPO to epoxidize oleic acid (while no activity was detected for the I153V variant). The rationale for these results was revealed by the substrate positioning in the above computational simulations, which predict a shorter distance between the oleic acid double bond and the oxygen atom of the peroxide-activated heme (compound I) in the I153T variant than in the native enzyme, promoting epoxidation. In contrast, the I153F/S156F double mutation fully prevents the approach of oleic acid in the bent conformation required for double-bond epoxidation, although its (sub)terminal hydroxylation was predicted and experimentally confirmed. The I153T mutation also increased the UPO selectivity on polyunsaturated fatty acid epoxidation, strongly reducing the ratio

Las lignina peroxidasas (LiPs) han recibido especial atención en el contexto de las biorrefinerías de lignocelulosa por su capacidad para oxidar directamente la lignina.Hasta ahora, estas enzimas solo se habían identificado en basidiomicetos del orden Polyporales, que incluye la mayoría de las especies degradadoras de madera.Tras la secuenciación del genoma de Agrocybe pediades [5] identificamos la primera enzima ligninolítica de esta familia en un hongo del orden Agaricales (ApeLiP) degradador de hojarasca. Su estructura cristalográfica reveló que contiene un grupo hemo como centro activo y un triptófano catalítico posicionado en su superficie (Trp166). La caracterización bioquímica de la enzima nativa y de una variante carente de dicho triptófano confirmó que este residuo está involucrado en la oxidación directa de sustratos aromáticos no-fenólicos de alto potencial redox y de compuestos fenólicos, incluyendo monómeros y dímeros modelos de lignina y distintos colorantes. Además, mediante espectroscopía de flujo detenido y 2D-NMR se confirmó que este residuo es responsable de la oxidación directa de lignina real, tanto de angiospermas como de gimnospermas, con una eficiencia igual o superior al de otras peroxidasas ligninolíticas conocidas. La caracterización fue complementada con el estudio de los potenciales de reducción de los intermediarios del ciclo catalítico y con estudios de estabilidad. Así, se observó una activación por peróxido similar a la de otras peroxidasas, pero un menor potencial redox en el paso limitante de la reacción comparado con otras LiPs, así como una alta estabilidad a pH básico. Todos estos estudios demuestran que en hongos Agaricales también existen enzimas relevantes para el reciclado del carbono en la naturaleza que además pueden aprovecharse para su aplicación biotecnológica.

Pleurotus eryngii es un hongo de prados y pastizales de interés biotecnológico por su capacidad para transformar el material lignocelulósico no leñoso. En este estudio combinamos análisis transcriptómicos, exoproteómicos y metabolómicos con objeto de ofrecer una explicación sobre los aspectos enzimáticos relacionados con la degradación de la paja de trigo. Durante la fase temprana de crecimiento encontramos un conjunto de enzimas extracelulares inducidas y constitutivas formado por glicosil hidrolasas, polisacárido liasas y carbohidrato esterasas activas sobre polisacáridos,lacasas activas sobre lignina, y una cantidad sorprendente de aril-alcohol oxidasas (AAOs). A tiempos largos identificamos una mayor diversidad y abundancia de enzimas, representada por oxidorreductasas implicadas en la despolimerización de celulosa y lignina, muchas de ellas inducidas desde la fase temprana de crecimiento. Estas enzimas oxidativas incluyeron monooxigenasas líticas de polisacáridos (LPMOs), celobiosa deshidrogenasa implicada en la activación de las LPMOs, y peroxidasas ligninolíticas (principalmente manganeso peroxidasas), junto a una gran abundancia de AAOs productoras de H2O2. Algunas de las enzimas más relevantes activas sobre polisacáridos aparecieron unidas a módulos de unión a celulosa. Esto se relacionó con el hábitat de P. eryngii.También elucidamos aspectos del catabolismo intracelular de compuestos aromáticos, un tema poco investigado en los basidiomicetos degradadores de lignina. Este enfoque multiómico revela que, aunque la descomposición de la paja de trigo no se traduce en grandes cambios (de acuerdo con análisis de 2D-NMR, entre otros), se produce la activación de enzimas hidrolíticas y oxidativas de gran interés biotecnológico en procesos dirigidos al aprovechamiento de la biomasa vegetal.

Wood extractives, solvent-soluble fractions of woody biomass, are considered to be a factor impeding or excluding fungal colonization on the freshly harvested conifers. Among wood decay fungi, the basidiomycete Phlebiopsis gigantea has evolved a unique enzyme system to efficiently transform or degrade conifer extractives but little is known about the mechanism(s). In this study, to clarify the mechanism(s) of softwood degradation, we examined the transcriptome, proteome, and metabolome of P. gigantea when grown on defined media containing microcrystalline cellulose and pine sapwood extractives. Beyond the conventional enzymes often associated with cellulose, hemicellulose and lignin degradation, an array of enzymes implicated in the metabolism of softwood lipophilic extractives such as fatty and resin acids, steroids and glycerides was significantly up-regulated. Among these, a highly expressed and inducible lipase is likely responsible for lipophilic extractive degradation, based on its extracellular location and our characterization of the recombinant enzyme. Our results provide insight into physiological roles of extractives in the interaction between wood and fungi. © 2021, The Author(s).

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat–straw transformation. Up-regulated and constitutive glycoside–hydrolases, polysaccharide–lyases, and carbohydrate–esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl–alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H2O2-producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Selective oxyfunctionalizations of aliphatic compounds are difficult chemical reactions, where enzymes can play an important role due to their stereo- and regio-selectivity and operation under mild reaction conditions. P450 monooxygenases are well-known biocatalysts that mediate oxyfunctionalization reactions in different living organisms (from bacteria to humans). Unspecific peroxygenases (UPOs), discovered in fungi, have arisen as “dream biocatalysts” of great biotechnological interest because they catalyze the oxyfunctionalization of aliphatic and aromatic compounds, avoiding the necessity of expensive cofactors and regeneration systems, and only depending on H2O2 for their catalysis. Here, we summarize recent advances in aliphatic oxyfunctionalization reactions by UPOs, as well as the molecular determinants of the enzyme structures responsible for their activities, emphasizing the differences found between well-known P450s and the novel fungal peroxygenases. © 2021 The Authors

Epoxide metabolites from n-3 and n-6 polyunsaturated fatty acids arouse interest thanks to their physiological and pharmacological activities. Their chemical synthesis has significant drawbacks, and enzymes emerge as an alternative with potentially higher selectivity and greener nature. Conversion of eleven eicosanoid, docosanoid, and other n-3/n-6 fatty acids into mono-epoxides by fungal unspecific peroxygenases (UPOs) is investigated, with emphasis on the Agrocybe aegerita (AaeUPO) and Collariella virescens (rCviUPO) enzymes. GC-MS revealed the strict regioselectivity of the n-3 and n-6 reactions with AaeUPO and rCviUPO, respectively, yielding 91%-quantitative conversion into mono-epoxides at the last double bond. Then, six of these mono-epoxides were obtained at mg-scale, purified and further structurally characterized by 1H, 13C and HMBC NMR. Moreover, chiral HPLC showed that the n-3 epoxides were also formed (by AaeUPO) with total S/R enantioselectivity (ee > 99%) while the n-6 epoxides (from rCviUPO reactions) were formed in nearly racemic mixtures. The high regio- and enantioselectivity of several of these reactions unveils the synthetic utility of fungal peroxygenases in fatty acid epoxidation.

Lignin biodegradation has been extensively studied in white-rot fungi, which largely belong to order Polyporales. Among the enzymes that wood-rotting polypores secrete, lignin peroxidases (LiPs) have been labeled as the most efficient. Here, we characterize a similar enzyme (ApeLiP) from a fungus of the order Agaricales (with ~13,000 described species), the soil-inhabiting mushroom Agrocybe pediades. X-ray crystallography revealed that ApeLiP is structurally related to Polyporales LiPs, with a conserved heme-pocket and a solvent-exposed tryptophan. Its biochemical characterization shows that ApeLiP can oxidize both phenolic and non-phenolic lignin model-compounds, as well as different dyes. Moreover, using stopped-flow rapid spectrophotometry and 2D-NMR, we demonstrate that ApeLiP can also act on real lignin. Characterization of a variant lacking the above tryptophan residue shows that this is the oxidation site for lignin and other high redox-potential substrates, and also plays a role in phenolic substrate oxidation. The reduction potentials of the catalytic-cycle intermediates were estimated by stopped-flow in equilibrium reactions, showing similar activation by H2O2, but a lower potential for the rate-limiting step (compound-II reduction) compared to other LiPs. Unexpectedly, ApeLiP was stable from acidic to basic pH, a relevant feature for application considering its different optima for oxidation of phenolic and nonphenolic compounds.

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.

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 beta-ether linkages (83 %), whereas those from the two Acacia barks have similar compositions (H/G/S ratio of approximate to 5:50:45), with a predominance of beta-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 beta-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.

Cereal brans constitute an unexploited source of valuable cell wall polysaccharides and phenolics. Sequential subcritical water extraction (SWE) of wheat, barley and rye bran enables the isolation of cell wall polysaccharides with targeted molecular structures. The extracts switched from glucan-rich to higher contents of feruloylated arabinoxylan (F-AX) with extraction time, resulting in significant radical scavenging activity. Structurally, wheat and rye F-AXs exhibited mostly single C(O)-3 arabinose substitutions, whereas barley bran F-AX showed a complex substitution pattern. The recalcitrance of the insoluble residues after SWE was revealed by the enrichment of F-AX populations with di-substituted arabinose units, and the presence of hydroxycinnamic acids, lignin and proteins. This study demonstrates the versatility of SWE to extract cell wall polysaccharides from cereal brans with tailored molecular structures and radical scavenging activity, with potential use in food and biomedical applications.

Lignin is conventionally defined as being formed by the oxidative polymerization of three main monolignols, p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, that are derived from the general phenylpropanoid biosynthetic pathway. Many other phenolic compounds that are also derived from the phenylpropanoid pathway are also known to perform as genuine lignin monomers in many plants, as is the case of the monolignol ester conjugates, phenolic compounds arising from the truncated biosynthesis of monolignols, or ferulate esters. Recent investigations, however, have indicated that phenolic compounds arising from beyond the canonical phenylpropanoid pathway, namely flavonoids, hydroxystilbenes, and hydroxycinnamic amides, may also behave as authentic lignin monomers and are incorporated into the lignin in some plants, further challenging the traditional definition of lignin. This is the case of the flavone tricin that is incorporated into the lignin of grasses and other monocots, the hydroxystilbene piceatannol (together with resveratrol and isorhapontigenin, at lower levels) that has been found in the lignins of palm fruit shells, their respective O-glucosides (astringin, piceid, and isorhapontin) that are present in the lignin of Norway spruce bark, or the ferulic amides feruloyltyramine, incorporated into the lignin of tobacco and potato tubers, and diferuloylputrescine, which appears to be incorporated into maize kernel lignin. These valuable compounds are potentially available in high amounts and at low cost and may be obtained from the waste products from the processing of agricultural or forest biomass.

Enzyme-driven oxygenation reactions are in the spotlight for organic synthesis. In this regard, a hemethiolate unspecific peroxygenase (UPO) from the fungus Chaetomium globosum has recently proven to be a suitable catalyst for selective epoxidation of unsaturated fatty acids in the context of the bio-based industry, but this enzyme could not be expressed in Escherichia coli for directed mutagenesis studies. Here, a previously unknown UPO from the related Collariella virescens (synonym: Chaetomium virescens) was obtained by E. coli expression of a putative upo gene. The activity of the purified enzyme on unsaturated fatty acids with different lengths and unsaturation degrees was tested. The ability of C. virescens UPO to epoxidize these compounds increases in the order myristoleic acid (C14:1) < palmitoleic acid (C16:1) < oleic acid (C18:1) differing from that observed for the C. globosum UPO, which also forms less hydroxylated derivatives. Given the possibility to produce the C. virescens UPO in E. coli as a recombinant enzyme and its oxyfunctionalization ability, some mutated variants were obtained mimicking the active-site of C. globosum UPO and evaluated on 18-carbon unsaturated fatty acids. Results revealed that widening the heme-access channel of C. virescens UPO by substituting a phenylalanine residue (in a F88L variant) maintains the enzyme epoxidation activity, and reduces undesired hydroxylation side-reactions (from 34% to only 7% of linoleic acid products) approaching the oxyfunctionalization pattern obtained with C. globosum UPO, although maintaining the absence of diepoxides. Conversely, its partial occlusion by introducing a second phenylalanine residue (in a T158F variant) resulted in partial selective epoxidation of linoleic acid (C18:2), while the oleic acid epoxidation was prevented. The above results show how E. coli expression can speed up the availability of new UPOs, and the design of ad hoc variants of these self-sufficient monooxygenases.

Unspecific peroxygenases (UPOs) constitute a new family of fungal heme-thiolate enzymes with high biotechnological interest. Although several thousand genes encoding hypothetical UPO-type proteins have been identified in sequenced fungal genomes and other databases, only a handful of UPO enzymes have been experimentally characterized to date. Therefore, gene screening and heterologous expression from genetic databases is a priority in the search for ad-hoc UPOs for oxyfunctionalization reactions of interest. Very recently, Escherichia coli production (as a soluble and active enzyme) of a previously described basidiomycete UPO has been reported. Here, we explored this convenient heterologous expression system to obtain the protein products from several putative UPO genes available. In this way, two UPOs from the ascomycetes Collariella virescens (syn. Chaetomium virescens) and Daldinia caldariorum were successfully obtained, purified, and characterized. Comparison of their kinetic constants for oxidation of model substrates revealed 7-20 fold higher catalytic efficiency of the latter enzyme oxidizing simple aromatic compounds (such as veratryl alcohol, naphthalene and benzyl alcohol). Homology molecular models of these enzymes showed three conserved and two differing residues in the distal side of the heme (the latter representing two different positions of a phenylalanine residue). Interestingly, substitution of the C. virescens UPO Phe88 by the homologous residue in the D. caldariorum UPO resulted in a F88L variant with 5-21 fold higher efficiency oxidizing the above aromatic compounds.IMPORTANCE: UPOs catalyze regio- and stereo-selective oxygenations of both aromatic and aliphatic compounds. Similar reactions were previously described for cytochrome P450 monooxygenases, but UPOs have the noteworthy biotechnological advantage of being stable enzymes only requiring H2O2 to be activated. Both characteristics are related to the extracellular nature of UPOs as secreted

Novel laccases have promising and valuable applications in biorefineries. This investigation documents, for the first time, the potential of depolymerising and repolymerising lignin by the secretome, rich in laccases, from a newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111, for further saccharification and ethanolic fermentation steps. Proteomic analyses of the secretome of M. palmivorus show that laccases are the most predominant enzyme released by this fungus. The whole crude enzymatic broth is used for the delignification of lignin in Eucalyptus globulus wood, with the aim of enhancing the saccharification by cellulolytic and xylanolytic enzymes from Penicillium echinulatum S1M29. In addition, two different strategies, namely, laccase treatment before and after enzymatic hydrolysis, are employed to detoxify steam-exploded E. globulus wood. The objective is to increase the fermentative performance by removing substances formed during the feedstock pretreatment that can inhibit microbial fermentation. The E. globulus wood delignification results in a 31% decrease in the lignin content and a 10% increase in the glucose yield after hydrolysis. An important finding of the present work is the successful wood delignification in the absence of laccase mediators. This laccase-rich preparation also demonstrates its potential in removing the phenolic inhibitors present in steam-exploded E. globulus wood, increasing the ethanol yield by an additional 10%. Furthermore, it is important to highlight that these findings are achieved in the absence of commercial enzymes, making M. palmivorus laccases a potential candidate not only for the production of biofuels but also for the generation of lignin-derived aromatic compounds for different applications in the biotechnology industry. © 2020 Elsevier Ltd

A comparison of sequenced Agaricomycotina genomes suggests that efficient degradation of wood lignin was associated with the appearance of secreted peroxidases with a solvent-exposed catalytic tryptophan. This hypothesis is experimentally demonstrated here by resurrecting ancestral fungal peroxidases, after sequence reconstruction from genomes of extant white-rot Polyporales, and evaluating their oxidative attack on the lignin polymer by state-of-the-art analytical techniques. Rapid stopped-flow estimation of the transient-state constants for the 2 successive one-electron transfers from lignin to the peroxide-activated enzyme (k2app and k3app) showed a progressive increase during peroxidase evolution (up to 50-fold higher values for the rate-limiting k3app). The above agreed with 2-dimensional NMR analyses during steady-state treatments of hardwood lignin, showing that its degradation (estimated from the normalized aromatic signals of lignin units compared with a control) and syringyl-to-guaiacyl ratio increased with the enzyme evolutionary distance from the first peroxidase ancestor. More interestingly, the stopped-flow estimations of electron transfer rates also showed how the most recent peroxidase ancestors that already incorporated the exposed tryptophan into their molecular structure (as well as the extant lignin peroxidase) were comparatively more efficient at oxidizing hardwood (angiosperm) lignin, while the most ancestral “tryptophanless” enzymes were more efficient at abstracting electrons from softwood (conifer) lignin. A time calibration of the ancestry of Polyporales peroxidases localized the appearance of the first peroxidase with a solvent-exposed catalytic tryptophan to 194 ± 70 Mya, coincident with the diversification of angiosperm plants characterized by the appearance of dimethoxylated syringyl lignin units.

Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography–mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the channel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the ability of the MroUPO to epoxidize oleic acid (while no activity was detected for the I153V variant). The rationale for these results was revealed by the substrate positioning in the above computational simulations, which predict a shorter distance between the oleic acid double bond and the oxygen atom of the peroxide-activated heme (compound I) in the I153T variant than in the native enzyme, promoting epoxidation. In contrast, the I153F/S156F double mutation fully prevents the approach of oleic acid in the bent conformation required for double-bond epoxidation, although its (sub)terminal hydroxylation was predicted and experimentally confirmed. The I153T mutation also increased the UPO selectivity on polyunsaturated fatty acid epoxidation, strongly reducing the rati

Lignin is a phenylpropanoid polymer derived essentially from the oxidative radical coupling of three p-hydroxycinnamyl alcohols (so-called “monolignols”) differing in their degree of methoxylation, p-coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all deriving from the shikimate-derived monolignol biosynthetic pathway, have been found to behave as lignin monomers in many plants, including the monolignol ester conjugates (with acetate, p-coumarate, ferulate, and p-hydroxybenzoate) or the incompletely methylated monomers caffeyl and 5-hydroxyconiferyl alcohols, among others. However, recent studies have revealed that some phenolic compounds derived from other biosynthetic pathways also act as true lignin monomers participating in coupling and cross-coupling reactions during lignification in several plants. This is the case for the flavone tricin recently established as a monomer in the lignins from grasses and other monocots (and the flavanone naringenin found in the lignin in an fnsII rice mutant) or the newly discovered hydroxystilbenes (resveratrol, isorhapontigenin and piceatannol) that are implicated as monomers in the lignin of palm fruit endocarps. The discovery in lignins of ‘non-conventional’ phenolic precursors arising from other biosynthetic pathways expands the traditional definition of lignin, illustrates yet again the high metabolic plasticity of lignification, and reveals that any phenolic compound that is delivered to the cell wall may be oxidized and incorporated into the lignin polymer during lignification, subject exclusively to its chemical compatibility. On the other hand, these discoveries reveal that valuable flavonoids and hydroxystilbenes could also be obtained from agricultural resides (cereal straws or palm fruit shells) or from low-value lignin side-streams in lignocellulose processing mills, which will open new opportunities for the valorization of these residues that are considered as wastes.

The recently discovered unspecific peroxygenases (UPOs) from the ascomycetes Chaetomium globosum and Humicola insolens were capable of selectively hydroxylating isophorone to 4-hydroxyisophorone (4HIP) and 4-ketoisophorone (4KIP), which are substrates of interest for the pharmaceutical and flavor-and-fragrance sectors. The model UPO from the basidiomycete Agrocybe aegerita was less regioselective, forming 7-hydroxyisophorone (and 7-formylisophorone) in addition to 4HIP. However, it was the most stereoselective UPO yielding the S-enantiomer of 4HIP with 88% ee. Moreover, using H. insolens UPO full kinetic resolution of racemic HIP was obtained within only 15 min, with >75% recovery of the R-enantiomer. Surprisingly, the UPOs from two other basidiomycetes, Marasmius rotula and Coprinopsis cinerea, failed to transform isophorone. The different UPO selectivities were rationalized by computational simulations, in which isophorone and 4HIP were diffused into the enzymes using the adaptive PELE software, and the distances from heme-bound oxygen in H2O2-activated enzyme to different substrate atoms, and the corresponding binding energies were analyzed. Interestingly, for process upscaling, full conversion of 10 mM isophorone was achieved with H. insolens UPO within nine hours, with total turnover numbers up to 5500. These biocatalysts, which only require H2O2 for activation, may represent a novel, simple and environmentally-friendly route for the production of isophorone derivatives.

This work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H2O2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200000) than MroUPO (26000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (kcat/Km) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18O from H218O2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C.

Lignin is a phenylpropanoid polymer derived from the oxidative radical coupling of three p-hydroxycinnamyl alcohols (so-called “monolignols”) differing in their degree of methoxylation, p-coumaryl, coniferyl, and sinapyl alcohols. Several other phenolic compounds, all deriving from the shikimate-derived monolignol biosynthetic pathway, have been found to behave as lignin monomers in many plants, including the monolignol ester conjugates (with acetate, p-coumarate, ferulate, and p-hydroxybenzoate) or the incompletely methylated monomers caffeyl and 5-hydroxyconiferyl alcohols, among others. However, recent studies have revealed that some phenolic compounds derived from other biosynthetic pathways also act as true lignin monomers participating in coupling and cross-coupling reactions during lignification in several plants. This is the case for the flavone tricin recently established as a monomer in the lignins from grasses and other monocots (and the flavanone naringenin found in the lignin in an fnsII rice mutant) or the newly discovered hydroxystilbenes (resveratrol, isorhapontigenin and piceatannol) that are implicated as monomers in the lignin of palm fruit endocarps. The discovery in lignins of ‘non-conventional’ phenolic precursors arising from other biosynthetic pathways than the canonical monolignol biosynthetic pathway expands the traditional definition of lignin and reveals that any phenolic compound that is delivered to the cell wall may be oxidized and incorporated into the lignin polymer during lignification, subject exclusively to its chemical compatibility

La presente Tesis plantea el estudio de la oxifuncionalización enzimática de diferentes compuestos alifáticos lineales, principalmente alcanos y ácidos grasos, catalizada por una serie de peroxigenasas fúngicas denominadas peroxigenasas inespecíficas (UPO). Se han estudiado en mayor profundidad las reacciones catalizadas por la peroxigenasa del hongo Marasmius rotula (MroUPO) y también se han llevado a cabo estudios con la peroxigenasa de Chaetomium globosum (CglUPO), comparándose con la peroxigenasa salvaje de Agrocybe aegerita (AaeUPO) y la peroxigenasa recombinante de Coprinopsis cinerea (rCciUPO). Estas peroxigenasas, descubiertas recientemente, son capaces de oxidar una gran variedad de sustratos orgánicos con peróxido de hidrógeno (H2O2) como único co‐sustrato, presentando además de ésta, otras importantes ventajas frente a los citocromos P450 (P450), las enzimas hidroxilantes más versátiles. La evaluación de la conversión de los diferentes sustratos por las peroxigenasas así como la identificación de los productos de reacción se llevó a cabo mediante Cromatografía de Gases acoplada a Espectrometría de Masas (GC‐MS). Finalmente, con objeto de studiar y verificar el mecanismo de acción de estas reacciones enzimáticas, se llevaron a cabo estudios con peróxido de hidrógeno marcado con el isótopo de oxígeno 18 (H2 18O2). Los resultados de esta Tesis demuestran que MroUPO cataliza la oxifuncionalización selectiva de los sustratos estudiados, revelando importantes y novedosas diferencias con respecto a las otras dos peroxigenasas (AaeUPO y rCciUPO) estudiadas anteriormente. En estudios previos se había visto que estas dos últimas peroxigenasas catalizan la hidroxilación de ácidos grasos saturados en las posiciones subterminales (‐1 y ‐2) obteniéndose como productos principales los derivados monohidroxilados en dichas posiciones. Del mismo modo, estas peroxigenasas son capaces de hidroxilar las posiciones subterminales de los alcanos a ambos lados de la cadena hidr

Stopped-flow spectrophotometry enables us to estimate the rate constants for lignin oxidation by ligninolytic peroxidases following the electron-transfer from the "enzyme side" (heme reduction). Using this technique and water-soluble (hardwood and softwood) lignosulfonates as substrates, we were able to demonstrate that a solvent exposed tryptophan residue is strictly required for oxidation of nonphenolic (permethylated) lignin by ligninolytic peroxidases. Simultaneously, the use of 2D-NMR spectroscopy revealed that the tryptophan-Iess variants only cause negligible modification of lignin in long-term steady-state enzymatic treatments (and null modification when applied on nonphenolic lignin

Lignin is derived from the oxidative radical coupling of three main p-hydroxycinnamyl alcohols, the monolignols p-coumaryl, coniferyl, and sinapyl alcohols. Recently, we discovered in grass lignins the first phenolic monomer from outside the canonical lignin biosynthetic pathway, the flavone tricin. In this work, we report the discovery of another polyphenolic compound from yet another biosynthetic pathway, the hydroxystilbene piceatannol, which also acts as a true lignin monomer in palm fruit endocarps. Piceatannol was released from these lignins upon derivatization followed by reductive cleavage (DFRC), a degradative method that cleaves ß-ether bonds, indicating that at least a fraction of it is incorporated into the lignin through labile ether bonds. 2D-NMR revealed the occurrence of other structures involving piceatannol in the lignin polymer, and demonstrated that piceatannol acts as an authentic lignin monomer participating in coupling and cross-coupling reactions during lignification of palm fruit endocarps.

Macaúba (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) is a tropical palm tree from the Arecaceae family native to South America. This palm is considered a promising energy crop due to the high bioenergy production potential of its fruit and the possibility of integrated crop-livestock/forest-farming systems. The fruit has a high oil content, which has favorable characteristics for the production of biodiesel. In addition, from the residues generated in the processing of the fruits, it is possible to produce charcoal (from the shell) and densified briquettes (from the husk), both biofuels of high energy density. In this study, the lignins from different anatomical parts of the macaúba palm fruit, namely stalks, epicarp (husk), and endocarp (shell), were thoroughly analyzed by different analytical techniques, including twodimensional nuclear magnetic resonance (2D-NMR) spectroscopy, analytical pyrolysis coupled to gas chromatography and mass spectrometry (Py-GC/MS) and derivatization followed by reductive cleavage (DFRC). These lignins presented important differences among them, in both composition and structure. The lignin from stalks was enriched in Slignin units (S/G ratio 1.2) and ß-ethers (84% of the total), whereas the epicarp presented a highly condensed G-rich lignin (S/G ratio 0.2), consequently depleted in ß-ethers (65%). The endocarp, on the other hand, was characterized by the occurrence of significant amounts of hydroxystilbenes, particularly piceatannol, incorporated into the lignin structure, thus producing a greater accumulation of lignin that is more condensed and presumably contributing to endocarp hardening by reinforcing the cell wall. The results also indicated that piceatannol acts as a true lignin monomer participating in coupling and cross-coupling reactions during lignification of macaúba endocarp.

Methodological limitations difficult estimation of lignin oxidation rates by Iigninolytic peroxidases. Here, lignin oxidation was seen from the "enzyme side" by estimating the reduction rates of H202 activated peroxidase compounds I and 11, using rapid spectrophotometry. Removal of Trp164 in versatile peroxidase resulted in 4-100 fold slower rate-limiting compound-II reduction by watersoluble sulfonated lignins. Moreover, when methylated lignosulfonates were used, negligible transfer was found, confirming that residual reduction by native lignin was due to its phenolic moiety. In agreement with the transient-state kinetic data, low or null structural modification of lignin was found (by NMR) during steady-state treatment with the W164S variant compared with native VP. Similar results were obtained with lignin peroxidase. Therefore, we demonstrate for the first time that the surface tryptophan conserved in most LiPs and VPs is strictly required for oxidation of the nonphenolic moiety, which represents the major and more recalcitrant part of the lignin polymer.

Recently discovered fungal unspecific peroxygenases from Marasmius rotula and Chaetomium globosum catalyze the epoxidation of unsaturated fatty acids (FA) and FA methyl esters (FAME), unlike the well-known peroxygenases from Agrocybe aegerita and Coprinopsis cinerea. Reactions of a series of unsaturated FA and FAME with cis-configuration revealed high (up to 100%) substrate conversion and selectivity towards epoxidation, although some significant differences were observed between enzymes and substrates with the best results being obtained with the C. globosum enzyme. This and the M. rotula peroxygenase appear as promising biocatalysts for the environmentally-friendly production of reactive FA epoxides given their self-sufficient monooxygenase activity and the high conversion rate and epoxidation selectivity

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 p-hydroxycinnamates (ferulates—FAs and p-coumarates—pCAs) and major structural components during bioprocessing helps to discriminate the sources of recalcitrance in grasses and paves the way for the recovery of p-hydroxycinnamates, which have multiple applications. To address these subjects, we assessed sugarcane bagasse biorefining under alkalinesulfite 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 pCAs were also retained in pretreated material. After pretreatment and enzymatic hydrolysis, the residual solids contained mostly lignin and ester-linked pCAs, 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 pCAs; nevertheless, a significant fraction of the pCAs remained attached to the saponified solids, probably to lignin, through 4-O 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 pCAs remained in pretreated material. Ester-linked pCAs detected in pretreated material also resisted to the enzymatic hydrolysis step. O

Due to its well-known advantages Supercritical Fluid Extraction (SFE) have greatly expanded its application field as alternative to conventional extraction methods in the last decade. Most of these applications do not include the screening of all extractable components present in the solid matrix but individual extraction of specific classes of organics, such as pollutant families present in environmental samples. In the present work, we applied SFE to the extraction as a whole of lipids from Eucalyptus globulus wood. This is a new specific analytical problem, and, as usual in SFE applications, it was necessary to find empirically which parameters influence the extraction efficiency, since many different factors may affect the recovery of analytes. As an empirical approach to obtain reliable results in a reasonable time is an arduous and tedious task, we use a statistical approach. For practical reasons the operational parameters considered were temperature, pressure and the use of different concentrations of a modifier, methanol, to improve the extraction efficiency of supercritical carbon dioxide. Their influence on the extraction efficiency have been evaluated by using an optimized rotatable central composite design, which change the above three variables to result a 23 factorial design. The extracts obtained under different conditions were analyzed by OC using an improved methodology (Gutiérrez el al., this meeting). The main conclusion drawn is that lipids of the E globulus wood, including fatty acids, sterols and steryl esters among others, can be extracted by CO, in supercritical conditions, being the extracts qualitatively similar to those obtained by Soxhlet extraction.

In order lo meet growing demands for paper of increasing quality and in greater yields, a variety of chemical pulping processes has been developed, together with improved bleaching methods. Regardless of pulping and bleaching procedures, extractives from both softwood and hardwoods cause production and environmental problems in the pulp and paper industry. The accumulation of very small amounts of wood extractives can result in blockages easing a shutdown of operations. These blockages have long been a serious problem in the industry and are responsible for reduced levels of production higher equipment maintenance costs, higher operation costs, and an increased incidence of quality defects. The use of certain pulping procedures with some species can lead to a particularly serious accumulation that will cover the surfaces of various pans of the equipment of both the pulp and paper mill, as well as becoming entrained in the pulp. The increasing need for recirculation and conservation of water in pans of pulp and paper mills and the increasingly stringent controls of the composition of effluents lo meet environmental protection requirements is also leading lo an increase in pitch concentrations which likewise results in higher deposition. In this paper, we study the composition of the organic deposits accumulated during the kraft pulping of Eucalyptus globulus using two different bleaching procedures (ECF and TCF). The deposits were exhaustively extracted with acetone and analysed by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The residues, on the other hand, were studied by pyrolysis-gas chromatography-mass spectrometry (PyGC-MS) and pyrolysis-methylation. The knowledge of the chemical nature of these deposits will assist the control of this problem extraction.

An analytical procedure for the analysis of extractives from wood pulp and pitch deposits is described in this paper It comprises: i) The development of a solid phase extraction (SPE) method for the fractionation of wood and pitch extracts into less complex lipid fractions for a further characterization by gas chromatography-mass spectrometry, ü) The development of a rapid gas-chromatographic method that enables the analysis of a high number of samples in a short period. A fractionation method of wood extractives using solid-phase extraction (SPE) in aminopropyl cartridges was developed for the preparative scale isolation and separation of major lipid classes. The advantages of using SPE include smaller sample and solvent requirements and simpler use than with conventional solvent extraction techniques. Sample fractionation permits the obtention of different fractions which can be studied by using the most suitable conditions required for the analysis of each classes of compounds. A mixture of standard compounds (fatty acid, resin acid, sterol, steryl ester and triglyceride) was used to optimize the chromatographic conditions for the analysis of wood extractives. High temperature capillary columns (DB-5HT; J&W Scientific) of different lengths (from 25 to 2 m) were used. The comparative study of different column lengths and temperature programs showed that the short capillary column gas-chromatography is the most suitable procedure for a rapid analysis of wood extractives, since it enables elution and separation of a wide range of molecular weight compounds (from fatty acids to steryl esters and triglycerides) in the same chromatogram in a short period of time. Different examples of analysis of wood extractives from Eucalyptus globulus and Pinus sylvestris, pulps and pitch deposits in pulp mills, are shown in this paper.