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Angel T Martinez - One of the best experts on this subject based on the ideXlab platform.
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Reaction mechanisms and applications of Aryl-Alcohol Oxidase.
The Enzymes, 2020Co-Authors: Ana Serrano, Juan Carro, Angel T MartinezAbstract:Aryl-Alcohol Oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline Oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic perOxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing perOxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.
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structure guided evolution of aryl alcohol Oxidase from pleurotus eryngii for the selective oxidation of secondary benzyl alcohols
Advanced Synthesis & Catalysis, 2019Co-Authors: Javier Vinagonzalez, Angel T Martinez, Victor Guallar, Ana Serrano, Ferran Sancho, Diego Jimenezlalana, Miguel AlcaldeAbstract:This research was supported by the EU project FP7‐KBBE‐2013‐7‐613549‐INDOX, by the Spanish Government projects BIO2016‐79106‐R‐Lignolution, CTQ2016‐74959‐R (MINECO/FEDER, EU) and by the Comunidad de Madrid project Y2018/BIO4738‐EVOCHIMERA.
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Switching the substrate preference of fungal Aryl-Alcohol Oxidase: towards stereoselective oxidation of secondary benzyl alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Javier Viña-gonzalez, Ferran Sancho, Angel T MartinezAbstract:Oxidation of primary alcohols by Aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary Aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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switching the substrate preference of fungal aryl alcohol Oxidase towards stereoselective oxidation of secondary benzyl alcohols
Catalysis Science & Technology, 2019Co-Authors: Ana Serrano, Juan Carro, Miguel Alcalde, Victor Guallar, Ferran Sancho, Javier Vinagonzalez, Angel T MartinezAbstract:Oxidation of primary alcohols by Aryl-Alcohol Oxidase (AAO), a flavoenzyme that provides H2O2 to fungal perOxidases for lignin degradation in nature, is achieved by concerted hydroxyl proton transfer and stereoselective hydride abstraction from the pro-R benzylic position. In racemic secondary alcohols, the R-hydrogen abstraction would result in the selective oxidation of the S-enantiomer to the corresponding ketone. This stereoselectivity of AAO may be exploited for enzymatic deracemization of chiral mixtures and isolation of R-enantiomers of industrial interest by switching the enzyme activity from primary to secondary alcohols. A combination of computational simulations and mutagenesis has been used to produce AAO variants with increased activity on secondary alcohols, using the already available F501A variant of Pleurotus eryngii AAO as a starting point. Adaptive-PELE simulations for the diffusion of (S)-1-(p-methoxyphenyl)-ethanol in this variant allowed Ile500 to be identified as one of the key residues with a higher number of contacts with the substrate during its transition from the solvent to the active site. Substitution of Ile500 produced more efficient variants for the oxidation of several secondary alcohols, and the I500M/F501W double variant was able to fully oxidize (after 75 min) with high selectivity (ee >99%) the S-enantiomer of the model secondary Aryl-Alcohol (±)-1-(p-methoxyphenyl)-ethanol, while the R-enantiomer remained unreacted.
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Multiple implications of an active site phenylalanine in the catalysis of Aryl-Alcohol Oxidase
Scientific reports, 2018Co-Authors: Juan Carro, Patricia Ferreira, Milagros Medina, Victor Guallar, Pep Amengual-rigo, Ferran Sancho, Angel T MartinezAbstract:Aryl-Alcohol Oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.
Patricia Ferreira - One of the best experts on this subject based on the ideXlab platform.
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Multiple implications of an active site phenylalanine in the catalysis of Aryl-Alcohol Oxidase
Scientific reports, 2018Co-Authors: Juan Carro, Patricia Ferreira, Milagros Medina, Victor Guallar, Pep Amengual-rigo, Ferran Sancho, Angel T MartinezAbstract:Aryl-Alcohol Oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.
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Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass
Applied and environmental microbiology, 2016Co-Authors: Yann Mathieu, Patricia Ferreira, Francois Piumi, Craig B. Faulds, Richard Valli, Juan Carro Aramburu, Eric RecordAbstract:ABSTRACT Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol Oxidases (AAO) and glucose Oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger. Based on the enzyme9s activity and specificity, it was assigned to the glucose dehydrogenases (P. cinnabarinus GDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol Oxidase from Pleurotus eryngii. Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as Aryl-Alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual Oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.
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aromatic stacking interactions govern catalysis in aryl alcohol Oxidase
FEBS Journal, 2015Co-Authors: Patricia Ferreira, Juan Carro, Angel T Martinez, Kenneth W. Borrelli, Victor Guallar, Aitor Hernandezortega, Fatima Lucas, Beatriz Herguedas, Milagros MedinaAbstract:This is the peer reviewed version of the following article: [Ferreira, P., Hernandez-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martinez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in Aryl-Alcohol Oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html The version posted may not be updated or replaced with the final published version (the Version of Record).
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5-hydroxymethylfurfural conversion by fungal Aryl-Alcohol Oxidase and unspecific peroxygenase.
The FEBS journal, 2015Co-Authors: Juan Carro, Patricia Ferreira, Alicia Prieto, Ana Gutierrez, Jesús Jiménez-barbero, Ana Serrano, Leonor Rodríguez, Beatriz Balcells, Ana Ardá, Rene UllrichAbstract:Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal Aryl-Alcohol Oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H2O2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H2O2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O2 is the only co-substrate required, and water is the only by-product formed.
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Comparative analysis of gene repertoires associated with degradation of plant cell wall polymers and extractives in 21 fungal genomes.
2014Co-Authors: Chiaki Hori, Patricia Ferreira, Francisco J. Ruiz-dueñas, Takuya Ishida, Kiyohiko Igarashi, Masahiro Samejima, Hitoshi Suzuki, Emma Master, Benjamin Held, Paulo CanessaAbstract:(A) Principal component analysis (PCA) of 21 fungi using 73 CAZy and 12 AA families (Dataset S1). GMC oxidoreductases methanol Oxidase, glucose Oxidase and aryl alcohol Oxidase were excluded because confident functional assignments could not be made and/or their inclusion did not contribute to separation of white- and brown-rot species. (B) PCA of 21 fungi using genes encoding 14 enzymes involved in lipid metabolism (KEGG reference pathway 00071, Dataset S1). There is no significant segregation of white-rot and brown-rot fungi although P. gigantea was positioned in the third quadrant with B. adusta and P. carnosa. Symbols for white rot and brown rot fungi appear in blue and red, respectively. Tremella mesenterica is a mycoparasite. For raw data and contributions of the top 20 families see Dataset S1, Text S1 and Figures S2 and S3.
María Jesús Martínez - One of the best experts on this subject based on the ideXlab platform.
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new Oxidase from bjerkandera arthroconidial anamorph that oxidizes both phenolic and nonphenolic benzyl alcohols
Biochimica et Biophysica Acta, 2009Co-Authors: Elvira Romero, Patricia Ferreira, Angel T Martinez, María Jesús MartínezAbstract:Abstract A new flavoOxidase is described from a Bjerkandera arthroconidial anamorph. Its physicochemical characteristics, a monomeric enzyme containing non-covalently bound flavin adenine dinucleotide (FAD), and several catalytic properties, such as oxidation of aromatic and polyunsaturated aliphatic primary alcohols, are similar to those of Pleurotus eryngii Aryl-Alcohol Oxidase (AAO). However, it also efficiently oxidizes phenolic benzyl and cinnamyl alcohols that are typical substrates of vanillyl-alcohol Oxidase (VAO), a flavoOxidase from a different family, characterized by its multimeric nature and presence of covalently-bound FAD. The enzyme also differs from P. eryngii AAO by having extremely high efficiency oxidizing chlorinated benzyl alcohols (1000–1500 s− 1 mM− 1), a feature related to the different alcohol metabolites secreted by the Pleurotus and Bjerkandera species including chloroaromatics, and higher activity on aromatic aldehydes. What is even more intriguing is the fact that, the new Oxidase is optimally active at pH 6.0 on both p-anisyl and vanillyl alcohols, suggesting a mechanism for phenolic benzyl alcohol oxidation that is different from that described in VAO, which proceeds via the substrate phenolate anion formed at basic pH. Based on the above properties, and its ADP-binding motif, partially detected after N-terminus sequencing, the new enzyme is classified as a member of the GMC (glucose–methanol–choline Oxidase) oxidoreductase family oxidizing both AAO and VAO substrates.
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An anamorph of the white-rot fungus Bjerkandera adusta capable of colonizing and degrading compact disc components
FEMS microbiology letters, 2007Co-Authors: Elvira Romero, Angel T Martinez, Mariela Speranza, Javier García-guinea, María Jesús MartínezAbstract:A Geotrichum-like fungus isolated from a biodeteriorated compact disc (CD) was able to degrade in vitro the components of different CD types. The fungal hyphae inside the CD fragments grew through the aluminium layer and produced the solubilization of this metal. Furthermore, examination of CDs by scanning electron microscopy showed that the fungus was able to destroy the pits and lands structures grooved in the polycarbonate layer, confirming degradation of this aromatic polymer. The fungus secretes Aryl-Alcohol Oxidase and Mn2+-oxidizing perOxidase, two kinds of oxidoreductases characteristic of ligninolytic basidiomycetes. Analysis of the ITS region of ribosomal DNA, as well as the morphological characteristics, the lack of sexual forms and the profile of enzymes secreted in liquid medium identified the fungus as a Geotrichum-like anamorph of Bjerkandera adusta (Willd.) P. Karst.
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screening of 68 species of basidiomycetes for enzymes involved in lignin degradation
Fungal Biology, 1995Co-Authors: Fernando Pelaez, María Jesús Martínez, Angel T MartinezAbstract:A survey for enzymes involved in lignin degradation was carried out from 90 strains of 68 species of different groups of basidiomycetes. Laccase activity was found in 50% of the fungi tested, whereas Aryl-Alcohol Oxidase (AAO) and Mn-dependent perOxidase (MnP) were detected in 40% and 29% of species, respectively. Laccase activity of more than 200 U l−1 was obtained in cultures of Trametes versicolor, Phellinus torulosus, Cerrena unicolor and Pleurotus eryngii, whereas the AAO and MnP levels were comparatively lower, and lignin perOxidase was not detected in the different fungi tested. Previously known AAO-producing fungi, P. eryngii and Bjerkandera adusta, showed the highest AAO activities, and the enzyme was detected for the first time in fungi from very different taxonomic groups, including the gasteromycete Cyathus olla. In contrast to laccase and AAO, MnP seemed to be restricted to species of Aphyllophorales, with the exception of the tremellaceous fungus Exidia glandulosa. It is interesting to note that MnP was found in most Phellinus species, P. ribis, P. trivialis and P. torulosus producing the highest activities among the fungi studied. Simultaneous production of MnP and H2O2-producing AAO was found in several polyporaceous fungi, but the latter enzyme was absent from the above Phellinus species.
Martínez, Ángel T. - One of the best experts on this subject based on the ideXlab platform.
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved Aryl-Alcohol Oxidase
'Elsevier BV', 2021Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde Galeote MiguelAbstract:[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved Aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde MiguelAbstract:Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.Peer ReviewedPostprint (author's final draft
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Reaction mechanisms and applications of Aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Serrano Ana, Carro Juan, Martínez, Ángel T.Abstract:26 p.-7 fig.-2 tab. The Enzymes (2020)Aryl-Alcohol Oxidases (AAO) constitute a family of FAD-containing enzymes, included in the glucose-methanol-choline Oxidase/dehydrogenase superfamily of proteins. They are commonly found in fungi, where their eco-physiological role is to produce hydrogen peroxide that activates ligninolytic perOxidases in white-rot (lignin-degrading) basidiomycetes or to trigger the Fenton reactions in brown-rot (carbohydrate-degrading) basidiomycetes. These enzymes catalyze the oxidation of a plethora of aromatic, and some aliphatic, polyunsaturated alcohols bearing conjugated primary hydroxyl group. Besides, the enzymes show activity on the hydrated forms of the corresponding aldehydes. Some AAO features, such as the broad range of substrates that it can oxidize (with the only need of molecular oxygen as co-substrate) and its stereoselective mechanism, confer good properties to these enzymes as industrial biocatalysts. In fact, AAO can be used for different biotechnological applications, such as flavor synthesis, secondary alcohol deracemization and oxidation of furfurals for the production of furandicarboxylic acid as a chemical building block. Also, AAO can participate in processes of interest in the wood biorefinery and textile industries as an auxiliary enzyme providing hydrogen peroxide to ligninolytic or dye-decolorizing perOxidases. Both rational design and directed molecular evolution have been employed to engineer AAO for some of the above biotechnological applications.This work has been supported by the EnzOx2 project (H2020-BBI-PPP-2015-2-720297,https://www.enzox2.eu) of the European BBI-JU (https://www.bbi-europe.eu), the GenoBioref project (BIO2017-86559-R) of the Spanish Ministry of Economy, Industry and Competitiveness, co-financed by FEDER funds, and the PIE201620E081 CSIC project.Peer reviewe
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Laboratory evolution platform for aryl alcohol Oxidase in Saccharomyces cerevisiae
2019Co-Authors: Viña-gonzález Javier, Alcalde Galeote Miguel, González-pérez David, Martínez, Ángel T.Abstract:Trabajo presentado en el 3rd Multistep Enzyme Catalyzed Processes Congress, celebrado en Madrid (España) del 07 al 10 de abril de 2014.Among the ligninolytic oxidoreductases secreted by white-rot fungi, the aryl alcohol Oxidase (AAO) plays an outstanding role as H2O2 supplying enzyme during lignin decay1. With high enantioselectivity and broad substrate specificity, this flavo-enzyme is also a promising departure point for directed evolution studies towards different biotechnological fates, but the lack of heterologous functional expression levels precludes further advances in the field. In this study, the native signal peptide of AAO from Pleurotus eryngii was replaced by those of the mating α-factor, the toxin K1 Killer, as well as combinations of pre- and pro-regions from both leaders to achieve secretion inSaccharomyces cerevisiae2,3. AAO expression in yeast was measured with the help of an ad-hoc colorimetric-dual HTS-protocol based on the detection of H2O2 with a chemical (FOX) and an enzymatic assay (ABTS-HRP). All constructs were successfully processed and secreted by yeast showing extracellular AAO activities with several aromatic alcohols, which opens new paths for future developments.Peer Reviewe
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Complete oxidation of hydroxymethylfurfural to furandicarboxylic acid by Aryl-Alcohol Oxidase
'Springer Science and Business Media LLC', 2019Co-Authors: Serrano-lotina, Ana M., Calviño Eva, Carro Juan, Sánchez-ruiz María, Cañada F. Javier, Martínez, Ángel T.Abstract:[Background] 5-Hydroxymethylfurfural (HMF) is a highly valuable platform chemical that can be obtained from plant biomass carbohydrates. HMF can be oxidized to 2,5-furandicarboxylic acid (FDCA), which is used as a renewable substitute for the petroleum-based terephthalic acid in polymer production.[Results] Aryl-Alcohol Oxidase (AAO) from the white-rot fungus Pleurotus eryngii is able to oxidize HMF and its derivative 2,5-diformylfuran (DFF) producing formylfurancarboxylic acid (FFCA) thanks to its activity on benzylic alcohols and hydrated aldehydes. Here, we report the ability of AAO to produce FDCA from FFCA, opening up the possibility of full oxidation of HMF by this model enzyme. During HMF reactions, an inhibitory effect of the H2O2 produced in the first two oxidation steps was found to be the cause of the lack of AAO activity on FFCA. In situ monitoring of the whole reaction by 1H-NMR confirmed the absence of any unstable dead-end products, undetected in the HPLC analyses, that could be responsible for the incomplete conversion. The deleterious effect of H2O2 was confirmed by successful HMF conversion into FDCA when the AAO reaction was carried out in the presence of catalase. On the other hand, no H2O2 formation was detected during the slow FFCA conversion by AAO in the absence of catalase, in contrast to typical Oxidase reaction with HMF and DFF, suggesting an alternative mechanism as reported in some reactions of related flavo-Oxidases. Moreover, several active-site AAO variants that yield nearly complete conversion in shorter reaction times than the wild-type enzyme have been identified.[Conclusions] The use of catalase to remove H2O2 from the reaction mixture leads to 99% conversion of HMF into FDCA by AAO and several improved variants, although the mechanism of peroxide inhibition of the AAO action on the aldehyde group of FFCA is not fully understood.We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This work has been funded by the H2020 BBI-JU (https://www.bbi-europe.eu) project EnzOx2 (H2020-BBI-PPP-2015-2-720297; https://www.enzox2.eu) and the GENOBIOREF (BIO2017-86559-R) and CTQ2015-64597-C2-2-P projects of the Spanish Ministry of Economy, Industry and Competitiveness, co-financed by FEDER funds.Peer reviewe
Viña-gonzález Javier - One of the best experts on this subject based on the ideXlab platform.
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved Aryl-Alcohol Oxidase
'Elsevier BV', 2021Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde Galeote MiguelAbstract:[EN] Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA
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Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions
'American Chemical Society (ACS)', 2021Co-Authors: Gómez De Santos, Patricia, Lázaro Sofia, Viña-gonzález Javier, Hoang, Manh Dat, Sánchez-moreno Israel, Glieder Anton, Hollmann Frank, Alcalde Galeote MiguelAbstract:[EN] Fungal peroxygenases are deemed emergent biocatalysts for selective C¿H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-Oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal Aryl-Alcohol Oxidase that supplies H2O2in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol Oxidase fusions in the pharmaceutical and chemical sectors.This work was supported by the Comunidad de Madrid Synergy CAM Project Y2018/BIO-4738-EVOCHIMERA-CM, the Spanish Government Projects BIO2016-79106-R-Lignolution, PID2019-106166RB-100-OXYWAVE and the CSIC Project PIE-201580E042. P. Gomez de Santos is grateful to the Ministry of Science, Innovation and Universities (Spain) for her FPI contract (BES-2017- 080040)
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Biotechnological platforms for Aryl-Alcohol Oxidases by directed evolution
2021Co-Authors: Viña-gonzález JavierAbstract:Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 03-10-2019Esta tesis tiene embargado el acceso al texto completo hasta el 03-04-2021The Aryl-Alcohol Oxidase (AAO) is a fungal flavoenzyme that supplies H2O2 to the ligninolytic consortium during natural wood decay. Being active on a wide array of aromatic alcohols, this GMC Oxidase presents a highly enantioselective mechanism of great interest in organic synthesis processes. The most powerful strategy for the AAO to meet industrial standards is the engineering of its properties by directed evolution. In the present Doctoral Thesis, an evolutionary platform for the AAO from Pleurotus eryngii was developed in order to: (i) obtain functional expression in yeasts, (ii) design a secondary benzyl-alcohol Oxidase, and (iii) explore the enzymatic conversion of furfural derivatives. To achieve functional expression in Saccharomyces cerevisiae, the AAO gene was fused to different signal peptides including chimeric versions of the mating-α factor and the killer K1 toxin preprosequences. The platform for in vitro evolution was completed with a dual high-throughput screening assay to detect H2O2 that included a method based on the Fenton reaction. To enhance secretion, several libraries were created combining classical evolution (i.e. mutagenic PCR and DNA shuffling) with structure-guided evolution by MORPHING. The final secretion variant FX9, carried four mutations in the signal peptide and two substitutions in the mature protein including the consensus/ancestral H91N. The FX9 improved secretion up to 4.5 mg/L and presented high stability and kinetic values similar to the native enzyme. FX9 was cloned and expressed in Pichia pastoris maintaining expression levels and main biochemical properties. When the production was scaled-up in 5L fermenter, AAO production was increased to 25.5 mg/L. FX9 was further evolved to selectively oxidize secondary benzyl alcohols. The residual activity on chiral molecules was unlocked with the modulation for the catalytic pocket by combinatorial saturation mutagenesis. After four generations, that included a site-directed recombination step to polish mutations, LanDo variant harbored five new substitutions increasing the catalytic efficiency with 1-(p-methoxyphenyl)-ethanol in 3 orders of magnitude with a 99% ee. Exploring the transformation of 5-hydroxymethylfurfural (HMF) into furan-2,5-dicarboxylic acid (FDCA), FX9 acquired mutation F501W that improved catalytic efficiency on HMF 3-fold and showed for the first time the performance of three consecutive oxidations for the AAOLa aril-alcohol oxidasa (AAO) es una flavoenzima fúngica que suple H2O2 al consorcio ligninolítico durante la degradación natural de la madera. Siendo activa con una amplia variedad de alcoholes aromáticos, esta oxidasa GMC presenta un mecanismo altamente enantioselectivo de gran interés en procesos de síntesis orgánica. La estrategia más potente para adaptar a la AAO a estándares industriales es la ingeniería de sus propiedades mediante técnicas de evolución dirigida. En la presente Tesis Doctoral, una plataforma evolutiva para la AAO de Pleurotus eryngii fue desarrollada con el objetivo de: (i) obtener expresión funcional en levaduras, (ii) diseñar una aril-alcohol oxidasa activa con alcoholes secundarios, y (iii) explorar la conversión enzimática de derivados del furfural. Para obtener expresión funcional en Saccharomyces cerevisiae, el gen de la AAO se fusionó a diferentes péptidos señales incluyendo versiones quiméricas de las secuencias prepro del factor-α y la toxina killer K1. La plataforma para la evolución in vitro se completó con un ensayo dual de screening para la detección de H2O2 incluyendo un método basado en la reacción de Fenton. Para mejorar la secreción, se crearon varias librerías combinando evolución clásica (PCR mutagénica y DNA shuffling) con evolución focalizada con el método MORPHING. La variante final FX9, con alta estabilidad y constantes cinéticas similares a la enzima nativa, presentó cuatro mutaciones en el péptido señal y dos substituciones en la proteína madura incluyendo la consenso/ancestral H91N. FX9 se expresó en S. cerevisiae con valores de 4.5 mg/L y fue posteriormente clonada y expresada en Pichia pastoris a escala de fermentador de 5 L alcanzando niveles de secreción 25.5 mg/L y manteniendo sus propiedades bioquímicas generales. La variante FX9 fue sometida a posteriores ciclos de evolución, incluyendo el remodelado del bolsillo catalítico por mutagénesis saturada combinatorial, para la oxidación de alcoholes bencílicos secundarios. Las cinco mutaciones introducidas en la variante LanDo aumentaron la eficiencia catalítica con 1-(p-methoxyphenyl)-ethanol en 3 órdenes de magnitud con un 99 % ee. Explorando la transformación del 5-hydroxymethylfurfural (HMF) en furan-2,5-dicarboxylic acid (FDCA), FX9 adquirió la mutación F501W que mejoró 3 veces la eficiencia catalítica con HMF y demostró por primera vez la catálisis de 3 oxidaciones consecutivas para la AAOLa financiación que me ha permitido seguir mis estudios doctorales. Los proyectos europeos “Optimized oxidoreductases for medium and large scale industrial biotransformations (INDOX FP7-KBBE-2013-7-613549)” y “New enzymatic oxidation/oxyfunctionalization technologies for added value bio-based products. (ENZOX2 H2020-BBI-PPP-2015-2-720297)”. Los proyectos nacionales “Evolución dirigida de oxidoreductasas ligninolíticas modernas y ancestrales para el diseño de una levadura de podredumbre blanca (DEWRY BIO2013-43407-R)”, “Evolución dirigida y computacional de ligninasas (LIGNOLUTION BIO2016-79106-R)“ y “Química sintética mediante enzimas quiméricas de fusión diseñadas por evolución dirigida y computacional (EVOCHIMERA Y2018/BIO-4738)”
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Sequential oxidation of 5-hydroxymethylfurfural to furan-2,5-dicarboxylic acid by an evolved Aryl-Alcohol Oxidase
'Elsevier BV', 2020Co-Authors: Viña-gonzález Javier, Martínez, Ángel T., Guallar Víctor, Alcalde MiguelAbstract:Furan-2,5-dicarboxylic acid (FDCA) is a building block of biodegradable plastics that can be used to replace those derived from fossil carbon sources. In recent years, much interest has focused on the synthesis of FDCA from the bio-based 5-hydroxymethylfurfural (HMF) through a cascade of enzyme reactions. Aryl-Alcohol Oxidase (AAO) and 5-hydroxymethylfurfural Oxidase (HMFO) are glucose-methanol-choline flavoenzymes that may be used to produce FDCA from HMF through three sequential oxidations, and without the assistance of auxiliary enzymes. Such a challenging process is dependent on the degree of hydration of the original aldehyde groups and of those formed, the rate-limiting step lying in the final oxidation of the intermediate 5-formyl-furancarboxylic acid (FFCA) to FDCA. While HMFO accepts FFCA as a final substrate in the HMF reaction pathway, AAO is virtually incapable of oxidizing it. Here, we have engineered AAO to perform the stepwise oxidation of HMF to FDCA through its structural alignment with HMFO and directed evolution. With a 3-fold enhanced catalytic efficiency for HMF and a 6-fold improvement in overall conversion, this evolved AAO is a promising point of departure for further engineering aimed at generating an efficient biocatalyst to synthesize FDCA from HMF.This research was supported by the EU project H2020-BBI-PPP-2015-2-720297-ENZOX2, by the Spanish Government projects BIO2016-79106-R-Lignolution, and by the Comunidad de Madrid project Y2018/BIO4738-EVOCHIMERA.Peer ReviewedPostprint (author's final draft
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Evolved Peroxygenase-Aryl Alcohol Oxidase Fusions for Self-Sufficient Oxyfunctionalization Reactions
'American Chemical Society (ACS)', 2020Co-Authors: Gómez De Santos, Patricia, Lázaro Sofia, Viña-gonzález Javier, Hoang, Manh Dat, Sánchez-moreno Israel, Glieder Anton, Hollmann F., Alcalde MiguelAbstract:Fungal peroxygenases are deemed emergent biocatalysts for selective C-H bond oxyfunctionalization reactions. In this study, we have engineered a functional and stable self-sufficient chimeric peroxygenase-Oxidase fusion. The bifunctional biocatalyst carried a laboratory-evolved version of the fungal peroxygenase fused to an evolved fungal Aryl-Alcohol Oxidase that supplies H2O2 in situ. Enzyme fusion libraries with peptide linkers of different sizes and amino acid compositions were designed, while attached leader sequences favored secretion in yeast. The most promising functional enzyme fusions were characterized biochemically and further tested for the synthesis of dextrorphan, a metabolite of the antitussive drug dextromethorphan. This reaction system was optimized to control the aromatic alcohol transformation rate, and therefore the H2O2 supply, to achieve total turnover numbers of 62,000, the highest value reported for the biocatalytic synthesis of dextrorphan to date. Accordingly, our study opens an avenue for the use of peroxygenase-aryl alcohol Oxidase fusions in the pharmaceutical and chemical sectors. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work publicBT/Biocatalysi
