18 results on '"Habrylo O"'
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2. The specificity of pectate lyase VdPelB from Verticilium dahliae is highlighted by structural, dynamical and biochemical characterizations
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Safran, J. (Josip), Ung, V. (Vanessa), Bouckaert, J. (Julie), Habrylo, O. (Olivier), Molinié, R. (Roland), Fontaine, J-X. (Jean-Xavier), Lemaire, A. (Adrien), Voxeur, A. (Aline), Pilard, S. (Serge), Pau-Roblot, C. (Corinne), Mercadante, D. (Davide), Pelloux, J. (Jérôme), Sénéchal, F. (Fabien), Transfrontalière BioEcoAgro - UMR 1158 (BioEcoAgro), Université d'Artois (UA)-Université de Liège-Université de Picardie Jules Verne (UPJV)-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA), Université catholique de Lille (UCL)-Université catholique de Lille (UCL), Biologie des Plantes et Innovation - UR UPJV 3900 (BIOPI), Université de Picardie Jules Verne (UPJV)-Transfrontalière BioEcoAgro - UMR 1158 (BioEcoAgro), Université catholique de Lille (UCL)-Université catholique de Lille (UCL)-Université d'Artois (UA)-Université de Liège-Université du Littoral Côte d'Opale (ULCO)-Université de Lille-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-JUNIA (JUNIA), School of Chemical Sciences [Auckland], University of Auckland [Auckland], Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Jean-Pierre Bourgin (IJPB), AgroParisTech-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université de Picardie Jules Verne (UPJV), Plateforme Analytique (PFA), Université de Lille, CNRS, Transfrontalière BioEcoAgro - UMR 1158 [BioEcoAgro], Unité de Glycobiologie Structurale et Fonctionnelle (UGSF) - UMR 8576, Institut Charles Viollette (ICV) - ULR 7394, Université Paris Saclay (COmUE), and Université de Picardie Jules Verne [UPJV]
- Subjects
Verticillium dahliae ,Structural Biology ,Pectins ,[SDV.BV]Life Sciences [q-bio]/Vegetal Biology ,Pectate lyase ,Homogalacturonan ,Oligogalacturonides ,General Medicine ,Molecular Biology ,Biochemistry - Abstract
International audience; Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.
- Published
- 2023
3. Quantification of Penicillium camemberti and P. roqueforti mycelium by real-time PCR to assess their growth dynamics during ripening cheese
- Author
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Le Dréan, G., primary, Mounier, J., additional, Vasseur, V., additional, Arzur, D., additional, Habrylo, O., additional, and Barbier, G., additional
- Published
- 2010
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4. TACKLING HUMULUS LUPULUS FUNGAL DISEASES BY STUDYING A HOP CELL WALL / FUSARIUM GRAMINEARUM MODEL
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Phalip, V., primary, Forster, A., additional, Carapito, R., additional, Habrylo, O., additional, Jeltsch, J.-M., additional, and Hatsch, D., additional
- Published
- 2009
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5. Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.
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Hocq L, Habrylo O, Sénéchal F, Voxeur A, Pau-Roblot C, Safran J, Fournet F, Bassard S, Battu V, Demailly H, Tovar JC, Pilard S, Marcelo P, Savary BJ, Mercadante D, Njo MF, Beeckman T, Boudaoud A, Gutierrez L, Pelloux J, and Lefebvre V
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- Cell Wall metabolism, Mutation genetics, Pectins metabolism, Hydrogen-Ion Concentration, Hypocotyl genetics, Hypocotyl metabolism, Arabidopsis metabolism, Carboxylic Ester Hydrolases
- Abstract
Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2024
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6. Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.
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Safran J, Tabi W, Ung V, Lemaire A, Habrylo O, Bouckaert J, Rouffle M, Voxeur A, Pongrac P, Bassard S, Molinié R, Fontaine JX, Pilard S, Pau-Roblot C, Bonnin E, Larsen DS, Morel-Rouhier M, Girardet JM, Lefebvre V, Sénéchal F, Mercadante D, and Pelloux J
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- Pectins metabolism, Proteins metabolism, Cell Wall metabolism, Polygalacturonase genetics, Polygalacturonase metabolism, Arabidopsis metabolism
- Abstract
Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)
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- 2023
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7. Design of a Protein with Improved Thermal Stability by an Evolution-Based Generative Model.
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Tian P, Lemaire A, Sénéchal F, Habrylo O, Antonietti V, Sonnet P, Lefebvre V, Isa Marin F, Best RB, Pelloux J, and Mercadante D
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- Amino Acid Sequence, Protein Stability, Proteins chemistry
- Abstract
Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)
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- 2022
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8. Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana .
- Author
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Xu F, Gonneau M, Faucher E, Habrylo O, Lefebvre V, Domon JM, Martin M, Sénéchal F, Peaucelle A, Pelloux J, and Höfte H
- Abstract
The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro . Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V.)
- Published
- 2022
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9. Characterization of a novel strain of Aspergillus aculeatinus: From rhamnogalacturonan type I pectin degradation to improvement of fruit juice filtration.
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Duran Garzon C, Habrylo O, Lemaire A, Guillaume A, Carré Y, Millet C, Fourtot-Brun C, Trezel P, Le Blond P, Perrin A, Georgé S, Wagner M, Coutel Y, Levavasseur L, Pau-Roblot C, and Pelloux J
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- Carbohydrate Metabolism, Cellulases metabolism, Food Handling methods, Glycoside Hydrolases metabolism, Hydrolases metabolism, Malus, Pectins metabolism, Polygalacturonase metabolism, Proteomics, Aspergillus enzymology, Filtration methods, Fruit and Vegetable Juices, Fungal Proteins metabolism, Rhamnogalacturonans metabolism
- Abstract
Aspergillus spp. are well-known producers of pectinases commonly used in the industry. Aspergillus aculeatinus is a recently identified species but poorly characterized. This study aimed at giving a comprehensive characterization of the enzymatic potential of the O822 strain to produce Rhamnogalacturonan type I (RGI)-degrading enzymes. Proteomic analysis identified cell wall degrading enzymes (cellulases, hemicellulases, and pectinases) that accounted for 92 % of total secreted proteins. Twelve out of fifty proteins were identified as RGI-degrading enzymes. NMR and enzymatic assays revealed high levels of arabinofuranosidase, arabinanase, galactanase, rhamnogalacturonan hydrolases and rhamnogalacturonan acetylesterase activities in aqueous extracts. Viscosity assays carried out with RGI-rich camelina mucilage confirmed the efficiency of enzymes secreted by O822 to hydrolyze RGI, by decreasing viscosity by 70 %. Apple juice trials carried out at laboratory and pilot scale showed an increase in filtration flow rate and yield, paving the way for an industrial use of enzymes derived from A. aculeatinus., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
- Published
- 2021
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10. New insights into the specificity and processivity of two novel pectinases from Verticillium dahliae.
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Safran J, Habrylo O, Cherkaoui M, Lecomte S, Voxeur A, Pilard S, Bassard S, Pau-Roblot C, Mercadante D, Pelloux J, and Sénéchal F
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- Ascomycota genetics, Ascomycota pathogenicity, Carboxylic Ester Hydrolases chemistry, Carboxylic Ester Hydrolases genetics, Flax metabolism, Fungal Proteins chemistry, Fungal Proteins genetics, Kinetics, Models, Molecular, Pectins metabolism, Phylogeny, Plant Diseases microbiology, Plant Roots metabolism, Polygalacturonase chemistry, Polygalacturonase genetics, Static Electricity, Substrate Specificity, Ascomycota enzymology, Carboxylic Ester Hydrolases metabolism, Fungal Proteins metabolism, Polygalacturonase metabolism
- Abstract
Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55-70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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11. Three novel rhamnogalacturonan I- pectins degrading enzymes from Aspergillus aculeatinus: Biochemical characterization and application potential.
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Lemaire A, Duran Garzon C, Perrin A, Habrylo O, Trezel P, Bassard S, Lefebvre V, Van Wuytswinkel O, Guillaume A, Pau-Roblot C, and Pelloux J
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- Aspergillus genetics, Aspergillus metabolism, Cell Wall chemistry, Enzyme Stability, Fungal Proteins genetics, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Hydrogen-Ion Concentration, Kinetics, Magnetic Resonance Spectroscopy, Pichia genetics, Polysaccharide-Lyases genetics, Polysaccharide-Lyases metabolism, Recombinant Proteins metabolism, Temperature, Aspergillus enzymology, Fungal Proteins metabolism, Pectins metabolism
- Abstract
Rhamnogalaturonans I (RGI) pectins, which are a major component of the plant primary cell wall, can be recalcitrant to digestion by commercial enzymatic cocktails, in particular during fruit juice clarification process. To overcome these problems and get better insights into RGI degradation, three RGI degrading enzymes (RHG: Endo-rhamnogalacturonase; ABF: α-Arabinofuranosidases; GAN: Endo-β-1,4-galactanase) from Aspergillus aculeatinus were expressed in Pichia pastoris, purified and fully biochemically characterized. All three enzymes showed acidic pH optimum, and temperature optima between 40-50 °C. The Km values were 0.5 mg.ml
-1 , 1.64 mg.ml-1 and 3.72 mg.ml-1 for RHG, ABF, GAN, respectively. NMR analysis confirmed an endo-acting mode of action for RHG and GAN, and exo-acting mode for ABF. The application potential of these enzymes was assessed by measuring changes in viscosity of RGI-rich camelina mucilage, showing that RHG-GAN enzymes induced a decrease in viscosity by altering the structures of the RGI backbone and sidechains., (Copyright © 2020 Elsevier Ltd. All rights reserved.)- Published
- 2020
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12. The exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.
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Hocq L, Guinand S, Habrylo O, Voxeur A, Tabi W, Safran J, Fournet F, Domon JM, Mollet JC, Pilard S, Pau-Roblot C, Lehner A, Pelloux J, and Lefebvre V
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- Arabidopsis drug effects, Arabidopsis enzymology, Arabidopsis genetics, Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins pharmacology, Plant Roots metabolism, Plants, Genetically Modified, Pollen Tube drug effects, Polygalacturonase genetics, Polygalacturonase pharmacology, Saccharomycetales, Arabidopsis Proteins physiology, Pollen Tube physiology, Polygalacturonase physiology
- Abstract
Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.)
- Published
- 2020
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13. Oligogalacturonide production upon Arabidopsis thaliana - Botrytis cinerea interaction.
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Voxeur A, Habrylo O, Guénin S, Miart F, Soulié MC, Rihouey C, Pau-Roblot C, Domon JM, Gutierrez L, Pelloux J, Mouille G, Fagard M, Höfte H, and Vernhettes S
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- Arabidopsis Proteins metabolism, Botrytis metabolism, Gene Expression Regulation, Plant genetics, Pectins metabolism, Plant Diseases microbiology, Plant Leaves metabolism, Polygalacturonase metabolism, Signal Transduction, Arabidopsis metabolism, Botrytis pathogenicity, Hexuronic Acids metabolism
- Abstract
Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea Indeed, in contrast to previous reports, most OGs were acetyl- and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors., Competing Interests: The authors declare no conflict of interest.
- Published
- 2019
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14. The pectinases from Sphenophorus levis: Potential for biotechnological applications.
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Habrylo O, Evangelista DE, Castilho PV, Pelloux J, and Henrique-Silva F
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- Amino Acid Sequence genetics, Animals, Biotechnology, Carboxylic Ester Hydrolases genetics, Hydrogen-Ion Concentration, Pichia genetics, Polygalacturonase genetics, Saccharum chemistry, Carboxylic Ester Hydrolases chemistry, Pectins chemistry, Polygalacturonase chemistry, Weevils enzymology
- Abstract
Pectinases represent about one fifth of the enzyme worldwide market due their wide range of biotechnological applications. Current commercial pectinases are exclusively obtained from microbial sources, but here we report a pectin methylesterase (Sl-PME) and an endo-polygalacturonase (Sl-EPG) bioprospected from the sugarcane weevil, Sphenophorus levis, which revealed good potential for industrial applications. Sl-PME and Sl-EPG were overexpressed in Pichia pastoris, purified and enzymatically characterized. Sl-EPG presents optimal activity at pH 4-5 and 50 °C, showing that it can be used for juice extraction and clarification. On the other hand, Sl-PME presents optimal activity at pH 6-8 and 40 °C, and thus, suitable for both acidic and alkaline processing, such as coffee and tea fermentation. Sl-EPG shows V
max = 3.23 mM/min, KM = 2.4 g/L and kcat = 418.6 s-1 . While Sl-PME shows Vmax = 0.14 mM/min, KM = 4.1 g/L and kcat = 1.7 s-1 . A PG inhibitor (PGIP2) weakly interfered in the Sl-EPG activity and Sl-PME was not affected by a usual PME inhibitor. Moreover, these enzymes manifested synergistic action towards methylesterified pectin. Here, we propose these enzymes as novel alternative tools for the current commercial pectinases., (Copyright © 2018 Elsevier B.V. All rights reserved.)- Published
- 2018
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15. Structural and dynamical characterization of the pH-dependence of the pectin methylesterase-pectin methylesterase inhibitor complex.
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Sénéchal F, Habrylo O, Hocq L, Domon JM, Marcelo P, Lefebvre V, Pelloux J, and Mercadante D
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- Amino Acid Sequence genetics, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Carboxylic Ester Hydrolases antagonists & inhibitors, Carboxylic Ester Hydrolases genetics, Cell Membrane metabolism, Cell Wall metabolism, Gene Expression Regulation, Plant genetics, Hydrogen-Ion Concentration, Pectins metabolism, Plant Proteins metabolism, Protein Interaction Domains and Motifs, Arabidopsis Proteins metabolism, Carboxylic Ester Hydrolases chemistry, Carboxylic Ester Hydrolases metabolism
- Abstract
Pectin methylesterases (PMEs) catalyze the demethylesterification of pectin, one of the main polysaccharides in the plant cell wall, and are of critical importance in plant development. PME activity generates highly negatively charged pectin and mutates the physiochemical properties of the plant cell wall such that remodeling of the plant cell can occur. PMEs are therefore tightly regulated by proteinaceous inhibitors (PMEIs), some of which become active upon changes in cellular pH. Nevertheless, a detailed picture of how this pH-dependent inhibition of PME occurs at the molecular level is missing. Herein, using an interdisciplinary approach that included homology modeling, MD simulations, and biophysical and biochemical characterizations, we investigated the molecular basis of PME3 inhibition by PMEI7 in Arabidopsis thaliana Our complementary approach uncovered how changes in the protonation of amino acids at the complex interface shift the network of interacting residues between intermolecular and intramolecular. These shifts ultimately regulate the stability of the PME3-PMEI7 complex and the inhibition of the PME as a function of the pH. These findings suggest a general model of how pH-dependent proteinaceous inhibitors function. Moreover, they enhance our understanding of how PMEs may be regulated by pH and provide new insights into how this regulation may control the physical properties and structure of the plant cell wall., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2017
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16. Enzymatic cocktails produced by Fusarium graminearum under submerged fermentation using different lignocellulosic biomasses.
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Debeire P, Delalande F, Habrylo O, Jeltsch JM, Van Dorsselaer A, and Phalip V
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- Fusarium genetics, Fusarium growth & development, Glucose chemistry, Polysaccharides chemistry, Proteome genetics, Proteome metabolism, Substrate Specificity, Biomass, Fermentation, Fusarium enzymology, Lignin chemistry
- Abstract
Fusarium graminearum was grown on four lignocellulosic substrates (corn cobs, wheat bran, hop cell walls, and birchwood) and glucose as the sole carbon source. Proteomic studies performed on the resulting enzymatic cocktails highlighted a great diversity in the number and type of proteins secreted. The cell wall-degrading enzymes (CWDE) proportion varied greatly from 20% to 69%. Only one of the 57 CWDEs detected in this study was common to the five proteomes. In contrast, 35 CWDEs were specific to one proteome only. The polysaccharide-degradation activities were different depending on the cocktail and the polysaccharide used. F. graminearum strongly modifies the enzymatic cocktail it secretes as a function of the biomass used for growth., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)
- Published
- 2014
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17. The characterisation of xyloglucanase inhibitors from Humulus lupulus.
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Habrylo O, Forster A, Jeltsch JM, and Phalip V
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- Dose-Response Relationship, Drug, Enzyme Inhibitors chemistry, Glycoside Hydrolases metabolism, Humulus metabolism, Plant Proteins chemistry, Plant Proteins isolation & purification, Plant Proteins pharmacology, Structure-Activity Relationship, Enzyme Inhibitors isolation & purification, Enzyme Inhibitors pharmacology, Fungi enzymology, Glycoside Hydrolases antagonists & inhibitors, Humulus chemistry
- Abstract
Phytopathogenic fungi secrete a powerful arsenal of enzymes that are potentially active against each polysaccharide component of the plant cell wall. To defend themselves, plants synthetise a variety of molecules that inhibit the activity of cell wall-degrading enzymes. Xyloglucan-specific endoglucanase inhibitor proteins (XEGIPs) act specifically against the members of fungal glycoside hydrolase family 12 (GH12 in the CAZy database). In the present study, we describe the identification of three XEGIP homologues from hop (Humulus lupulus L.). When incubating each of the recombinant inhibitors with an enzymatic cocktail from Aspergillus aculeatus (Viscozyme®), the xyloglucan-degrading endoglucanase activity decreased to 15% and 5% for HlXEGIP1 and HlXEGIP2, respectively, whereas no inhibition of the Viscozyme® enzymes was observed for the third (also called HlXEGIP homologue 3, or HlXEGIPh3). Fungal enzymatic cocktails from 20 different species also showed xyloglucan-degrading endoglucanase activities, and most of them were inhibited by HlXEGIP1 and -2. Furthermore, a real time RT-PCR analysis revealed variations in the spatial distribution of the genes encoding the three inhibitors and differential expression during development and (a) biotic stress. The role of XEGIPs in the plant-fungus interaction is discussed, and a model suggesting a distinct role of these XEGIP homologues is proposed: HlXEGIP1 may act in cases of abiotic stress, while HlXEGIP2 reacts to biotic stress, and physiological development may be influenced by HlXEGIPh3., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
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18. Characterization of the four GH12 Endoxylanases from the plant pathogen Fusarium graminearum.
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Habrylo O, Song X, Forster A, Jeltsch JM, and Phalip V
- Subjects
- Cloning, Molecular, Endo-1,4-beta Xylanases genetics, Endo-1,4-beta Xylanases isolation & purification, Escherichia coli genetics, Fusarium genetics, Fusarium isolation & purification, Gene Expression, Glucans metabolism, Plant Diseases microbiology, Plants microbiology, Protein Binding, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, Substrate Specificity, Xylans metabolism, Endo-1,4-beta Xylanases metabolism, Fusarium enzymology
- Abstract
Four putative GH12 genes were found in the Fusarium graminearum genome. The corresponding proteins were expressed in Escherichia coli, purified, and evaluated. FGSG_05851 and FGSG_11037 displayed high activities towards xyloglucan (V(max) of 4 and 11 micronmol/min, respectively), whereas FGSG_07892 and FGSG_16349 were much less active with this substrate (0.081 and 0.004 micronmol/min, respectively). However, all four of these enzymes had a similar binding affinity for xyloglucan. Xyloglucan was the substrate preferred by FGSG_05851, in contrast to the three other enzymes, which preferred beta-glucan or lichenan. Therefore, FGSG_05851 is a xyloglucan-specific glucanase (E.C. 3.2.1.151) rather than an endoglucanase (E.C. 3.2.1.4) with broad substrate specificity. FGSG_11037 displayed a peculiar behavior in that the xyloglucan binding was highly cooperative, with a Hill coefficient of 2.5. Finally, FGSG_05851 essentially degraded xyloglucan into hepta-, octa-, and nonasaccharides, whereas the three other enzymes yielded hepta- and octa-saccharides as well as larger molecules.
- Published
- 2012
- Full Text
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