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Vera Meyer - One of the best experts on this subject based on the ideXlab platform.
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A Computational Modeling Approach Predicts Interaction of the Antifungal Protein AFP from Aspergillus giganteus with Fungal Membranes via Its γ-Core Motif.
mSphere, 2018Co-Authors: Tillmann Utesch, Vera Meyer, Alejandra De Miguel Catalina, Caspar Schattenberg, Norman Paege, Peter Schmieder, Eberhard Krause, Yinglong Miao, J. Andrew Mccammon, Sascha JungAbstract:Fungal pathogens kill more people per year globally than malaria or tuberculosis and threaten international food security through crop destruction. New sophisticated strategies to inhibit fungal growth are thus urgently needed. Among the potential candidate molecules that strongly inhibit fungal spore germination are small cationic, cysteine-stabilized proteins of the AFP family secreted by a group of filamentous Ascomycetes. Its founding member, AFP from Aspergillus giganteus, is of particular interest since it selectively inhibits the growth of filamentous fungi without affecting the viability of mammalian, plant, or bacterial cells. AFPs are also characterized by their high efficacy and stability. Thus, AFP can serve as a lead compound for the development of novel antifungals. Notably, all members of the AFP family comprise a γ-core motif which is conserved in all antimicrobial proteins from pro- and eukaryotes and known to interfere with the integrity of cytoplasmic plasma membranes. In this study, we used classical molecular dynamics simulations combined with wet laboratory experiments and nuclear magnetic resonance (NMR) spectroscopy to characterize the structure and dynamical behavior of AFP isomers in solution and their interaction with fungal model membranes. We demonstrate that the γ-core motif of structurally conserved AFP is the key for its membrane interaction, thus verifying for the first time that the conserved γ-core motif of antimicrobial proteins is directly involved in protein-membrane interactions. Furthermore, molecular dynamic simulations suggested that AFP does not destroy the fungal membrane by pore formation but covers its surface in a well-defined manner, using a multistep mechanism to destroy the membranes integrity.IMPORTANCE Fungal pathogens pose a serious danger to human welfare since they kill more people per year than malaria or tuberculosis and are responsible for crop losses worldwide. The treatment of fungal infections is becoming more complicated as fungi develop resistances against commonly used fungicides. Therefore, discovery and development of novel antifungal agents are of utmost importance.
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The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis
BMC microbiology, 2011Co-Authors: Ulrike Binder, Mojca Benčina, Andrea Eigentler, Vera Meyer, Florentine MarxAbstract:Background The antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.
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The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley
Applied microbiology and biotechnology, 2010Co-Authors: Hassan Barakat, Vera Meyer, Anja Spielvogel, Mahmoud E. Hassan, A I El-desouky, H. A. El-mansy, Frank Rath, Ulf StahlAbstract:Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.
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The antifungal protein AFP from Aspergillus giganteus inhibits chitin synthesis in sensitive fungi.
Applied and environmental microbiology, 2007Co-Authors: Silke Hagen, Florentine Marx, Arthur F. J. Ram, Vera MeyerAbstract:The antifungal protein AFP from Aspergillus giganteus is highly effective in restricting the growth of major human- and plant-pathogenic filamentous fungi. However, a fundamental prerequisite for the use of AFP as an antifungal drug is a complete understanding of its mode of action. In this study, we performed several analyses focusing on the assumption that the chitin biosynthesis of sensitive fungi is targeted by AFP. Here we show that the N-terminal domain of AFP (amino acids 1 to 33) is sufficient for efficient binding of AFP to chitin but is not adequate for inhibition of the growth of sensitive fungi. AFP susceptibility tests and SYTOX Green uptake experiments with class III and class V chitin synthase mutants of Fusarium oxysporum and Aspergillus oryzae showed that deletions made the fungi less sensitive to AFP and its membrane permeabilization effect. In situ chitin synthase activity assays revealed that chitin synthesis is specifically inhibited by AFP in sensitive fungi, indicating that AFP causes cell wall stress and disturbs cell integrity. Further evidence that there was AFP-induced cell wall stress was obtained by using an Aspergillus niger reporter strain in which the cell wall integrity pathway was strongly induced by AFP.
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Alkaline pH-induced up-regulation of the afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus is not mediated by the transcription factor PacC: possible involvement of calcineurin
Molecular genetics and genomics : MGG, 2005Co-Authors: Vera Meyer, Anja Spielvogel, Laura Funk, Joan Tilburn, Herbert N. Arst, Ulf StahlAbstract:The afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus has a prototypical alkaline gene expression pattern, which suggests that the gene might be under the control of the ambient pH-dependent zinc-finger transcription factor PacC. This notion is corroborated by the presence in the upstream region of afp of two putative PacC binding sites, afpP1 and afpP2, which are specifically recognised by the PacC protein of A. nidulans in vitro. However, in this report we provide several lines of evidence to show that pH-dependent up-regulation of afp is not mediated by transcriptional activation through PacC. (1) The temporal expression pattern of the A. giganteus pacC gene does not parallel the accumulation of the afp mRNA during cultivation. (2) Inactivation of afpP1 and afpP2 did not reduce promoter activity under alkaline conditions, as determined from the relative wild-type and mutant afp::lacZ reporter activities in A. nidulans. (3) Reporter activities in acidity- and alkalinity-mimicking mutant strains are inconsistent with a positive role for PacC in afp expression. (4) In A. giganteus, the pH-dependent increase in afp mRNA and AFP levels can be completely prevented by the calcineurin inhibitor FK506, suggesting that the calcineurin signalling pathway might control the in vivo activation of the afp promoter by alkaline pH.
Blanca San Segundo - One of the best experts on this subject based on the ideXlab platform.
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Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris.
Protein expression and purification, 2009Co-Authors: Belén López-garcía, Ana Moreno, Blanca San Segundo, Vivian De Los Ríos, James M. Manning, José G. Gavilanes, Álvaro Martínez-del-pozoAbstract:Abstract The mould Aspergillus giganteus produces a basic, low molecular weight protein (AFP) showing in vitro and in vivo antifungal properties against important plant pathogens. AFP is secreted as an inactive precursor containing an amino-terminal extension of six amino acids ( lf -AFP) which is later removed to produce the active protein. The molecular basis to explain this behavior and the features that determine the fungal specificity of this protein are not completely solved. In this work, the mature AFP (AFP∗) and a version of AFP with an extended amino-terminal (proAFP) have been cloned and produced in the yeast Pichia pastoris . The two proteins have been purified to homogeneity and characterized from structural and functional points of view. Recombinant AFP∗ produced is practically indistinguishable from the natural fungal protein in terms of its spectroscopic and antifungal properties while proAFP is mostly inactive under identical assay conditions. The availability of an active AFP protein produced in P. pastoris will permit investigation of the mode of action and targeting specificity of AFP by using site-directed mutagenesis approaches.
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Biotechnologically relevant enzymes and proteins. Antifungal mechanism of the Aspergillus giganteus AFP against the rice blast fungus Magnaporthe grisea.
Applied microbiology and biotechnology, 2006Co-Authors: Ana Moreno, Álvaro Martínez Del Pozo, Blanca San SegundoAbstract:The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.
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Pathogen-Induced Production of the Antifungal AFP Protein from Aspergillus giganteus Confers Resistance to the Blast Fungus Magnaporthe grisea in Transgenic Rice
Molecular plant-microbe interactions : MPMI, 2005Co-Authors: Ana Moreno, Mar Rufat, Gisela Peñas, Joaquima Messeguer, Juan Manuel Bravo, Montserrat Estopà, Blanca San SegundoAbstract:Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the β-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus.
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Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea
Plant Molecular Biology, 2004Co-Authors: María Coca, Álvaro Martínez Del Pozo, Cristina Bortolotti, Mar Rufat, Gisela Peñas, Ramón Eritja, Didier Tharreau, Joaquima Messeguer, Blanca San SegundoAbstract:The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea . In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T_2 homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea .
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Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea.
Phytopathology, 2003Co-Authors: Ana Moreno, Álvaro Martínez Del Pozo, M. Borja, Blanca San SegundoAbstract:Moreno, A. B., Martinez del Pozo, A., Borja, M., and San Segundo, B. 2003. Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea. Phytopathology 93:1344-1353. Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic lowmolecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases. Additional keyword: Pelargonium.
Ulf Stahl - One of the best experts on this subject based on the ideXlab platform.
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The antifungal protein AFP from Aspergillus giganteus prevents secondary growth of different Fusarium species on barley
Applied microbiology and biotechnology, 2010Co-Authors: Hassan Barakat, Vera Meyer, Anja Spielvogel, Mahmoud E. Hassan, A I El-desouky, H. A. El-mansy, Frank Rath, Ulf StahlAbstract:Secondary growth is a common post-harvest problem when pre-infected crops are attacked by filamentous fungi during storage or processing. Several antifungal approaches are thus pursued based on chemical, physical, or bio-control treatments; however, many of these methods are inefficient, affect product quality, or cause severe side effects on the environment. A protein that can potentially overcome these limitations is the antifungal protein AFP, an abundantly secreted peptide of the filamentous fungus Aspergillus giganteus. This protein specifically and at low concentrations disturbs the integrity of fungal cell walls and plasma membranes but does not interfere with the viability of other pro- and eukaryotic systems. We thus studied in this work the applicability of AFP to efficiently prevent secondary growth of filamentous fungi on food stuff and chose, as a case study, the malting process where naturally infested raw barley is often to be used as starting material. Malting was performed under lab scale conditions as well as in a pilot plant, and AFP was applied at different steps during the process. AFP appeared to be very efficient against the main fungal contaminants, mainly belonging to the genus Fusarium. Fungal growth was completely blocked after the addition of AFP, a result that was not observed for traditional disinfectants such as ozone, hydrogen peroxide, and chlorine dioxide. We furthermore detected reduced levels of the mycotoxin deoxynivalenol after AFP treatment, further supporting the fungicidal activity of the protein. As AFP treatments did not compromise any properties and qualities of the final products malt and wort, we consider the protein as an excellent biological alternative to combat secondary growth of filamentous fungi on food stuff.
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Alkaline pH-induced up-regulation of the afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus is not mediated by the transcription factor PacC: possible involvement of calcineurin
Molecular genetics and genomics : MGG, 2005Co-Authors: Vera Meyer, Anja Spielvogel, Laura Funk, Joan Tilburn, Herbert N. Arst, Ulf StahlAbstract:The afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus has a prototypical alkaline gene expression pattern, which suggests that the gene might be under the control of the ambient pH-dependent zinc-finger transcription factor PacC. This notion is corroborated by the presence in the upstream region of afp of two putative PacC binding sites, afpP1 and afpP2, which are specifically recognised by the PacC protein of A. nidulans in vitro. However, in this report we provide several lines of evidence to show that pH-dependent up-regulation of afp is not mediated by transcriptional activation through PacC. (1) The temporal expression pattern of the A. giganteus pacC gene does not parallel the accumulation of the afp mRNA during cultivation. (2) Inactivation of afpP1 and afpP2 did not reduce promoter activity under alkaline conditions, as determined from the relative wild-type and mutant afp::lacZ reporter activities in A. nidulans. (3) Reporter activities in acidity- and alkalinity-mimicking mutant strains are inconsistent with a positive role for PacC in afp expression. (4) In A. giganteus, the pH-dependent increase in afp mRNA and AFP levels can be completely prevented by the calcineurin inhibitor FK506, suggesting that the calcineurin signalling pathway might control the in vivo activation of the afp promoter by alkaline pH.
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New insights into the target site and mode of action of the antifungal protein of Aspergillus giganteus
Research in microbiology, 2005Co-Authors: Torsten Theis, Ulf Stahl, Florentine Marx, Willibald Salvenmoser, Vera MeyerAbstract:The antifungal protein (AFP) secreted by Aspergillus giganteus exerts growth inhibitory effects on various filamentous fungi. In order to obtain more information on the mode of action of AFP, we used transmission electron microscopy in this study to compare the cellular ultrastructure of the AFP-sensitive Aspergillus niger and of the AFP-resistant Penicillium chrysogenum upon AFP treatment. Furthermore, AFP was localized by immunogold staining in both fungi. Severe membrane alterations in A. niger were observed, whereas the membrane of P. chrysogenum was not affected after treatment with AFP. The protein localized predominantly to a cell wall attached outer layer which is probably composed of glycoproteins, as well as to the cell wall of A. niger. It was found to accumulate within defined areas of the cell wall, pointing towards a specific interaction of AFP with cell wall components. In contrast, very little protein was bound to the outer layer and cell wall of P. chrysogenum. For future applications of AFP as an antimycotic drug, the mode of action of the protein was further characterized. The protein was found to act in a dose-dependent manner: it was fungistatic when applied at concentrations below the minimal inhibitory concentration, but fungicidal at higher concentrations. Using an in vivo model system, we were able to finally show that AFP indeed prevented the infection of tomato roots (Lycopersicon esculentum) by the plant-pathogenic fungus Fusarium oxysporum f. sp. lycopersici.
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Comparison of different transformation methods for Aspergillus giganteus
Current Genetics, 2003Co-Authors: Vera Meyer, Dirk Mueller, Till Strowig, Ulf StahlAbstract:Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus , the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens -mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus , due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10^8 protoplasts/µg DNA and was enhanced to 140-fold by A. tumefaciens -mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.
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The antifungal protein from Aspergillus giganteus causes membrane permeabilization.
Antimicrobial agents and chemotherapy, 2003Co-Authors: Torsten Theis, Vera Meyer, M. Wedde, Ulf StahlAbstract:We investigated the inhibitory effects of the antifungal protein (AFP) from Aspergillus giganteus on the growth of several filamentous fungi. For this purpose, the MICs of AFP were determined and ranged from 0.1 μg/ml for Fusarium oxysporum to 200 μg/ml for Aspergillus nidulans. The antifungal activity of AFP was diminished in the presence of cations. We were able to show that incubation of AFP-sensitive fungi with the protein resulted in membrane permeabilization using an assay based on the uptake of the fluorescent dye SYTOX Green. No permeabilization by AFP could be detected at concentrations below the species-specific MIC. Furthermore, AFP-induced permeabilization could readily be detected after 5 min of incubation. Localization experiments with fluorescein isothiocyanate-labeled AFP and immunofluorescence staining with an AFP-specific antibody supported the observation that the protein interacts with membranes. After treatment of AFP-sensitive fungi with AFP, the protein was localized at the plasma membrane, whereas it was mainly detected inside the cells of AFP-resistant fungi. We conclude from these data that the growth-inhibitory effect of AFP is caused by permeabilization of the fungal membranes.
Eleonora Cano Carmona - One of the best experts on this subject based on the ideXlab platform.
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Research Article Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate
2016Co-Authors: During Pectin Degradation, Eleonora Cano Carmona, Danielle Biscaro Pedrolli, Department Of Bioprocess, Biotechnology School Of Pharmaceutical SciencesAbstract:Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking. 1
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Purification and Characterization of a Unique Pectin Lyase from Aspergillus giganteus Able to Release Unsaturated Monogalacturonate during Pectin Degradation
Enzyme research, 2014Co-Authors: Danielle Biscaro Pedrolli, Eleonora Cano CarmonaAbstract:A pectin lyase, named PLIII, was purified to homogeneity from the culture filtrate of Aspergillus giganteus grown in submerged culture containing orange peel waste as carbon source. PLIII was able to digest apple pectin and citrus pectins with different degrees of methyl esterification. Interestingly, the PLIII activity was stimulated in the presence of some divalent cations including Pb2+ and was not significantly affected by Hg2+. Like other pectin lyases, PLIII is stimulated by but is not dependent on Ca2+. The main soluble product released during the degradation of pectic substances promoted by the PLIII is compatible with an unsaturated monogalacturonate. PLIII is a unique enzyme able to release unsaturated monogalacturonate as the only soluble product during the degradation of pectic substances; therefore, PLIII was classified as an exo-pectin lyase. To our knowledge, this is the first characterization of an exo-pectin lyase. The PLIII described in this work is potentially useful for ethanol production from pectin-rich biomass, besides other common applications for alkaline pectinases like preparation of textile fibers, coffee and tea fermentation, vegetable oil extraction, and the treatment of pulp in papermaking.
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Purification and characterization of the exopolygalacturonase produced by Aspergillus giganteus in submerged cultures
Journal of Industrial Microbiology & Biotechnology, 2010Co-Authors: Danielle Biscaro Pedrolli, Eleonora Cano CarmonaAbstract:Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications, such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited maximal activity at pH 6.0 and 55–60°C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min at 40, 50 and 55°C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was enhanced in the presence of β-mercaptoethanol, dithiothreitol, Co^2+, Mn^2+, Mg^2+, NH_4 ^+, and Na^+ and was resistant to inhibition by Pb^2+.
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Studies on Productivity and Characterization of Polygalacturonase from Aspergillus giganteus Submerged Culture Using Citrus Pectin and Orange Waste
Applied Biochemistry and Biotechnology, 2008Co-Authors: Danielle Biscaro Pedrolli, Eleni Gomes, Rubens Monti, Eleonora Cano CarmonaAbstract:Polygalacturonases are part of the group of enzymes involved in pectin degradation. The aim of this work was to investigate some of the factors affecting polygalacturonase production by an Aspergillus giganteus strain and to characterize this pectinolytic activity. Several carbon sources, both pure substances and natural substrates, were tested in standing cultures, and the best results were obtained with orange bagasse and purified citrus pectin. On citrus pectin as sole carbon source, the highest extracellular activity (9.5 U/ml and 40.6 U/mg protein) was obtained in 4.5-day-old cultures shaken at 120 rpm, pH 3.5 and 30°C, while on orange bagasse, the highest extracellular activity (48.5 U/ml and 78.3 U/mg protein) was obtained in 3.5-day-old cultures shaken at 120 rpm, pH 6.0 and 30°C. Optimal polygalacturonase activity was observed in assays conducted at pH 5.5–6.5 and 55–60°C. The activity showed good thermal stability, with half-lives of 90 and 30 min when incubated at 55 and 60°C, respectively. High stability was observed from pH 4.5 to 8.5; more than 90% of the activity remained after 24 h in this pH range.
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Purification and characterization of xylanases from Aspergillus giganteus.
Folia microbiologica, 2004Co-Authors: Mauricio Batista Fialho, Eleonora Cano CarmonaAbstract:A strain ofAspergillus giganteus cultivated in a medium with xylan produced two xylanases (xylanase I and II) which were purified to homogeneity. Their molar mass, estimated by SDS-PAGE, were 21 and 24 kDa, respectively. Both enzymes are glycoproteins with 50 °C temperature optimum; optimum pH was 6.0–6.5 for xylanase I and 6.0 for xylanase II. At 50 °C xylanase I exhibited higher thermostability than xylanase II. Hg2+, Cu2+ and SDS were strong inhibitors, 1,4-dithiothreitol stimulated the reaction of both enzymes. Both xylanases are xylan-specific; kinetic parameters indicated higher efficiency in the hydrolysis of oat spelts xylan. In hydrolysis of this substrate, xylotriose, xylotetraose and larger xylooligosaccharides were released and hence the enzymes were classified as endoxylanases.
Álvaro Martínez Del Pozo - One of the best experts on this subject based on the ideXlab platform.
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A peptide of nine amino acid residues from α‐sarcin cytotoxin is a membrane‐perturbing structure
The journal of peptide research : official journal of the American Peptide Society, 2009Co-Authors: José M. Mancheño, Álvaro Martínez Del Pozo, Mercedes Oñaderra, Juan Pablo Albar, José G. GavilanesAbstract:A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131-139 sequence region of the cytotoxic protein alpha-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire alpha-helical conformation in the presence of trifluoroethanol, but display beta-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in beta-structure. The obtained data are discussed in terms of the involvement of the 131-139 stretch of alpha-sarcin in its interaction with lipid membranes.
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Biotechnologically relevant enzymes and proteins. Antifungal mechanism of the Aspergillus giganteus AFP against the rice blast fungus Magnaporthe grisea.
Applied microbiology and biotechnology, 2006Co-Authors: Ana Moreno, Álvaro Martínez Del Pozo, Blanca San SegundoAbstract:The mold Aspergillus giganteus produces a basic, low molecular weight protein showing antifungal properties against economically important plant pathogens, the AFP (Antifungal Protein). In this study, we investigated the mechanisms by which AFP exerts its antifungal activity against Magnaporthe grisea. M. grisea is the causal agent of rice blast, one of the most devastating diseases of cultivated rice worldwide. AFP was purified from the extracellular medium of A. giganteus cultures. The AFP protein was found to induce membrane permeabilization in M. grisea cells. Electron microscopy studies revealed severe cellular degradation and damage of plasma membranes in AFP-treated fungal cells. AFP however failed to induce membrane permeabilization on rice or human HeLa cells. Furthermore, AFP enters the fungal cell and targets to the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye. Finally, AFP binds to nucleic acids, including M. grisea DNA. Our results suggest that the combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP determines its potent antifungal activity against M. grisea. These results are discussed in relation to the potential of the AFP protein to enhance crop protection against fungal diseases.
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Transgenic Rice Plants Expressing the Antifungal AFP Protein from Aspergillus giganteus Show Enhanced Resistance to the Rice Blast Fungus Magnaporthe Grisea
Plant Molecular Biology, 2004Co-Authors: María Coca, Álvaro Martínez Del Pozo, Cristina Bortolotti, Mar Rufat, Gisela Peñas, Ramón Eritja, Didier Tharreau, Joaquima Messeguer, Blanca San SegundoAbstract:The Aspergillus giganteus antifungal protein (AFP), encoded by the afp gene, has been reported to possess in vitro antifungal activity against various economically important fungal pathogens, including the rice blast fungus Magnaporthe grisea . In this study, transgenic rice ( Oryza sativa ) constitutively expressing the afp gene was generated by Agrobacterium -mediated transformation. Two different DNA constructs containing either the afp cDNA sequence from Aspergillus or a chemically synthesized codon-optimized afp gene were introduced into rice plants. In both cases, the DNA region encoding the signal sequence from the tobacco AP24 gene was N-terminally fused to the coding sequence of the mature AFP protein. Transgenic rice plants showed stable integration and inheritance of the transgene. No effect on plant morphology was observed in the afp -expressing rice lines. The inhibitory activity of protein extracts prepared from leaves of afp plants on the in vitro growth of M. grisea indicated that the AFP protein produced by the trangenic rice plants was biologically active. Several of the T_2 homozygous afp lines were challenged with M. grisea in a detached leaf infection assay. Transformants exhibited resistance to rice blast at various levels. Altogether, the results presented here indicate that AFP can be functionally expressed in rice plants for protection against the rice blast fungus M. grisea .
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Activity of the Antifungal Protein from Aspergillus giganteus Against Botrytis cinerea.
Phytopathology, 2003Co-Authors: Ana Moreno, Álvaro Martínez Del Pozo, M. Borja, Blanca San SegundoAbstract:Moreno, A. B., Martinez del Pozo, A., Borja, M., and San Segundo, B. 2003. Activity of the antifungal protein from Aspergillus giganteus against Botrytis cinerea. Phytopathology 93:1344-1353. Botrytis blight (gray mold), caused by Botrytis cinerea, is one of the most widely distributed diseases of ornamental plants. In geranium plants, gray mold is responsible for important losses in production. The mold Aspergillus giganteus is known to produce and secrete a basic lowmolecular-weight protein, the antifungal protein (AFP). Here, the antifungal properties of the Aspergillus AFP against various B. cinerea isolates obtained from naturally infected geranium plants were investigated. AFP strongly inhibited mycelial growth as well as conidial germination of B. cinerea. Microscopic observations of fungal cultures treated with AFP revealed reduced hyphal elongation and swollen hyphal tips. Washout experiments in which B. cinerea was incubated with AFP for different periods of time and then washed away revealed a fungicidal activity of AFP. Application of AFP on geranium plants protected leaves against Botrytis infection. Cecropin A also was active against this pathogen. An additive effect against the fungus was observed when AFP was combined with cecropin A. These results are discussed in relation to the potential of the afp gene to enhance crop protection against B. cinerea diseases. Additional keyword: Pelargonium.
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the antifungal protein afp of Aspergillus giganteus is an oligonucleotide oligosaccharide binding ob fold containing protein that produces condensation of dna
Journal of Biological Chemistry, 2002Co-Authors: Álvaro Martínez Del Pozo, Valle Lacadena, José M. Mancheño, Mercedes Oñaderra, Nieves Olmo, José G. GavilanesAbstract:The antifungal protein AFP is a small polypeptide of 51 amino acid residues secreted by Aspergillus giganteus. Its potent activity against phytopathogenic fungi converts AFP in a promising tool in plant protection. However, no data have been reported regarding the mode of action of AFP. The three-dimensional structure of this protein, a small and compact beta barrel composed of five highly twisted antiparallel beta strands, displays the characteristic features of the oligonucleotide/oligosaccharide binding (OB fold) structural motif. A comparison of the structures of AFP and OB fold-containing proteins shows this structural similarity despite the absence of any significant sequence similarity. AFP, like most OB fold-containing proteins, binds nucleic acids. The protein promotes charge neutralization and condensation of DNA as demonstrated by electrophoretic mobility shift and ethidium bromide displacement assays. Nucleic acid produces quenching of the protein fluorescence emission. This nucleic acid interacting ability of AFP may be related to the antifungal activity of this small polypeptide.
