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Vassilis Bouriotis - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of a Chitin Deacetylase gene cda2 from saccharomyces cerevisiae in escherichia coli purification and characterization of the cobalt dependent recombinant enzyme
Enzyme and Microbial Technology, 2003Co-Authors: Aggeliki Martinou, Dimitris Koutsioulis, Vassilis BouriotisAbstract:Abstract The Chitin Deacetylase gene CDA2 from Saccharomyces cerevisiae has been cloned and expressed in Escherichia coli and the recombinant enzyme purified to homogeneity and further characterized. The enzyme exhibits an apparent molecular mass of 35 kDa. When glycol Chitin is used as substrate the optimum temperature for enzyme activity is 50 °C and the pH optimum is 8.0. The enzyme requires at least two N -acetyl- d -glucosamine residues (chitobiose) for catalysis and is inhibited by acetate. The presence of CoCl 2 proved to be essential for enzyme activity. Seven amino acids could be eliminated from the C-terminus without a significant loss of enzyme activity. However, truncation of 12 or more amino acids from the N-terminus or 27 amino acids from the C-terminus resulted in complete loss of enzyme activity.
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Expression, purification, and characterization of a cobalt-activated Chitin Deacetylase (Cda2p) from Saccharomyces cerevisiae.
Protein Expression and Purification, 2002Co-Authors: Aggeliki Martinou, Dimitris Koutsioulis, Vassilis BouriotisAbstract:Chitin Deacetylase (Cda2p) (EC 3.5.1.41) from Saccharomyces cerevisiae has been purified from vegetative cells grown in galactose and further characterized. The enzyme is a glycoprotein with an apparent molecular mass of ∼43 kDa and a carbohydrate content of approximately 18% by weight. With glycol Chitin as substrate, the optimum temperature for enzyme activity is 50°C and the pH optimum is 8.0. The enzyme requires at least two N-acetyl-d-glucosamine residues (chitobiose) for catalysis and is partially inhibited by acetate. Deglycosylation of the enzyme causes total loss of enzyme activity, which can be restored by the addition of CoCl2.
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Mode of action of Chitin Deacetylase from Mucor rouxii on N‐acetylchitooligosaccharides
European Journal of Biochemistry, 2001Co-Authors: Iason Tsigos, Aggeliki Martinou, Nathalie Zydowicz, Alain Domard, Vassilis BouriotisAbstract:The mode of action of Chitin Deacetylase from the fungus Mucor rouxii on N-acetylchitooligosaccharides with a degree of polymerization 1–7 has been elucidated. Identification of the sequence of Chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo-glycosidases in conjunction with HPLC. The results were further verified by 1H-NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate Chitin oligomers with a degree of polymerization lower than three. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and hepta-N-acetylchitoheptaose the reducing-end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing-end residue of all Chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.
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mode of action of Chitin Deacetylase from mucor rouxii on n acetylchitooligosaccharides
FEBS Journal, 2001Co-Authors: Iason Tsigos, Aggeliki Martinou, Nathalie Zydowicz, Alain Domard, Vassilis BouriotisAbstract:The mode of action of Chitin Deacetylase from the fungus Mucor rouxii on N-acetylchitooligosaccharides with a degree of polymerization 1–7 has been elucidated. Identification of the sequence of Chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo-glycosidases in conjunction with HPLC. The results were further verified by 1H-NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate Chitin oligomers with a degree of polymerization lower than three. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and hepta-N-acetylchitoheptaose the reducing-end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing-end residue of all Chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.
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Yeast ascospore wall assembly requires two Chitin Deacetylase isozymes.
FEBS Letters, 1999Co-Authors: Anna Christodoulidou, Peter Briza, Adi Ellinger, Vassilis BouriotisAbstract:Chitin Deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two Chitin Deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the Chitin Deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.
Catherine Texier - One of the best experts on this subject based on the ideXlab platform.
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The putative Chitin Deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore‐wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
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the putative Chitin Deacetylase of encephalitozoon cuniculi a surface protein implicated in microsporidian spore wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
Damien Brosson - One of the best experts on this subject based on the ideXlab platform.
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The putative Chitin Deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore‐wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
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the putative Chitin Deacetylase of encephalitozoon cuniculi a surface protein implicated in microsporidian spore wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
Christian P. Vivarès - One of the best experts on this subject based on the ideXlab platform.
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The putative Chitin Deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore‐wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
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the putative Chitin Deacetylase of encephalitozoon cuniculi a surface protein implicated in microsporidian spore wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
Gérard Prensier - One of the best experts on this subject based on the ideXlab platform.
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The putative Chitin Deacetylase of Encephalitozoon cuniculi: A surface protein implicated in microsporidian spore‐wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.
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the putative Chitin Deacetylase of encephalitozoon cuniculi a surface protein implicated in microsporidian spore wall formation
Fems Microbiology Letters, 2005Co-Authors: Damien Brosson, Lauriane Kuhn, Gérard Prensier, Christian P. Vivarès, Catherine TexierAbstract:Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick cell wall composed of glycoproteins and Chitin. Despite an extensive description of the fibrillar structure of this wall, very little is known about its protein components and deposit mechanisms. In this study on the human pathogen Encephalitozoon cuniculi, we identify by mass spectrometry the target of polyclonal antibodies previously raised against a 33-kDa protein located at the outer face of the parasite plasma membrane. This 254-amino acid protein is encoded by the ECU11_0510 open reading frame and presents two isoforms of 33 and 55 kDa. Sequence analysis supports an assignment to the polysaccharide Deacetylase family with a suspected Chitin Deacetylase activity (EcCDA). As demonstrated by TEM studies, EcCDA is present at the plasma membrane of the early stages of E. cuniculi life-cycle. At the sporoblast stage, the enzyme accumulates especially in paramural bodies which are convolutions of the plasma membrane opened to the wall. The identification of an EcCDA homologue in the insect parasite Antonospora locustae (ex Nosema locustae) suggests a widespread distribution of this enzyme among Microsporidia. This characterization of a new microsporidian surface protein creates new perspectives to understand spore wall formation and spore resistance.