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Marcel Asther - One of the best experts on this subject based on the ideXlab platform.
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Fusion of a family 1 carbohydrate binding module of Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulp biobleaching
Journal of Biotechnology, 2009Co-Authors: Holly Ravalason, Eric Record, Marcel Asther, Isabelle Herpoël-gimbert, Frédérique Bertaud, Sacha Grisel, Sandra De Weert, Cees A.m.j.j. Van Den Honde, Michel Petit-conil, Jean-claude SigoillotAbstract:Pycnoporus cinnabarinus laccase was fused to the C-terminal linker and carbohydrate binding module (CBM) of Aspergillus niger cellobiohydrolase B (CBHB). The chimeric enzyme of molecular mass 100 kDa was successfully produced in A. niger. Laccase-CBM was further purified to determine its main biochemical properties. The Michaelis-Menten constant and pH activity profile were not modified, but the chimeric enzyme was less thermostable than either the P. cinnabarinus laccase or the recombinant laccase produced in the same strain. Laccase-CBM was able to bind to a cellulosic substrate and, to a greater extent, to softwood kraft pulp. Binding to the pulp was shown to be mainly time and temperature-dependent. Laccase-CBM was further investigated for its softwood kraft pulp biobleaching potential and compared with the P. cinnabarinus laccase. Addition of a CBM was shown to greatly improve the delignification capabilities of the laccase in the presence of 1-hydroxybenzotriazole (HBT). In addition, ClO2 reduction using 5 U of chimeric enzyme per gram of pulp was almost double than that observed using 20 U of P. cinnabarinus laccase per gram of pulp. We demonstrated that conferring a carbohydrate binding capability to the laccase could significantly enhance its biobleaching properties
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Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica
FEMS Yeast Research, 2005Co-Authors: Catherine Madzak, Marcel Asther, Serge Moukha, Ludovic Otterbein, Mohamed Chamkha, Claude Gaillardin, Jean-marie BeckerichAbstract:Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mg l(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications. (c) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
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Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica
Fems Yeast Research, 2004Co-Authors: Catherine Madzak, Marcel Asther, Serge Moukha, Ludovic Otterbein, Mohamed Chamkha, Claude Gaillardin, Jean-marie BeckerichAbstract:Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications.
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Highly Efficient Production of Laccase by the Basidiomycete Pycnoporus cinnabarinus
Applied and environmental microbiology, 2004Co-Authors: Alexandra M.c.r. Alves, Eric Record, Marcel Asther, Anne Lomascolo, Karin Scholtmeijer, Joseph G.h. Wessels, Han A.b. WöstenAbstract:Filamentous fungi belonging to the homobasidiomycetes offer great potential for industrial and medical applications. They secrete proteins into their culture media with activities or in amounts that are not found in other fungi. For instance, homobasidiomycetes produce various metalloenzymes, such as laccases, which are attractive candidates for a wide variety of applications. These enzymes degrade a large number of recalcitrant pollutants and are a biological and environmentally friendly alternative to the highly contaminating pulping and bleaching treatments of the paper and pulp industries (3, 4). Until now, the expression of basidiomycete metalloenzymes in ascomycete production systems such as Aspergillus ssp. and Trichoderma reesei has had limited success (6). Therefore, basidiomycetes should be developed as hosts for large-scale protein production. The white rot fungus Pycnoporus cinnabarinus is an attractive candidate in this respect. This basidiomycete was selected for its ability to efficiently degrade lignin and to transform lignin-derived compounds such as ferulic acid into vanillin (9, 11, 22). P. cinnabarinus has a simple ligninolytic system. Neither lignin peroxidase nor manganese peroxidase activity has been detected, but laccase is produced (9). Two laccase genes have been cloned, i.e., lcc3-1 or the allelic form lac1 (10, 23) and lcc3-2 (34). Until now, transformation procedures and expression systems for P. cinnabarinus were not available. This was part of the subject of this study. Classical and molecular genetics have been well established for Schizophyllum commune, which can be considered a model system for the homobasidiomycetes. S. commune was transformed to phleomycin and hygromycin resistance by use of the regulatory sequences of the GPD (glyceraldehyde-3-phosphate dehydrogenase) gene (26, 27). Apart from the GPD promoter, the SC3 promoter can also be used for high-level gene expression (36). The former promoter is constitutively expressed, whereas the monokaryon-specific SC3 promoter is expressed only after a few days of growth. mRNA accumulation in S. commune does not only depend on the promoter used but also depends on the presence of introns in or near the coding sequence of the gene (18, 26). Moreover, AT-rich regions within the coding sequence cause premature termination, resulting in truncated mRNAs (28). Full-length mRNAs have been produced by increasing the GC content in such a region (26). For this study, a transformation and expression system for P. cinnabarinus was developed. This system was used to produce high levels of the homologous laccase lac1.
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Highly efficient production of laccase by the basidiomycete Pycnoporus cinnabarinus
Applied and Environmental Microbiology, 2004Co-Authors: Alexandra M.c.r. Alves, Eric Record, Marcel Asther, Anne Lomascolo, Karin Scholtmeijer, Joseph G.h. Wessels, Han A.b. WöstenAbstract:An efficient transformation and expression system was developed for the industrially relevant basidiomycete Pycnoporus cinnabarinus. This was used to transform a laccase-deficient monokaryotic strain with the homologous lac1 laccase gene placed under the regulation of its own promoter or that of the SC3 hydrophobin gene or the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene of Schizophyllum commune. SC3-driven expression resulted in a maximal laccase activity of 107 nkat ml–1 in liquid shaken cultures. This value was about 1.4 and 1.6 times higher in the cases of the GPD and lac1 promoters, respectively. lac1-driven expression strongly increased when 25 g of ethanol liter–1 was added to the medium. Accordingly, laccase activity increased to 1,223 nkat ml–1. These findings agree with the fact that ethanol induces laccase gene expression in some fungi. Remarkably, lac1 mRNA accumulation and laccase activity also strongly increased in the presence of 25 g of ethanol liter–1 when lac1 was expressed behind the SC3 or GPD promoter. In the latter case, a maximal laccase activity of 1,393 nkat ml–1 (i.e., 360 mg liter–1) was obtained. Laccase production was further increased in transformants expressing lac1 behind its own promoter or that of GPD by growth in the presence of 40 g of ethanol liter–1. In this case, maximal activities were 3,900 and 4,660 nkat ml–1, respectively, corresponding to 1 and 1.2 g of laccase per liter and thus representing the highest laccase activities reported for recombinant fungal strains. These results suggest that P. cinnabarinus may be a host of choice for the production of other proteins as well.
Anne Lomascolo - One of the best experts on this subject based on the ideXlab platform.
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Identification of an Oligosaccharide Dehydrogenase from Pycnoporus cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose
2021Co-Authors: Gabriele Cerutti, David Navarro, Anne Lomascolo, Francois Piumi, Elena Gugole, Linda Celeste Montemiglio, Annick Turbé-doan, Dehbia Chena, Cécile Exertier, Ida FredaAbstract:Abstract Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.
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the genome of the white rot fungus Pycnoporus cinnabarinus a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown
BMC Genomics, 2014Co-Authors: Anthony Levasseur, Mireille Haon, Anne Lomascolo, Francois Piumi, Olivier Chabrol, Francisco J Ruizduenas, Eva Boukhrisuzan, Ursula Kues, Claude Murat, Isabelle BenoitAbstract:Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
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The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown
BMC Genomics, 2014Co-Authors: Anthony Levasseur, Mireille Haon, Anne Lomascolo, Francois Piumi, Olivier Chabrol, Ursula Kues, Claude Murat, Francisco J Ruiz-dueñas, Eva Boukhris-uzan, Isabelle BenoitAbstract:Background Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. Results The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. Conclusions With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
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BMC Genomics - The genome of the white-rot fungus Pycnoporus cinnabarinus: a basidiomycete model with a versatile arsenal for lignocellulosic biomass breakdown.
BMC genomics, 2014Co-Authors: Anthony Levasseur, Anne Lomascolo, Francois Piumi, Olivier Chabrol, Ursula Kues, Claude Murat, Francisco J Ruiz-dueñas, Eva Boukhris-uzan, Arthur F. J. Ram, Mireille HaonAbstract:Saprophytic filamentous fungi are ubiquitous micro-organisms that play an essential role in photosynthetic carbon recycling. The wood-decayer Pycnoporus cinnabarinus is a model fungus for the study of plant cell wall decomposition and is used for a number of applications in green and white biotechnology. The 33.6 megabase genome of P. cinnabarinus was sequenced and assembled, and the 10,442 predicted genes were functionally annotated using a phylogenomic procedure. In-depth analyses were carried out for the numerous enzyme families involved in lignocellulosic biomass breakdown, for protein secretion and glycosylation pathways, and for mating type. The P. cinnabarinus genome sequence revealed a consistent repertoire of genes shared with wood-decaying basidiomycetes. P. cinnabarinus is thus fully equipped with the classical families involved in cellulose and hemicellulose degradation, whereas its pectinolytic repertoire appears relatively limited. In addition, P. cinnabarinus possesses a complete versatile enzymatic arsenal for lignin breakdown. We identified several genes encoding members of the three ligninolytic peroxidase types, namely lignin peroxidase, manganese peroxidase and versatile peroxidase. Comparative genome analyses were performed in fungi displaying different nutritional strategies (white-rot and brown-rot modes of decay). P. cinnabarinus presents a typical distribution of all the specific families found in the white-rot life style. Growth profiling of P. cinnabarinus was performed on 35 carbon sources including simple and complex substrates to study substrate utilization and preferences. P. cinnabarinus grew faster on crude plant substrates than on pure, mono- or polysaccharide substrates. Finally, proteomic analyses were conducted from liquid and solid-state fermentation to analyze the composition of the secretomes corresponding to growth on different substrates. The distribution of lignocellulolytic enzymes in the secretomes was strongly dependent on growth conditions, especially for lytic polysaccharide mono-oxygenases. With its available genome sequence, P. cinnabarinus is now an outstanding model system for the study of the enzyme machinery involved in the degradation or transformation of lignocellulosic biomass.
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Role of ethanol on growth, laccase production and protease activity in Pycnoporus cinnabarinus ss3
Enzyme and Microbial Technology, 2007Co-Authors: Juan Carlos Meza, Jean-claude Sigoillot, Anne Lomascolo, Richard Auria, Laurence CasalotAbstract:Laccase production by the strain Pycnoporus cinnabarinus ss3 was studied in a solid-state culture on sugar-cane bagasse using chemical compounds as inducers (ethanol, methanol, veratryl alcohol and ferulic acid). Laccase productions were about 5- to 8.5-fold higher than non-induced cultures. Liquid-culture experiments with 14C-labeled ethanol were conducted. Ninety-eight percent of the initial amount of 14C from ethanol was recovered as 14CO2, 14C-biomass and soluble 14C-compounds (mainly ethanol). The amount of 14C in the biomass was only 6.8% of the total carbon consumed by P. cinnabarinus, in absence of maltose, representing only 2.8% of added ethanol (1.1% and 1.6% in presence of maltose, respectively). Ethanol was poorly used as carbon and energy sources for P. cinnabarinus growth and other carbon sources present in the liquid medium (yeast extract and sodium tartrate) were preferentially degraded. Time-courses of laccase activity and gene expression were monitored in column in presence or in absence of ethanol vapors. Analyses showed a perfect correlation between the activity and the amount of transcript. After 16 days of ethanol ventilation through the column, the ethanol flow was stopped. Immediately, both laccase activity and gene expression decreased, but started to increase again as soon as the ventilation was restored. In parallel, the effect of ethanol on protease activity in P. cinnabarinus was measured. Presence of ethanol led to an inhibition of protease activity. Therefore ethanol plays a regulatory role on two elements (gene-expression and protease-activity levels) that are both in favor of an increase in laccase production by the fungus
Sami Sayadi - One of the best experts on this subject based on the ideXlab platform.
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decolorization of semisolid olive residues of alperujo during the solid state fermentation by phanerochaete chrysosporium trametes versicolor Pycnoporus cinnabarinus and aspergillus niger
Biochemical Engineering Journal, 2007Co-Authors: Fathi Aloui, Nabil Abid, Sevastianos Roussos, Sami SayadiAbstract:Abstract Studies were carried out on decolorization of semisolid olive mill residues called “alperujo” (AL), by four strains of Phanerochaete chrysosporium, Trametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger in solid state frementation (SSF). Fungal strains were selected by their ability to grow on olive mill waste water. The treatment of AL with P. chrysosporium led to higher removal of organic matter and decolorization than P. cinnabarinus and T. versicolor . The strain A. niger leads to a relative improvement of the biodegradability of OMW. Using P. chrysosporium, the treatment of AL substrate in SSF showed an efficient decolorization and an appreciable COD and phenolic content removal only in the presence of a support. Sugarcane bagasse used as support improved the oxygen transfer in the culture. The extracellular fluid of P. chrysosporium (composed mainly of LiP) exhibited high ability to decolorize AL showing the efficiency of the enzyme produced. A percentage of sugarcane bagasse equal to 30% was an optimal condition to improve the growth and the decolorization of AL. The results showed good prospects of using the three basidiomycetes, in particular P. chrysosporium , for the Decolorization of AL.
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Decolorization of semisolid olive residues of “alperujo” during the solid state fermentation by Phanerochaete chrysosporium, Trametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger
Biochemical Engineering Journal, 2007Co-Authors: Fathi Aloui, Nabil Abid, Sevastianos Roussos, Sami SayadiAbstract:Abstract Studies were carried out on decolorization of semisolid olive mill residues called “alperujo” (AL), by four strains of Phanerochaete chrysosporium, Trametes versicolor, Pycnoporus cinnabarinus and Aspergillus niger in solid state frementation (SSF). Fungal strains were selected by their ability to grow on olive mill waste water. The treatment of AL with P. chrysosporium led to higher removal of organic matter and decolorization than P. cinnabarinus and T. versicolor . The strain A. niger leads to a relative improvement of the biodegradability of OMW. Using P. chrysosporium, the treatment of AL substrate in SSF showed an efficient decolorization and an appreciable COD and phenolic content removal only in the presence of a support. Sugarcane bagasse used as support improved the oxygen transfer in the culture. The extracellular fluid of P. chrysosporium (composed mainly of LiP) exhibited high ability to decolorize AL showing the efficiency of the enzyme produced. A percentage of sugarcane bagasse equal to 30% was an optimal condition to improve the growth and the decolorization of AL. The results showed good prospects of using the three basidiomycetes, in particular P. chrysosporium , for the Decolorization of AL.
Laurence Lesage-meessen - One of the best experts on this subject based on the ideXlab platform.
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Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant
World Journal of Microbiology and Biotechnology, 2003Co-Authors: Isabel Estrada Alvarado, Eric Record, Marcel Asther, David Navarro, Laurence Lesage-meessenAbstract:Fungal biotransformation of p -coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p -coumaric acid. Preliminary experiments showed no toxicity of both p -coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^−1. Feeding 450 mg p -coumaric acid l^−1 into P. cinnabarinus cultures grown on 20 g l^−1 glucose medium resulted in the production of 257 mg caffeic acid l^−1with a molar yield of 21%.
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Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, selected for the biotransformation of ferulic acid to vanillin, are also able to produce cell wall polysaccharide-degrading enzymes and feruloyl esterases
Enzyme and Microbial Technology, 2001Co-Authors: E Bonnina, Marcel Asther, Laurence Lesage-meessen, Magali Brunel, Y Gouy, J.f. ThibaultAbstract:Abstract The filamentous fungal strains Aspergillus niger I-1472 and Pycnoporus cinnabarinus MUCL39533, previously selected for the bioconversion of ferulic acid to vanillic acid and vanillin respectively, were grown on sugar beet pulp. A large spectrum of polysaccharide-degrading enzymes was produced by A. niger and very few levels of feruloyl esterases were found. In contrast, P. cinnabarinus culture filtrate contained low amount of polysaccharide-degrading enzymes and no feruloyl esterases. In order to enhance feruloyl esterases in A. niger cultures, feruloylated oligosaccharide-rich fractions were prepared from sugar beet pulp or cereal bran and used as carbon sources. Number of polysaccharide-degrading enzymes were induced. Feruloyl esterases were much higher in maize bran-based medium than in sugar beet pulp-based medium, demonstrating the ability of carbon sources originating from maize to induce the synthesis of feruloyl esterases. Thus, A. niger I-1472 could be interesting to release ferulic acid from sugar beet pulp or maize bran.
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Selection of Pycnoporus cinnabarinus strains for laccase production.
FEMS microbiology letters, 2000Co-Authors: Isabelle Herpoël, Jean-claude Sigoillot, Serge Moukha, Laurence Lesage-meessen, Marcel AstherAbstract:A comparison of Pycnoporus cinnabarinus strains for laccase production was carried out. A dikaryotic strain, I-937 strain, producing a high level of laccase (9500 U l−1) was selected. The study of the life cycle in vitro of this dikaryotic strain led to isolation of monokaryons. Forty-eight monokaryotic strains were isolated and screened for laccase production. One of these strains, ss3, produced a higher level of laccase than the parental strain I-937. The maximum production reached 29 000 U l−1 in medium supplemented with ferulic acid.
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Design of a fungal bioprocess for vanillin production from vanillic acid at scalable level by Pycnoporus cinnabarinus
Journal of bioscience and bioengineering, 2000Co-Authors: Christelle Stentelaire, Benoit Colonna Ceccaldi, Laurence Lesage-meessen, Olivier Bernard, Georges Bastin, Julie Oddou, Marcel AstherAbstract:The biotechnological process of vanillin production from vanillic acid by Pycnoporus cinnabarinus was scaled-up at the laboratory level. Vanillin production was studied in two types of bioreactors, a mechanically agitated and an air-lift bioreactor. In the mechanically agitated bioreactor where vanillin was produced in greater quantities, oxygen availability was studied during the growth and production phases. A maximal aeration rate (90l/h equivalent to 0.83 volume of air/volume of medium/min or vvm) during the growth phase and a minimal aeration rate (30 l/h equivalent to 0.28 vvm) during the production phase were necessary to increase vanillin production to 1260 mg/l. Vanillic acid bioconversion to vanillin occurred under the conditions of reduced dissolved oxygen concentration, gentle agitation, high carbon dioxide production and low specific growth rate. However, under these conditions, vanillin production was accompanied by a significant amount of methoxyhydroquinone. Vanillin over a concentration of 1000 mg/l was shown to be highly toxic to the growth of P. cinnabarinus on agar medium. The application of selective XAD-2 resin led to a reduction of vanillin concentration in the medium, thus limiting its toxicity towards the fungal biomass as well as the formation of unwanted by-products such as methoxyhydroquinone and allowed the concentration of vanillin produced to reach 1575 mg/l.
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Enzymic release of cellobiose from sugar beet pulp, and its use to favour vanillin production in Pycnoporus cinnabarinus from vanillic acid
Carbohydrate Polymers, 2000Co-Authors: Estelle Bonnin, Marcel Asther, Laurence Lesage-meessen, H Grangé, J.f. ThibaultAbstract:Abstract This study was undertaken in order to optimise the release of cellobiose from sugar beet pulp by enzymic treatment, and to evaluate the effect of this cellobiose in activating the vanillin production by filamentous fungi. Cellulose-rich residues were obtained from sugar beet pulp by a pectinase treatment, and they were then hydrolysed by a commercial cellulase. Various pretreatments (drying, grinding) and storage were applied, in order to modify the physical state of the cellulose-rich residues, such as degree of crystallinity of cellulose. After cellulase treatment, 51–64% of the initial cellulose was degraded into cellobiose and glucose, depending on the pretreatment. Whatever the pretreatment applied, cellobiose was maximally produced after 2–4 h of hydrolysis. Thereafter, cellobiose was degraded by the β-glucosidase present in the commercial cellulase, but this activity could be inhibited by the addition of d -glucono-δ-lactone. Only in some cases, could the extent of degradation of cellulose be related to the physical state of cellulose-rich residues. The best conditions were used to produce large quantities of cellobiose in order to test its influence in the bioconversion of vanillic acid into vanillin by the filamentous fungus, Pycnoporus cinnabarinus . Addition of cellobiose to a 3-day-old culture yielded a 3.3-fold increase in vanillin production when compared to a control culture devoid of cellobiose.
Claudia Eggert - One of the best experts on this subject based on the ideXlab platform.
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Cloning and characterization of a second laccase gene from the lignin-degrading basidiomycete Pycnoporus cinnabarinus.
Gene, 1999Co-Authors: Ulrike Temp, Uwe Zierold, Claudia EggertAbstract:Abstract The gene lcc3-2 encoding a second laccase of the white-rot fungus Pycnoporus cinnabarinus has been cloned, sequenced, and characterized. The isolated gene consists of 2840 bp, with the coding region interrupted by ten introns and flanked by an upstream region in which putative CAAT and TATA boxes were identified. The cDNA of lcc3-2 contains an open reading frame of 1563 bp. The deduced mature laccase protein consisted of 498 amino acids and was preceded by a signal peptide of 23 amino acids. The sequence of lcc3-2 reveals 73% similarity on the protein level to the previously characterized lcc3-1 . The new laccase gene shares highest similarity to lcc1 from Trametes villosa (75%), and lcc2 from the unidentified basidiomycete CECT 20197 (75%). The calculated isoelectric point (p I ) of 6.1 for the gene product LCC3-2 was in good agreement with the experimentally determined p I of a laccase secreted by P. cinnabarinus grown on cellulose. Transcription analysis using competitive reverse transcription (RT)-PCR showed that lcc3-2 was expressed in glucose and cellulose containing cultures. However, in contrast to lcc3-1 , lcc3-2 transcription was not increased in response to 2,5-xylidine.
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novel interaction between laccase and cellobiose dehydrogenase during pigment synthesis in the white rot fungus Pycnoporus cinnabarinus
Applied and Environmental Microbiology, 1999Co-Authors: Ulrike Temp, Claudia EggertAbstract:When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.
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molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus
Applied and Environmental Microbiology, 1998Co-Authors: Claudia Eggert, Ulrike Temp, Karlerik Eriksson, Peter R Lafayette, Jeffrey F D DeanAbstract:It was recently shown that the white rot basidiomycete Pycnoporus cinnabarinus secretes an unusual set of phenoloxidases when it is grown under conditions that stimulate ligninolysis (C. Eggert, U. Temp, and K.-E. L. Eriksson, Appl. Environ. Microbiol. 62:1151-1158, 1996). In this report we describe the results of a cloning and structural analysis of the laccase-encoding gene (lcc3-1) expressed by P. cinnabarinus during growth under xylidine-induced conditions. The coding region of the genomic laccase sequence, which is preceded by the eukaryotic promoter elements TATA and CAATA, spans more than 2,390 bp. The corresponding laccase cDNA was identical to the genomic sequence except for 10 introns that were 50 to 60 bp long. A sequence analysis indicated that the P. cinnabarinus lcc3-1 product has a Phe residue at a position likely to influence the reduction-oxidation potential of the enzyme's type 1 copper center. The P. cinnabarinus lcc3-1 sequence was most similar to the sequence encoding a laccase from Coriolus hirsutus (level of similarity, 84%).
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molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus
Applied and Environmental Microbiology, 1998Co-Authors: Claudia Eggert, Ulrike Temp, Karlerik Eriksson, Peter R Lafayette, Jeffrey F D DeanAbstract:By definition, laccases (p-diphenol:O2 oxidoreductase; EC 1.10.3.2) catalyze the oxidation of p-diphenols and the concurrent reduction of dioxygen to water, although the actual substrate specificities of laccases are often quite broad and vary with the enzyme source (11, 29). Laccases are members of the blue copper oxidase enzyme family characterized by having four cupric (Cu2+) ions coordinated such that each of the known magnetic species (type 1, type 2, and type 3) is associated with a single polypeptide chain. The Cu2+-binding domains are highly conserved in the blue copper oxidases, and the crystallographic structure of ascorbate oxidase, another member of this enzyme class, has provided a good model for the structure of the laccase active site (30, 31). This model has been supported by the results of numerous studies of the electron transfer reactions that occur between cupric ions during catalysis (35, 39, 40). In contrast to our understanding of the electron transfer reactions that occur in laccases, relatively little is known about the physiological functions of these enzymes. Laccases have been implicated in pigmentation (1, 9), fruiting body formation (26), and pathogenicity (7, 45), as well as in lignin degradation (41) and biosynthesis (27). Very few of these functions have been experimentally proven, and only because of the availability of multiple gene sequences and crystallographic data has it been possible to speculate about how structure-function relationships may be important in the specific roles played by these enzymes (46). Some of this speculation has involved attempts to address the apparent contradictory functions of laccases in the synthesis and breakdown of lignin (3, 11). To better understand the role of laccases in lignin degradation by white rot fungi, we studied the ligninolytic system of Pycnoporus cinnabarinus, a basidiomycete that produces an unusual set of ligninolytic enzymes. Just a single isoform of laccase, but no lignin peroxidase (LiP) or manganese peroxidase (MnP), was produced by this organism under conditions that stimulated lignin degradation (13). We wanted to determine more completely the pattern of phenoloxidase production in P. cinnabarinus, so the primary objective of this study was to analyze the structure of the P. cinnabarinus laccase gene and determine whether there are multiple laccase genes in the P. cinnabarinus genome.
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laccase is essential for lignin degradation by the white rot fungus Pycnoporus cinnabarinus
FEBS Letters, 1997Co-Authors: Claudia Eggert, Ulrike Temp, Karlerik ErikssonAbstract:The white-rot fungus, Pycnoporus cinnabarinus, provides an excellent model organism to elucidate the controversial role of laccase in lignin degradation. P. cinnabarinus produces laccase in one isoform as the predominant phenoloxidase in ligninolytic cultures, and neither LiP nor MnP are secreted. Yet, P. cinnabarinus degrades lignin very efficiently. In the present work, we show that laccase-less mutants of P. cinnabarinus were greatly reduced in their ability to metabolize 14C ring-labeled DHP. However, 14CO2 evolution in these mutant cultures could be restored to levels comparable to those of the wild-type cultures by addition of purified P. cinnabarinus laccase. This clearly indicates that laccase is absolutely essential for lignin degradation by P. cinnabarinus.
