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Ramesh Chander Kuhad - One of the best experts on this subject based on the ideXlab platform.
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fungal pretreatment improves amenability of lignocellulosic material for its saccharification to sugars
Carbohydrate Polymers, 2014Co-Authors: Deepa Deswal, Preeti Nandal, Rishi Gupta, Ramesh Chander KuhadAbstract:Abstract The sugarcane bagasse was biologically pretreated with three white-Rot fungi; PleuRotus florida, Coriolopsis caperata RCK 2011 and Ganoderma sp. rckk-02, individually under solid-state fermentation. P. florida, C. caperata RCK 2011 and Ganoderma sp. rckk-02 degraded lignin up to 7.91, 5.48 and 5.58%, respectively. The lignocellulolytic enzymes produced by these fungi were also monitored during solid state fermentation of sugarcane bagasse. The fungal fermented sugarcane bagasse when hydrolyzed with crude cellulases from Brown-Rot Fungus, Fomitopsis sp. RCK2010, released comparatively 1.5–2.4 fold higher sugars than in case of untreated sugarcane bagasse. The study demonstrated that white-Rot fungal pretreatment improved the amenability of plant material for enzymatic hydrolysis.
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Enhanced exoglucanase production by Brown Rot Fungus Fomitopsis sp. RCK2010 and its application for cellulose saccharification.
Applied Biochemistry and Biotechnology, 2012Co-Authors: Deepa Deswal, Rishi Gupta, Ramesh Chander KuhadAbstract:Exoglucanase production by Brown Rot Fungus Fomitopsis sp. RCK2010 was optimized under solid-state fermentation using Plackett–Burman design (PBD) and response surface methodology (RSM). Four fermentation variables (moisture, inoculum level, casein, and Triton X-100) were identified to effect cellulase production significantly by PBD, which were further optimized using RSM of central composite design. An overall 130 % increase in enzyme production was achieved by the optimization of variables using statistical approaches. Moreover, crude cellulase from Fomitopsis sp. RCK2010 was applied to saccharify pretreated Prosopis juliflora (cellulosic fraction), which resulted in the release of 327.35 mg/g of reducing sugars that could further be utilized for bioethanol production.
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optimization of cellulase production by a Brown Rot Fungus fomitopsis sp rck2010 under solid state fermentation
Bioresource Technology, 2011Co-Authors: Deepa Deswal, Yogender Pal Khasa, Ramesh Chander KuhadAbstract:Abstract Culture conditions for enhanced cellulase production from a newly isolated Brown Rot Fungus, Fomitopsis sp. RCK2010 were optimized under solid state fermentation. An initial pH of 5.5 and moisture ratio of 1:3.5 (solid:liquid) were found to be optimal for maximum enzyme production. Of the different carbon sources tested wheat bran gave the maximum production of CMCase (71.526 IU/g), FPase (3.268 IU/g), and β-glucosidase (50.696 IU/g). Among the nitrogen sources, urea caused maximum production of CMCase (81.832 IU/g), where as casein and soyabean meal gave the highest FPase (4.682 IU/g) and β-glucosidase (69.083 IU/g) production, respectively. Among amino acids tested glutamic acid gave the highest production for CMCase (84.127 IU/g); however 4-hydroxy- l -proline stimulated maximum FPase production (6.762 IU/g). Saccharification of pretreated rice straw and wheat straw by crude enzyme extract from Fomitopsis sp. RCK2010 resulted in release of 157.160 and 214.044 mg/g of reducing sugar, respectively.
Barry Goodell - One of the best experts on this subject based on the ideXlab platform.
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Transcriptome analysis of the Brown Rot Fungus Gloeophyllum trabeum during lignocellulose degradation.
PloS one, 2020Co-Authors: Kiwamu Umezawa, Barry Goodell, Mai Niikura, Yuka Kojima, Makoto YoshidaAbstract:Brown Rot fungi have great potential in biorefinery wood conversion systems because they are the primary wood decomposers in coniferous forests and have an efficient lignocellulose degrading system. Their initial wood degradation mechanism is thought to consist of an oxidative radical-based system that acts sequentially with an enzymatic saccharification system, but the complete molecular mechanism of this system has not yet been elucidated. Some studies have shown that wood degradation mechanisms of Brown Rot fungi have diversity in their substrate selectivity. Gloeophyllum trabeum, one of the most studied Brown Rot species, has broad substrate selectivity and even can degrade some grasses. However, the basis for this broad substrate specificity is poorly understood. In this study, we performed RNA-seq analyses on G. trabeum grown on media containing glucose, cellulose, or Japanese cedar (Cryptomeria japonica) as the sole carbon source. Comparison to the gene expression on glucose, 1,129 genes were upregulated on cellulose and 1,516 genes were upregulated on cedar. Carbohydrate Active enZyme (CAZyme) genes upregulated on cellulose and cedar media by G. trabeum included glycoside hyrolase family 12 (GH12), GH131, carbohydrate esterase family 1 (CE1), auxiliary activities family 3 subfamily 1 (AA3_1), AA3_2, AA3_4 and AA9, which is a newly reported expression pattern for Brown Rot fungi. The upregulation of both terpene synthase and cytochrome P450 genes on cedar media suggests the potential importance of these gene products in the production of secondary metabolites associated with the chelator-mediated Fenton reaction. These results provide new insights into the inherent wood degradation mechanism of G. trabeum and the diversity of Brown Rot mechanisms.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown Rot Fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown-Rot, through degradation of the primary cell wall.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown-Rot Fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown Rot through degradation of the primary cell wall.
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Lignocellulosic polysaccharides and lignin degradation by wood decay fungi: the relevance of nonenzymatic Fenton-based reactions
Journal of Industrial Microbiology & Biotechnology, 2011Co-Authors: Valdeir Arantes, Timothy R Filley, Adriane M. F. Milagres, Barry GoodellAbstract:The Brown Rot Fungus Wolfiporia cocos and the selective white Rot Fungus Perenniporia medulla - panis produce peptides and phenolate-derivative compounds as low molecular weight Fe^3+-reductants. Phenolates were the major compounds with Fe^3+-reducing activity in both fungi and displayed Fe^3+-reducing activity at pH 2.0 and 4.5 in the absence and presence of oxalic acid. The chemical structures of these compounds were identified. Together with Fe^3+ and H_2O_2 (mediated Fenton reaction) they produced oxygen radicals that oxidized lignocellulosic polysaccharides and lignin extensively in vitro under conditions similar to those found in vivo. These results indicate that, in addition to the extensively studied Gloeophyllum trabeum —a model Brown Rot Fungus—other Brown Rot fungi as well as selective white Rot fungi, possess the means to promote Fenton chemistry to degrade cellulose and hemicellulose, and to modify lignin. Moreover, new information is provided, particularly regarding how lignin is attacked, and either repolymerized or solubilized depending on the type of fungal attack, and suggests a new pathway for selective white Rot degradation of wood. The importance of Fenton reactions mediated by phenolates operating separately or synergistically with carbohydrate-degrading enzymes in Brown Rot fungi, and lignin-modifying enzymes in white Rot fungi is discussed. This research improves our understanding of natural processes in carbon cycling in the environment, which may enable the exploration of novel methods for bioconversion of lignocellulose in the production of biofuels or polymers, in addition to the development of new and better ways to pRotect wood from degradation by microorganisms.
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Oxidation of 2-keto-4-thiomethylbutyric acid (KTBA) by iron-binding compounds produced by the wood-decaying Fungus Gloeophyllum trabeum
Fems Microbiology Letters, 2002Co-Authors: Vikas Chandhoke, Barry Goodell, Jody Jellison, Frank A FeketeAbstract:The ability of iron-binding compounds isolated from the Brown-Rot Fungus Gloeophyllum trabeum to carry out one-electron oxidation reactions was established using a model substrate, 2-keto-4-thiomethylbutyric acid (KTBA). The oxidation reaction was monitored by measuring the amount of ethylene produced from the substrate by gas chromatography. The extent of the reaction was found to be influenced by the concentration of the chelators, and by iron and manganese.
Gry Alfredsen - One of the best experts on this subject based on the ideXlab platform.
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accessibility of hydroxyl groups in furfurylated wood at different weight percent gains and during rhodonia placenta decay
European Journal of Wood and Wood Products, 2019Co-Authors: Greeley Beck, Callum A S Hill, Pierre Martin Cocher, Gry AlfredsenAbstract:Deuterium exchange was used to measure accessible hydroxyl (OH) groups in furfurylated wood at different weigh percent gains (WPGs) and at different levels of mass loss caused by exposure to the Brown-Rot Fungus Rhodonia placenta. The results from the deuterium exchange method showed that OH accessibility in furfurylated samples did not change with increasing WPGs. However, OH accessibility in furfurylated samples increased significantly after initiation of decay at commercial treatment level (mean WPG 32%), which is attributed to degradation of crystalline cellulose regions and formation of new OH groups in lignin and the furfuryl alcohol polymer.
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acetylation of pinus radiata delays hydrolytic depolymerisation by the Brown Rot Fungus rhondonia placenta
International Biodeterioration & Biodegradation, 2018Co-Authors: Greeley Beck, Olav Hegnar, Carl Gunnar Fossdal, Gry AlfredsenAbstract:Abstract Acetylation of wood can provide pRotection against wood deteriorating fungi, but the exact degradation mechanism remains unclear. The aim of this study was to determine the effect of acetylation of Pinus radiata wood (weight percent gain 13, 17 and 21%) on the expression of genes involved in decay by Brown-Rot Fungus Rhodonia placenta. Gene expression analysis using qRT-PCR captured incipient to advanced decay stages. As expected the initiation of decay was delayed as a result the degree of acetylation. However, once decay was established, the rate of degradation in acetylated samples was similar to that of unmodified wood. This suggests a delay in decay rather than an absolute pRotection threshold at higher acetylation levels. In accordance with previous studies, the oxidative system of R. placenta was more active in wood with higher degrees of acetylation and expression of cellulose active enzymes was delayed for acetylated samples compared to untreated samples. The reason for the delay in the latter might be because of the slower diffusion rate in acetylated wood or that partially acetylated cellobiose may be less effective in triggering production of saccharification enzymes. Enzymes involved in hemicellulose and pectin degradation have previously not been focused on in studies of degradation of acetylated wood. Surprisingly, CE16 carbohydrate esterase, assumed to be involved in deacetylation of carbohydrates, was expressed significantly more in untreated samples compared to highly acetylated samples. We hypothesise that this enzyme might be regulated through a negative feedback system, where acetic acid supresses the expression. The up-regulation of two expansin genes in acetylated samples suggests that their function, to loosen the cell wall, is needed more in acetylated wood due the physical bulking of the cell wall. In this study, we demonstrate that acetylation affects the expression of specific target genes not previously reported, resulting in delayed initiation of decay. Thus, targeting these degradation mechanisms can contribute to improving wood pRotection systems.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown Rot Fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown-Rot, through degradation of the primary cell wall.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown-Rot Fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown Rot through degradation of the primary cell wall.
Jody Jellison - One of the best experts on this subject based on the ideXlab platform.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum , tend to have few LPMOs and information on these enzymes is scarce. The genome of G. trabeum encodes four AA9 LPMOs, whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of Gt LPMO9A seem to be produced, a single domain variant, Gt LPMO9A-1, and a longer variant, Gt LPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct Gt LPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses, using HPAEC-PAD and MS, showed that Gt LPMO9A-2 is active on cellulose, carboxymethylcellulose and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, Gt LPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of Gt LPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, Nc LPMO9C from Neurospora crassa , revealed that Gt LPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurments also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of Nc LPMO9C. Experiments with co-polymeric substrates showed an inhibitory effect of hemicellulose-coating on cellulolytic LPMO activity and did not reveal additional activities of Gt LPMO9A-2. These results provide insight into the LPMO-potential of G. trabeum and provide a novel sensitive method, measurement of dynamic viscosity, for monitoring LPMO activity. Importance Currently, there are only a few methods available to analyze end-products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here we present an alternative and sensitive method based on measurement of dynamic viscosity, for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses such as xyloglucan. We have used both this novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown Rot Fungus. This enzyme, Gt LPMO9A-2, differs from previously characterized LPMOs, in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. Gt LPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibres together. The xyloglucan-degrading potential of Gt LPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown-Rot, through degradation of the primary cell wall.
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a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the Brown Rot Fungus gloeophyllum trabeum and its action on cellulose xyloglucan complexes
Applied and Environmental Microbiology, 2016Co-Authors: Yuka Kojima, Barry Goodell, Kiyohiko Igarashi, Jody Jellison, Gry Alfredsen, Dejan Petrovic, Aniko Varnai, Takuya Ishida, Naoki Sunagawa, Bjorge WesterengAbstract:ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-Rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are only a few methods available to analyze end products of lytic polysaccharide monooxygenase (LPMO) activity, the most common ones being liquid chromatography and mass spectrometry. Here, we present an alternative and sensitive method based on measurement of dynamic viscosity for real-time continuous monitoring of LPMO activity in the presence of water-soluble hemicelluloses, such as xyloglucan. We have used both these novel and existing analytical methods to characterize a xyloglucan-active LPMO from a Brown-Rot Fungus. This enzyme, GtLPMO9A-2, differs from previously characterized LPMOs in having broad substrate specificity, enabling almost random cleavage of the xyloglucan backbone. GtLPMO9A-2 acts preferentially on free xyloglucan, suggesting a preference for xyloglucan chains that tether cellulose fibers together. The xyloglucan-degrading potential of GtLPMO9A-2 suggests a role in decreasing wood strength at the initial stage of Brown Rot through degradation of the primary cell wall.
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Characterization of a transplasma membrane redox system of the Brown Rot Fungus Gloeophyllum trabeum
International Biodeterioration & Biodegradation, 2004Co-Authors: Jody JellisonAbstract:The transplasma membrane redox system of the Brown Rot Fungus Gloeophyllum trabeum was characterized with ferricyanide reduction kinetics. Nitrogen deficiency did not statistically affect the ferricyanide reduction rate, which depended on initial ferricyanide concentration and initial mycelial mass. The reduction rate increased with pH above pH 5.0, and was statistically lower in HEPES buffer (pH 8.0) than in potassium phosphate buffer (pH 8.0). Carbonyl cyanide m-chloromethoxyphenyl hydrazone, 2,4-dinitrophenol and sodium azide were efficient inhibitors of the transplasma membrane redox system. The quinone reducing activity of extracellular, membrane-bound, and intracellular quinone reductases of G. trabeum was tested, extracellular quinone reducing activity was not observed under the examined culture conditions. Quinone reduction by mycelium and the intracellular enzyme showed different kinetic constants. The Fungus produced constitutive intracellular benzoquinone reductases, which are NAD(P)H-dependent.
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Oxidation of 2-keto-4-thiomethylbutyric acid (KTBA) by iron-binding compounds produced by the wood-decaying Fungus Gloeophyllum trabeum
Fems Microbiology Letters, 2002Co-Authors: Vikas Chandhoke, Barry Goodell, Jody Jellison, Frank A FeketeAbstract:The ability of iron-binding compounds isolated from the Brown-Rot Fungus Gloeophyllum trabeum to carry out one-electron oxidation reactions was established using a model substrate, 2-keto-4-thiomethylbutyric acid (KTBA). The oxidation reaction was monitored by measuring the amount of ethylene produced from the substrate by gas chromatography. The extent of the reaction was found to be influenced by the concentration of the chelators, and by iron and manganese.
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two way translocation of cations by the Brown Rot Fungus gloeophyllum trabeum
International Biodeterioration & Biodegradation, 1997Co-Authors: Jon H Connolly, Jody JellisonAbstract:Abstract Wood decay fungi are known to translocate elements, but the details regarding mechanisms and specificity of translocation are not well understood. Gloeophyllum trabeum was grown on blocks of red spruce sapwood that had been previously soaked in salt solutions of CaCl 2 , KCl, NaCl, MgCl 2 and MgSO 4 . Decay-associated weight loss in most salted blocks was decreased by 80%. However, the concentration of the salting cations (Ca, K, Na, Mg) was reduced after 6 weeks of decomposition resulting from fungal activity. During decay, exchangeable ions lost during soaking treatments were replenished simultaneously with the depletion of the excess salting cations. These results indicate that wood decay fungi are capable of selectively translocating metabolically important mineral nutrients from the soil into the wood, while translocating interfering quantities of other cations out of the wood to external depositories.
Takefumi Hattori - One of the best experts on this subject based on the ideXlab platform.
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oxalate efflux transporter from the Brown Rot Fungus fomitopsis palustris
Applied and Environmental Microbiology, 2010Co-Authors: Tomoki Watanabe, Nobukazu Shitan, Shiro Suzuki, Toshiaki Umezawa, Mikio Shimada, Kazufumi Yazaki, Takefumi HattoriAbstract:An oxalate-fermenting Brown Rot Fungus, Fomitopsis palustris, secretes large amounts of oxalic acid during wood decay. Secretion of oxalic acid is indispensable for the degradation of wood cell walls, but almost nothing is known about the transport mechanism by which oxalic acid is secreted from F. palustris hyphal cells. We characterized the mechanism for oxalate transport using membrane vesicles of F. palustris. Oxalate transport in F. palustris was ATP dependent and was strongly inhibited by several inhibitors, such as valinomycin and NH(4)(+), suggesting the presence of a secondary oxalate transporter in this Fungus. We then isolated a cDNA, FpOAR (Fomitopsis palustris oxalic acid resistance), from F. palustris by functional screening of yeast transformants with cDNAs grown on oxalic acid-containing plates. FpOAR is predicted to be a membrane pRotein that possesses six transmembrane domains but shows no similarity with known oxalate transporters. The yeast transformant possessing FpOAR (FpOAR-transformant) acquired resistance to oxalic acid and contained less oxalate than the control transformant. Biochemical analyses using membrane vesicles of the FpOAR-transformant showed that the oxalate transport property of FpOAR was consistent with that observed in membrane vesicles of F. palustris. The quantity of FpOAR transcripts was correlated with increasing oxalic acid accumulation in the culture medium and was induced when exogenous oxalate was added to the medium. These results strongly suggest that FpOAR plays an important role in wood decay by acting as a secondary transporter responsible for secretion of oxalate by F. palustris.
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involvement of fptrp26 a thioredoxin related pRotein in oxalic acid resistance of the Brown Rot Fungus fomitopsis palustris
FEBS Letters, 2007Co-Authors: Tomoki Watanabe, Nobukazu Shitan, Toshiaki Umezawa, Mikio Shimada, Kazufumi Yazaki, Takefumi HattoriAbstract:Brown-Rot Fungus Fomitopsis palustris grows vigorously at high concentrations of oxalic acid (OA), which is fungal metabolite during wood decay. We isolated a cDNA FpTRP26 from F. palustris by functional screening of yeast transformants with cDNAs grown on plates containing OA. FpTRP26 conferred a specific resistance to OA on the transformant. OA-content in transformants grown with 2 mM OA decreased by 65% compared to that of the control. The amount of FpTRP26 transcript in F. palustris amplified with increasing OA-accumulation, and was maintained at high levels even in the stationary phase. Its transcription in F. palustris was inducible in response to exogenously added OA. These results suggest that FpTRP26 is involved in the OA-resistance in F. palustris.
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a metabolic role of the glyoxylate and tricarboxylic acid cycles for development of the copper tolerant Brown Rot Fungus fomitopsis palustris
Fems Microbiology Letters, 2002Co-Authors: Jeongjun Yoon, Takefumi Hattori, Mikio ShimadaAbstract:Fruit bodies of the copper-tolerant Brown-Rot Fungus Fomitopsis palustris were produced in liquid medium for the first time. To induce fruit body formation of this Fungus, it was important to inoculate the liquid medium with mycelia grown on potato dextrose agar plates and also to adjust the initial pH of the medium to 5.0. The metabolic role of the glyoxylate and tricarboxylic acid cycles during fungal development in the liquid culture was investigated in relation to oxalate biosynthesis. The enzymes for the glyoxylate cycle and oxalate biosynthesis in mycelium showed greater activities at the vegetative growth stage than at the fruiting stage. The ratios of isocitrate dehydrogenase activity to isocitrate lyase activity in mycelium were 0.3 and 4.0 at the vegetative and fruiting stage, respectively. Thus, isocitrate lyase of the glyoxylate cycle played a more important role in oxalate synthesis at the earlier stage of the cultivation, whereas isocitrate dehydrogenase played a major role in glutamate synthesis during fruit body formation.
