Sona Garajova - Academia.edu (original) (raw)

Papers by Sona Garajova

Research paper thumbnail of MOESM1 of Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6

Additional file 1. Production, purification and characterization of lytic polysaccharide monooxyg... more Additional file 1. Production, purification and characterization of lytic polysaccharide monooxygenases secreted by Pestalotiopsis sp. NCi6.

Research paper thumbnail of Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type

Scientific Reports

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of th... more Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery. Plant cell walls constitute the largest renewable source of biomass on Earth that can supply environmental benefits for the production of fuel, chemicals and materials. They are composed by lignocellulose made from three main polymers (cellulose, hemicelluloses, lignin) assembling as a network whose structural and chemical complexity hampers hydrolysis of cellulose by enzymes and microorganisms 1. In addition, cell walls display high variability depending on genetic and environmental factors, as well as plant tissue and cell types 2-4. Understanding plant cell wall complexity during lignocellulosic bioconversion is therefore important to identify critical features impacting hydrolysis, for optimising pretreatments of biomass 5 and enzyme cocktails 6. Investigation of the dynamics of lignocellulose hydrolysis requires physicochemical characterization and multiscale visualization 7,8. Combined approach using multiple microscopic techniques including scanning electron microscopy, atomic force microscopy, light microscopy, immunocytochemistry and microspectrometry can be used to monitor microstructural and topochemical heterogeneity of plant cell walls and their recalcitrance at tissue, cell and subcellular levels 9-15. In addition, spatial and temporal imaging of enzymes distribution within lignocellulose substrates can be carried out by means of immunoprobe or fluorescent labelled protein 16-18 to study accessibility at the molecular scale 19,20. However real time imaging of cell wall microstructure and enzyme distribution during bioconversion still remains challenging. Enzymatic degradation of cellulose and hemicelluloses involves several types of enzymes, namely glycoside hydrolases that work synergistically 21,22. To overcome the recalcitrance of plant polysaccharides, filamentous fungi and bacteria secrete lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of glycoside bonds 23-25. In industrial processes, addition of LPMOs to cellulolytic cocktails leads to the reduction of the enzyme loading required for efficient saccharification of lignocellulosic biomass 26. These powerful enzymes are copper-containing enzymes classified within the auxiliary activity (AA) class of the CAZy database (www.cazy. org 27) in AA9-AA11 and AA13 families. AA9 LPMOs are mostly active on cellulose but some have been shown to act on xyloglucan and glucomannan 25,28. Despite their recognized boosting effect on biomass hydrolysis, AA9 LPMOs activity has been essentially investigated on model substrates with only sparse studies focusing on the insoluble fraction of the substrate that show their disruptive action at the surface of cellulosic fibers 29-31. To our

Research paper thumbnail of Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

Scientific reports, Jan 10, 2017

Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakd... more Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LP...

Research paper thumbnail of Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

Scientific Reports, 2016

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of ly... more The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

Research paper thumbnail of Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6

Biotechnology for biofuels, 2016

Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" e... more Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stabilit...

Research paper thumbnail of Incorporation of β-(1,6)-linked glucooligosaccharides (pustulooligosaccharides) into plant cell wall structures

Chemical Papers, 2012

Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotran... more Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of X...

Research paper thumbnail of Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Carbohydrate Research, 2011

Research paper thumbnail of Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

Biologia, 2009

The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate p... more The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others, purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol). The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.

Research paper thumbnail of Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: Isolation and characterization of the major form

Plant Physiology and …, 2010

Research paper thumbnail of Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts

Glycoconjugate Journal, 2010

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleav... more Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during

Research paper thumbnail of Characterization of two partially purified xyloglucan endotransglycosylases from parsley (Petroselinum crispum) roots

Biologia, 2008

Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from t... more Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from the protein mixture obtained from parsley roots and partially characterized. Both forms were glycoproteins differing in their specific activities but other features were almost the same. Activity and stability of both enzymes in broad pH region were observed with two pH optima, one at acidic pH (5.8) and the second one at basic pH (8.8). The enzymes behaved as typical transglycosylases since no activity was observed in the absence of xyloglucan oligosaccharides in the viscometric assay. Small hetero-transglycosylating activities were observed when hydroxyethyl-or carboxymethyl-celluloses instead of xyloglucan as donor substrate were used as well as when cello-oligosaccharides instead of xyloglucan oligosaccharides were used as the acceptor substrate.

Research paper thumbnail of MOESM1 of Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6

Additional file 1. Production, purification and characterization of lytic polysaccharide monooxyg... more Additional file 1. Production, purification and characterization of lytic polysaccharide monooxygenases secreted by Pestalotiopsis sp. NCi6.

Research paper thumbnail of Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type

Scientific Reports

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of th... more Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery. Plant cell walls constitute the largest renewable source of biomass on Earth that can supply environmental benefits for the production of fuel, chemicals and materials. They are composed by lignocellulose made from three main polymers (cellulose, hemicelluloses, lignin) assembling as a network whose structural and chemical complexity hampers hydrolysis of cellulose by enzymes and microorganisms 1. In addition, cell walls display high variability depending on genetic and environmental factors, as well as plant tissue and cell types 2-4. Understanding plant cell wall complexity during lignocellulosic bioconversion is therefore important to identify critical features impacting hydrolysis, for optimising pretreatments of biomass 5 and enzyme cocktails 6. Investigation of the dynamics of lignocellulose hydrolysis requires physicochemical characterization and multiscale visualization 7,8. Combined approach using multiple microscopic techniques including scanning electron microscopy, atomic force microscopy, light microscopy, immunocytochemistry and microspectrometry can be used to monitor microstructural and topochemical heterogeneity of plant cell walls and their recalcitrance at tissue, cell and subcellular levels 9-15. In addition, spatial and temporal imaging of enzymes distribution within lignocellulose substrates can be carried out by means of immunoprobe or fluorescent labelled protein 16-18 to study accessibility at the molecular scale 19,20. However real time imaging of cell wall microstructure and enzyme distribution during bioconversion still remains challenging. Enzymatic degradation of cellulose and hemicelluloses involves several types of enzymes, namely glycoside hydrolases that work synergistically 21,22. To overcome the recalcitrance of plant polysaccharides, filamentous fungi and bacteria secrete lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of glycoside bonds 23-25. In industrial processes, addition of LPMOs to cellulolytic cocktails leads to the reduction of the enzyme loading required for efficient saccharification of lignocellulosic biomass 26. These powerful enzymes are copper-containing enzymes classified within the auxiliary activity (AA) class of the CAZy database (www.cazy. org 27) in AA9-AA11 and AA13 families. AA9 LPMOs are mostly active on cellulose but some have been shown to act on xyloglucan and glucomannan 25,28. Despite their recognized boosting effect on biomass hydrolysis, AA9 LPMOs activity has been essentially investigated on model substrates with only sparse studies focusing on the insoluble fraction of the substrate that show their disruptive action at the surface of cellulosic fibers 29-31. To our

Research paper thumbnail of Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

Scientific reports, Jan 10, 2017

Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakd... more Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LP...

Research paper thumbnail of Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

Scientific Reports, 2016

The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of ly... more The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi.

Research paper thumbnail of Salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus Pestalotiopsis sp. NCi6

Biotechnology for biofuels, 2016

Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" e... more Lytic polysaccharide monooxygenases (LPMOs) belong to the "auxiliary activities (AA)" enzyme class of the CAZy database. They are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. To date, most LPMOs have been characterized from terrestrial fungi, but novel fungal LPMOs isolated from more extreme environments such as an estuary mangrove ecosystem could offer enzymes with unique properties in terms of salt tolerance and higher stability under harsh condition. Two LPMOs secreted by the mangrove-associated fungus Pestalotiopsis sp. NCi6 (PsLPMOA and PsLPMOB) were expressed in the yeast Pichia pastoris and produced in a bioreactor with >85 mg L(-1) for PsLPMOA and >260 mg L(-1) for PsLPMOB. Structure-guided homology modeling of the PsLPMOs showed a high abundance of negative surface charges, enabling enhanced protein stabilit...

Research paper thumbnail of Incorporation of β-(1,6)-linked glucooligosaccharides (pustulooligosaccharides) into plant cell wall structures

Chemical Papers, 2012

Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotran... more Protein extract of germinating nasturtium (Tropaeolum majus) seeds containing xyloglucan endotransglycosylase (xyloglucan xyloglucosyl transferase, EC 2.4.1.207, abbreviated XET) exhibited the heterotransglycosylating activity with donor/acceptor substrate pair xyloglucan/sulphorhodamine labelled pustulooligosaccharides (XG/PUOS-SR) in a dot blot assay. The heterotransglycosylating activity was confirmed by the substrate-product changes during transglycosylation by HPLC size-exclusion chromatography. Another donor substrate capable of being coupled with PUOS-SR was cellulose, probably owing to its structural similarity to xyloglucan. Surprisingly, microscopic comparison of the incorporation of the labelled xyloglucan nonasaccharide XGO9-SR (specific substrate for XET) and PUOS-SR into the cell wall structures clearly showed differences in their binding to specific cell structures: the primary cell wall and the plasma membrane. These findings indicate the existence in nasturtium of X...

Research paper thumbnail of Effect of the label of oligosaccharide acceptors on the kinetic parameters of nasturtium seed xyloglucan endotransglycosylase (XET)

Carbohydrate Research, 2011

Research paper thumbnail of Oligogalacturonate hydrolase with unique substrate preference from the pulp of parsley roots

Biologia, 2009

The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate p... more The main form of pectate hydrolases in the cell wall of parsley roots showed a unique substrate preference of a plant exopolygalacturonase because it clearly preferred the substrates with degree of polymerization about 10. This form was separated from the others, purified and characterized. Enzyme exhibited sharp pH optimum corresponding to pH 4.7, molecular mass 53.5 kDa, and isoelectric point 5.3. It was stable at 50°C in 2-h assay and had optimum of temperature at 60°C (activation energy being 37.0 kJ/mol). The interaction with concanavalin A indicated the glycosylation of enzyme. Substrates were cleaved from the non-reducing end.

Research paper thumbnail of Xyloglucan endotransglycosylases (XETs) from germinating nasturtium (Tropaeolum majus) seeds: Isolation and characterization of the major form

Plant Physiology and …, 2010

Research paper thumbnail of Polysaccharide microarrays for high-throughput screening of transglycosylase activities in plant extracts

Glycoconjugate Journal, 2010

Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleav... more Polysaccharide transglycosylases catalyze disproportionation of polysaccharide molecules by cleaving glycosidic linkages in polysaccharide chains and transferring their cleaved portions to hydroxyl groups at the non-reducing ends of other polysaccharide or oligosaccharide molecules. In plant cell walls, transglycosylases have a potential to catalyze both cross-linking of polysaccharide molecules and grafting of newly arriving polysaccharide molecules into the cell wall structure during

Research paper thumbnail of Characterization of two partially purified xyloglucan endotransglycosylases from parsley (Petroselinum crispum) roots

Biologia, 2008

Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from t... more Two forms of xyloglucan endotransglycosylase differing in isoelectric points were isolated from the protein mixture obtained from parsley roots and partially characterized. Both forms were glycoproteins differing in their specific activities but other features were almost the same. Activity and stability of both enzymes in broad pH region were observed with two pH optima, one at acidic pH (5.8) and the second one at basic pH (8.8). The enzymes behaved as typical transglycosylases since no activity was observed in the absence of xyloglucan oligosaccharides in the viscometric assay. Small hetero-transglycosylating activities were observed when hydroxyethyl-or carboxymethyl-celluloses instead of xyloglucan as donor substrate were used as well as when cello-oligosaccharides instead of xyloglucan oligosaccharides were used as the acceptor substrate.