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Yves Briers - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the substrate specificity of α-L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Analysis of the substrate specificity of -L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
Satoshi Kaneko - One of the best experts on this subject based on the ideXlab platform.
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crystal structure and characterization of the glycoside hydrolase family 62 α l Arabinofuranosidase from streptomyces coelicolor
Journal of Biological Chemistry, 2014Co-Authors: Tomoko Maehara, Koichi Harazono, M Michikawa, Zui Fujimoto, Hitomi Ichinose, Satoshi KanekoAbstract:α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-Arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-Arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp202 and Glu361 were catalytic residues, and Trp270, Tyr461, and Asn462 were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn462 and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.
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the structure and function of an arabinan specific alpha 1 2 Arabinofuranosidase identified from screening the activities of bacterial gh43 glycoside hydrolases
Journal of Biological Chemistry, 2011Co-Authors: Alan Cartmell, Hitomi Ichinose, Satoshi Kaneko, Lauren S Mckee, Maria J Pena, Johan Larsbrink, Harry Brumer, Richard J Lewis, Anders Viksonielsen, Harry J. GilbertAbstract:Abstract Reflecting the diverse chemistry of plant cell walls, microorganisms that degrade these composite structures synthesize an array of glycoside hydrolases. These enzymes are organized into sequence-, mechanism-, and structure-based families. Genomic data have shown that several organisms that degrade the plant cell wall contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. Here we report the biochemical properties of the GH43 enzymes of a saprophytic soil bacterium, Cellvibrio japonicus, and a human colonic symbiont, Bacteroides thetaiotaomicron. The data show that C. japonicus uses predominantly exo-acting enzymes to degrade arabinan into arabinose, whereas B. thetaiotaomicron deploys a combination of endo- and side chain-cleaving glycoside hydrolases. Both organisms, however, utilize an arabinan-specific α-1,2-Arabinofuranosidase in the degradative process, an activity that has not previously been reported. The enzyme can cleave α-1,2-arabinofuranose decorations in single or double substitutions, the latter being recalcitrant to the action of other Arabinofuranosidases. The crystal structure of the C. japonicus arabinan-specific α-1,2-Arabinofuranosidase, CjAbf43A, displays a five-bladed β-propeller fold. The specificity of the enzyme for arabinan is conferred by a surface cleft that is complementary to the helical backbone of the polysaccharide. The specificity of CjAbf43A for α-1,2-l-arabinofuranose side chains is conferred by a polar residue that orientates the arabinan backbone such that O2 arabinose decorations are directed into the active site pocket. A shelflike structure adjacent to the active site pocket accommodates O3 arabinose side chains, explaining how the enzyme can target O2 linkages that are components of single or double substitutions.
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crystal structure of an exo 1 5 α l Arabinofuranosidase from streptomyces avermitilis provides insights into the mechanism of substrate discrimination between exo and endo type enzymes in glycoside hydrolase family 43
Journal of Biological Chemistry, 2010Co-Authors: Zui Fujimoto, Tomoko Maehara, Mariko Honda, Hitomi Ichinose, Motomitsu Kitaoka, Satoshi KanekoAbstract:Exo-1,5-α-l-Arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-Arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.
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an α l Arabinofuranosidase β d xylosidase from immature seeds of radish raphanus sativus l
Journal of Experimental Botany, 2006Co-Authors: Toshihisa Kotake, Satoshi Kaneko, Koji Tsuchiya, Tsutomu Aohara, Tomoyuki Konishi, Kiyohiko Igarashi, Masahiro Samejima, Yoichi TsumurayaAbstract:The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for their physiological functions and undergo rapid turnover in vivo. Degradation of the carbohydrate moieties of AGPs seems to occur by concerted action of several glycosidases, among them a-L-Arabinofuranosidase, b-Dgalactosidase, and b-D-glucuronidase. Here, a bifunctional a-L-Arabinofuranosidase/b-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses a-L-arabinofuranosyl residues of the carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The gene, designated RsAraf1 ,c ontained an open reading frame of 2343 bp (780 amino acids), including a putative signal sequence (33 amino acids) at the N-terminus. RsAraf1 is highly similar to barley a-L-Arabinofuranosidase/b-D-xylosidases and belongs to family 3 of the glycosyl hydrolases based on sequence homology. Southern blot analysis revealed that several related genes exist in the radish genome. RsAraf1 is expressed throughout seed development and weakly expressed in young seedlings. It was found that a-L-Arabinofuranosidase activity in a cell-wall protein fraction prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1 was significantly higher than that in a wild-type protein fraction; the crude enzyme preparation released L-arabinose from radish AGPs as well as a-(1!5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl residues in the cell walls of transgenic plants was significantly decreased. These results indicate that RsAraf1 encodes a bifunctional a-L-Arabinofuranosidase/b-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.
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An α-L-Arabinofuranosidase/β-D-xylosidase from immature seeds of radish (Raphanus sativus L.)
Journal of experimental botany, 2006Co-Authors: Toshihisa Kotake, Satoshi Kaneko, Koji Tsuchiya, Tsutomu Aohara, Tomoyuki Konishi, Kiyohiko Igarashi, Masahiro Samejima, Yoichi TsumurayaAbstract:The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for their physiological functions and undergo rapid turnover in vivo. Degradation of the carbohydrate moieties of AGPs seems to occur by concerted action of several glycosidases, among them a-L-Arabinofuranosidase, b-Dgalactosidase, and b-D-glucuronidase. Here, a bifunctional a-L-Arabinofuranosidase/b-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses a-L-arabinofuranosyl residues of the carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The gene, designated RsAraf1 ,c ontained an open reading frame of 2343 bp (780 amino acids), including a putative signal sequence (33 amino acids) at the N-terminus. RsAraf1 is highly similar to barley a-L-Arabinofuranosidase/b-D-xylosidases and belongs to family 3 of the glycosyl hydrolases based on sequence homology. Southern blot analysis revealed that several related genes exist in the radish genome. RsAraf1 is expressed throughout seed development and weakly expressed in young seedlings. It was found that a-L-Arabinofuranosidase activity in a cell-wall protein fraction prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1 was significantly higher than that in a wild-type protein fraction; the crude enzyme preparation released L-arabinose from radish AGPs as well as a-(1!5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl residues in the cell walls of transgenic plants was significantly decreased. These results indicate that RsAraf1 encodes a bifunctional a-L-Arabinofuranosidase/b-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.
Emma R Master - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the substrate specificity of α-L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Analysis of the substrate specificity of -L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Constructing Arabinofuranosidases for dual arabinoxylan debranching activity
Biotechnology and bioengineering, 2017Co-Authors: Weijun Wang, Maija Tenkanen, Nikola Andric, Cody Sarch, Bruno T. Silva, Emma R MasterAbstract:Enzymatic conversion of arabinoxylan requires α-L-Arabinofuranosidases able to remove α-L-arabinofuranosyl residues (α-L-Araf) from both mono- and double-substituted D-xylopyranosyl residues (Xylp) in xylan (i.e., AXH-m and AXH-d activity). Herein, SthAbf62A (a family GH62 α-L-Arabinofuranosidase with AXH-m activity) and BadAbf43A (a family GH43 α-L-Arabinofuranosidase with AXH-d3 activity), were fused to create SthAbf62A_BadAbf43A and BadAbf43A_SthAbf62A. Both fusion enzymes displayed dual AXH-m,d and synergistic activity toward native, highly branched wheat arabinoxylan (WAX). When using a customized arabinoxylan substrate comprising mainly α-(1 → 3)-L-Araf and α-(1 → 2)-L-Araf substituents attached to disubstituted Xylp (d-2,3-WAX), the specific activity of the fusion enzymes was twice that of enzymes added as separate proteins. Moreover, the SthAbf62A_BadAbf43A fusion removed 83% of all α-L-Araf from WAX after a 20 hr treatment. 1 H NMR analyses further revealed differences in SthAbf62A_BadAbf43 rate of removal of specific α-L-Araf substituents from WAX, where 9.4 times higher activity was observed toward d-α-(1 → 3)-L-Araf compared to m-α-(1 → 3)-L-Araf positions.
Jean-guy Berrin - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the substrate specificity of α-L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Analysis of the substrate specificity of -L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Comparative analyses of Podospora anserina secretomes reveal a large array of lignocellulose-active enzymes.
Applied Microbiology and Biotechnology, 2014Co-Authors: Laetitia Poidevin, Jean-guy Berrin, Chloé Bennati-granier, Anthony Levasseur, Isabelle Herpoël-gimbert, Didier Chevret, Pedro M. Coutinho, Bernard Henrissat, Senta Heiss-blanquet, Eric RecordAbstract:This study investigated the enzymatic capacity of several P. anserina secretomes obtained via induction by various carbon sources. P. anserina secretomes displayed similar cellulase, xylanase and pectinase activity and greater Arabinofuranosidase, arabinanase and galactanase activity than the levels found in T reesei. The secretomes were also tested for their capacity to supplement a T. reesei cocktail. Four of them improved the saccharification yield of steam exploded wheat straw by 14 to 48%. Our data strongly suggests that part of the improvement was due to complementary enzymes produced by P. anserina. Proteomic analysis of secretomes produced with Avicel and sugar beet pulp provided novel insights into the enzymatic arsenal of this fungus producing cellulases from families GH6 and 7 as well as producing GH10 xylanases, CE1 esterases, GH43 Arabinofuranosidases and AA1 laccase-like multicopper oxidases. A striking point was the extensive production of lytic polysaccharide monooxygenases specifically in sugar beet-induced cultures.
Kim Kataja - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the substrate specificity of α-L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria João Maurício Da Fonseca, Edita Jurak, Kim Kataja, A Van Landschoot, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different α-L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two α-L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 α-L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 α-L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two α-L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 α-L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted β-D-xylosyl residues, whereas a GH43 α-L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.
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Analysis of the substrate specificity of -L-Arabinofuranosidases by DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis
Applied Microbiology and Biotechnology, 2018Co-Authors: Maria Joao Mauricio Da Fonseca, Edita Jurak, Kim Kataja, Tom Desmet, Emma R Master, Jean-guy Berrin, Ingeborg Stals, Anita Van Landschoot, Yves BriersAbstract:Carbohydrate-active enzyme discovery is often not accompanied by experimental validation, demonstrating the need for techniques to analyze substrate specificities of carbohydrate-active enzymes in an efficient manner. DNA sequencer-aided fluorophore-assisted carbohydrate electrophoresis (DSA-FACE) is utmost appropriate for the analysis of glycoside hydrolases that have complex substrate specificities. DSA-FACE is demonstrated here to be a highly convenient method for the precise identification of the specificity of different -L-Arabinofuranosidases for (arabino)xylo-oligosaccharides ((A)XOS). The method was validated with two -L-Arabinofuranosidases (EC 3.2.1.55) with well-known specificity, specifically a GH62 -L-Arabinofuranosidase from Aspergillus nidulans (AnAbf62A-m2,3) and a GH43 -L-Arabinofuranosidase from Bifidobacterium adolescentis (BaAXH-d3). Subsequently, application of DSA-FACE revealed the AXOS specificity of two -L-Arabinofuranosidases with previously unknown AXOS specificities. PaAbf62A, a GH62 -L-Arabinofuranosidase from Podospora anserina strain S mat+, was shown to target the O-2 and the O-3 arabinofuranosyl monomers as side chain from mono-substituted -D-xylosyl residues, whereas a GH43 -L-Arabinofuranosidase from a metagenomic sample (AGphAbf43) only removes an arabinofuranosyl monomer from the smallest AXOS tested. DSA-FACE excels ionic chromatography in terms of detection limit for (A)XOS (picomolar sensitivity), hands-on and analysis time, and the analysis of the degree of polymerization and binding site of the arabinofuranosyl substituent.