{"id":{"repo_id":"aachen","oai_identifier":"oai:publications.rwth-aachen.de:49897"},"canonical_url":"https://search.dev.ndltd.org/etd/aachen/oai:publications.rwth-aachen.de:49897","repository":{"repo_id":"aachen","name":"RWTH Aachen University","base_url":"https://publications.rwth-aachen.de/oai2d"},"display":{"title":"Production and characterization of the recombinant wheat chitinase Wch1 and generation of chitin-specific antibodies","abstract":"The purpose of this study was the production and characterization of the wheat chitinase Wch1 with respect to its enzymatic activity as well as the physico-chemical characteristics of generated chitooligomers. The second focus of this work concerned the generation of chitin/chitosan-specific monoclonal antibodies to establish an ELISA assay allowing simple and rapid detection of chitooligosaccharides released from the degradation of chitin substrate in a fermenter. This assay would constitute a valuable tool for monitoring as well as quality assessment of the oligomers throughout the of the production process. The strategy for the generation of chitin-specific antibodies consisted in using peptide mimetics as substitutes of the insoluble and non-immunogenic chitin polymer. For this purpose, three peptide libraries were panned against wheat germ agglutinin (WGA) or chitin-binding domain (CBD). Five heptameric linear peptides were selected after showing specificity to the GlcNAc binding site on both WGA and D. stramonium lectin in an inhibition ELISA. All peptides were coupled to ovalbumin as the protein carrier and injected into mice. Splenocytes from WGA7 G2-immunized mouse were used for generation of hybridoma clones secreting WGA7 G2-specific monoclonal antibodies. Two clones, mAbachG25 and mAbachG211 showed binding to highly acetylated chitosan polymer and were able to bind to the hyphal cell walls of F. graminearum as demonstrated by immunofluorescence microscopy assay. Surprisingly, both antibodies displayed a complete loss of binding capacity towards the polymer but not toward the peptide after affinity purification. This result suggested WGA7 G2 mimicry was based on functional rather than on a structural resemblance of chitin binding to WGA, thus making the possibility of generating strong chitin-specific binders very difficult. Although further structural studies are required to understand the molecular basis of mAbachG25 and mAbachG211 interactions with WGA and chitin, this study is the first report of hybridoma-generated monoclonal antibodies specifically binding to chitin. The cDNA fragment of Wch1 was cloned into three different bacterial expression vectors fused at the N-terminal end of both a his6 tag and a Strep-tag II and as a C-terminal fusion with the maltose binding protein (MBP). Furthermore, the cDNA gene was cloned into plant expression vectors, enabling accumulation of the recombinant protein in the plant cell apoplast (Wch1-apo) and endoplasmatic reticulum (Wch1-ER). Quantification of protein yields as well as enzymatic units produced by each construct lead to the conclusion that the apoplastic space of tobacco leaves was the most appropriate compartment for production of recombinant Wch1. The pH and temperature optima for Wch1-apo enzymatic activity, as estimated with a colorimetric assay using CM-chitin-RBV as substrate were found to be pH 5 and 50ºC, respectively. The enzyme produced oligomers with degrees of polymerization ranging from DP2 to DP>40 but was unable to hydrolyze chitin oligomers that were shorter than the pentamer, thus, indicating an endo-chitinase activity. Moreover, inhibition of mycelia growth of F. graminearum suggests a role of Wch1 in antifungal resistance both by degrading fungal cell wall as well by releasing chitooligosaccharide elicitors of plant defense mechanisms. From the deduced amino acid sequence, Wch1 enzyme was found to belong to family 19 chitinases. The enzymatic mode of action resolved by TLC, HPLC and 1HNMR analysis of released oligomers following hydrolysis of (GlcNAc)6 showed that Wch1 inverts the anomeric conformation of newly formed reducing ends and possessed the subsite structure (-3)(-2)(-1)(+1)(+2)(+3). However, unexpected data from the physico-chemical analysis of oligomers resulting from the extended depolymerization of highly acetylated chitosan revealed an exclusive preference of Wch1 to acetylated units at the (-1) residues. This substrate preference is a feature exclusively attributed to family 18 chitinases and differed strongly with the mechanism described for family 19 chitinases. The data presented in this study clearly demonstrate that Wch1 is a novel family 19 chitinase in its hydrolytic mechanism. Its stability in a wide range of temperatures and pH as well as its high specificity for GlcNAc residues makes it a promising candidate for upscaled enzymatic depolymerization of chitin and chitosan to provide mixtures of oligosaccharides with predicted chemical composition having defined biological functions.","abstract_html":"The purpose of this study was the production and characterization of the wheat chitinase Wch1 with respect to its enzymatic activity as well as the physico-chemical characteristics of generated chitooligomers. The second focus of this work concerned the generation of chitin/chitosan-specific monoclonal antibodies to establish an ELISA assay allowing simple and rapid detection of chitooligosaccharides released from the degradation of chitin substrate in a fermenter. This assay would constitute a valuable tool for monitoring as well as quality assessment of the oligomers throughout the of the production process. The strategy for the generation of chitin-specific antibodies consisted in using peptide mimetics as substitutes of the insoluble and non-immunogenic chitin polymer. For this purpose, three peptide libraries were panned against wheat germ agglutinin (WGA) or chitin-binding domain (CBD). Five heptameric linear peptides were selected after showing specificity to the GlcNAc binding site on both WGA and D. stramonium lectin in an inhibition ELISA. All peptides were coupled to ovalbumin as the protein carrier and injected into mice. Splenocytes from WGA7 G2-immunized mouse were used for generation of hybridoma clones secreting WGA7 G2-specific monoclonal antibodies. Two clones, mAbachG25 and mAbachG211 showed binding to highly acetylated chitosan polymer and were able to bind to the hyphal cell walls of F. graminearum as demonstrated by immunofluorescence microscopy assay. Surprisingly, both antibodies displayed a complete loss of binding capacity towards the polymer but not toward the peptide after affinity purification. This result suggested WGA7 G2 mimicry was based on functional rather than on a structural resemblance of chitin binding to WGA, thus making the possibility of generating strong chitin-specific binders very difficult. Although further structural studies are required to understand the molecular basis of mAbachG25 and mAbachG211 interactions with WGA and chitin, this study is the first report of hybridoma-generated monoclonal antibodies specifically binding to chitin. The cDNA fragment of Wch1 was cloned into three different bacterial expression vectors fused at the N-terminal end of both a his6 tag and a Strep-tag II and as a C-terminal fusion with the maltose binding protein (MBP). Furthermore, the cDNA gene was cloned into plant expression vectors, enabling accumulation of the recombinant protein in the plant cell apoplast (Wch1-apo) and endoplasmatic reticulum (Wch1-ER). Quantification of protein yields as well as enzymatic units produced by each construct lead to the conclusion that the apoplastic space of tobacco leaves was the most appropriate compartment for production of recombinant Wch1. The pH and temperature optima for Wch1-apo enzymatic activity, as estimated with a colorimetric assay using CM-chitin-RBV as substrate were found to be pH 5 and 50ºC, respectively. The enzyme produced oligomers with degrees of polymerization ranging from DP2 to DP>40 but was unable to hydrolyze chitin oligomers that were shorter than the pentamer, thus, indicating an endo-chitinase activity. Moreover, inhibition of mycelia growth of F. graminearum suggests a role of Wch1 in antifungal resistance both by degrading fungal cell wall as well by releasing chitooligosaccharide elicitors of plant defense mechanisms. From the deduced amino acid sequence, Wch1 enzyme was found to belong to family 19 chitinases. The enzymatic mode of action resolved by TLC, HPLC and 1HNMR analysis of released oligomers following hydrolysis of (GlcNAc)6 showed that Wch1 inverts the anomeric conformation of newly formed reducing ends and possessed the subsite structure (-3)(-2)(-1)(+1)(+2)(+3). However, unexpected data from the physico-chemical analysis of oligomers resulting from the extended depolymerization of highly acetylated chitosan revealed an exclusive preference of Wch1 to acetylated units at the (-1) residues. This substrate preference is a feature exclusively attributed to family 18 chitinases and differed strongly with the mechanism described for family 19 chitinases. The data presented in this study clearly demonstrate that Wch1 is a novel family 19 chitinase in its hydrolytic mechanism. Its stability in a wide range of temperatures and pH as well as its high specificity for GlcNAc residues makes it a promising candidate for upscaled enzymatic depolymerization of chitin and chitosan to provide mixtures of oligosaccharides with predicted chemical composition having defined biological functions.","abstract_has_math":false,"creators":["Agdour, Siham"],"institution":"Publikationsserver der RWTH Aachen University","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Fischer, Rainer"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2007,"date_issued":"2007","date_published":"2007","updated_at":"2026-07-30T19:40:16Z","subjects":["info:eu-repo/classification/ddc/570","Chitinase","Antikörper","Chitin","Chitosan","Biowissenschaften, Biologie","family 19","antibodies","peptid mimicry"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112465%22"],"render_values":[{"text":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112465%22","href":"https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112465%22","code":true}]}]},"links":{"outbound_url":"https://publications.rwth-aachen.de/record/49897","outbound_label":"Repository record","outbound_source":"dc:identifier"},"source_record":{"url":"https://publications.rwth-aachen.de/oai2d?verb=GetRecord&metadataPrefix=oai_dc&identifier=oai%3Apublications.rwth-aachen.de%3A49897","prefix":"oai_dc"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Fischer, Rainer"]},{"key":"dc:creator","label":"Author","values":["Agdour, Siham"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:coverage","label":"Dc Coverage","values":["DE"]},{"key":"dc:date","label":"Dc Date","values":["2007"]},{"key":"dc:publisher","label":"Institution","values":["Publikationsserver der RWTH Aachen University"]},{"key":"dc:relation","label":"Dc Relation","values":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-21470"]},{"key":"dc:type","label":"Dc Type","values":["info:eu-repo/semantics/doctoralThesis","info:eu-repo/semantics/publishedVersion"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["info:eu-repo/classification/ddc/570","Chitinase","Antikörper","Chitin","Chitosan","Biowissenschaften, Biologie","family 19","antibodies","peptid mimicry"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["info:eu-repo/semantics/openAccess"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://publications.rwth-aachen.de/record/49897","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112465%22"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["The purpose of this study was the production and characterization of the wheat chitinase Wch1 with respect to its enzymatic activity as well as the physico-chemical characteristics of generated chitooligomers. The second focus of this work concerned the generation of chitin/chitosan-specific monoclonal antibodies to establish an ELISA assay allowing simple and rapid detection of chitooligosaccharides released from the degradation of chitin substrate in a fermenter. This assay would constitute a valuable tool for monitoring as well as quality assessment of the oligomers throughout the of the production process. The strategy for the generation of chitin-specific antibodies consisted in using peptide mimetics as substitutes of the insoluble and non-immunogenic chitin polymer. For this purpose, three peptide libraries were panned against wheat germ agglutinin (WGA) or chitin-binding domain (CBD). Five heptameric linear peptides were selected after showing specificity to the GlcNAc binding site on both WGA and D. stramonium lectin in an inhibition ELISA. All peptides were coupled to ovalbumin as the protein carrier and injected into mice. Splenocytes from WGA7 G2-immunized mouse were used for generation of hybridoma clones secreting WGA7 G2-specific monoclonal antibodies. Two clones, mAbachG25 and mAbachG211 showed binding to highly acetylated chitosan polymer and were able to bind to the hyphal cell walls of F. graminearum as demonstrated by immunofluorescence microscopy assay. Surprisingly, both antibodies displayed a complete loss of binding capacity towards the polymer but not toward the peptide after affinity purification. This result suggested WGA7 G2 mimicry was based on functional rather than on a structural resemblance of chitin binding to WGA, thus making the possibility of generating strong chitin-specific binders very difficult. Although further structural studies are required to understand the molecular basis of mAbachG25 and mAbachG211 interactions with WGA and chitin, this study is the first report of hybridoma-generated monoclonal antibodies specifically binding to chitin. The cDNA fragment of Wch1 was cloned into three different bacterial expression vectors fused at the N-terminal end of both a his6 tag and a Strep-tag II and as a C-terminal fusion with the maltose binding protein (MBP). Furthermore, the cDNA gene was cloned into plant expression vectors, enabling accumulation of the recombinant protein in the plant cell apoplast (Wch1-apo) and endoplasmatic reticulum (Wch1-ER). Quantification of protein yields as well as enzymatic units produced by each construct lead to the conclusion that the apoplastic space of tobacco leaves was the most appropriate compartment for production of recombinant Wch1. The pH and temperature optima for Wch1-apo enzymatic activity, as estimated with a colorimetric assay using CM-chitin-RBV as substrate were found to be pH 5 and 50ºC, respectively. The enzyme produced oligomers with degrees of polymerization ranging from DP2 to DP>40 but was unable to hydrolyze chitin oligomers that were shorter than the pentamer, thus, indicating an endo-chitinase activity. Moreover, inhibition of mycelia growth of F. graminearum suggests a role of Wch1 in antifungal resistance both by degrading fungal cell wall as well by releasing chitooligosaccharide elicitors of plant defense mechanisms. From the deduced amino acid sequence, Wch1 enzyme was found to belong to family 19 chitinases. The enzymatic mode of action resolved by TLC, HPLC and 1HNMR analysis of released oligomers following hydrolysis of (GlcNAc)6 showed that Wch1 inverts the anomeric conformation of newly formed reducing ends and possessed the subsite structure (-3)(-2)(-1)(+1)(+2)(+3). However, unexpected data from the physico-chemical analysis of oligomers resulting from the extended depolymerization of highly acetylated chitosan revealed an exclusive preference of Wch1 to acetylated units at the (-1) residues. This substrate preference is a feature exclusively attributed to family 18 chitinases and differed strongly with the mechanism described for family 19 chitinases. The data presented in this study clearly demonstrate that Wch1 is a novel family 19 chitinase in its hydrolytic mechanism. Its stability in a wide range of temperatures and pH as well as its high specificity for GlcNAc residues makes it a promising candidate for upscaled enzymatic depolymerization of chitin and chitosan to provide mixtures of oligosaccharides with predicted chemical composition having defined biological functions."]},{"key":"dc:source","label":"Dc Source","values":["Aachen : Publikationsserver der RWTH Aachen University VII, 144 S. : Ill., graph. Darst. (2007). = Aachen, Techn. Hochsch., Diss., 2007"]},{"key":"dc:title","label":"Title","values":["Production and characterization of the recombinant wheat chitinase Wch1 and generation of chitin-specific antibodies"]}]}],"canonical_facts":{"dc:contributor":["Fischer, Rainer"],"dc:coverage":["DE"],"dc:creator":["Agdour, Siham"],"dc:date":["2007"],"dc:description":["The purpose of this study was the production and characterization of the wheat chitinase Wch1 with respect to its enzymatic activity as well as the physico-chemical characteristics of generated chitooligomers. The second focus of this work concerned the generation of chitin/chitosan-specific monoclonal antibodies to establish an ELISA assay allowing simple and rapid detection of chitooligosaccharides released from the degradation of chitin substrate in a fermenter. This assay would constitute a valuable tool for monitoring as well as quality assessment of the oligomers throughout the of the production process. The strategy for the generation of chitin-specific antibodies consisted in using peptide mimetics as substitutes of the insoluble and non-immunogenic chitin polymer. For this purpose, three peptide libraries were panned against wheat germ agglutinin (WGA) or chitin-binding domain (CBD). Five heptameric linear peptides were selected after showing specificity to the GlcNAc binding site on both WGA and D. stramonium lectin in an inhibition ELISA. All peptides were coupled to ovalbumin as the protein carrier and injected into mice. Splenocytes from WGA7 G2-immunized mouse were used for generation of hybridoma clones secreting WGA7 G2-specific monoclonal antibodies. Two clones, mAbachG25 and mAbachG211 showed binding to highly acetylated chitosan polymer and were able to bind to the hyphal cell walls of F. graminearum as demonstrated by immunofluorescence microscopy assay. Surprisingly, both antibodies displayed a complete loss of binding capacity towards the polymer but not toward the peptide after affinity purification. This result suggested WGA7 G2 mimicry was based on functional rather than on a structural resemblance of chitin binding to WGA, thus making the possibility of generating strong chitin-specific binders very difficult. Although further structural studies are required to understand the molecular basis of mAbachG25 and mAbachG211 interactions with WGA and chitin, this study is the first report of hybridoma-generated monoclonal antibodies specifically binding to chitin. The cDNA fragment of Wch1 was cloned into three different bacterial expression vectors fused at the N-terminal end of both a his6 tag and a Strep-tag II and as a C-terminal fusion with the maltose binding protein (MBP). Furthermore, the cDNA gene was cloned into plant expression vectors, enabling accumulation of the recombinant protein in the plant cell apoplast (Wch1-apo) and endoplasmatic reticulum (Wch1-ER). Quantification of protein yields as well as enzymatic units produced by each construct lead to the conclusion that the apoplastic space of tobacco leaves was the most appropriate compartment for production of recombinant Wch1. The pH and temperature optima for Wch1-apo enzymatic activity, as estimated with a colorimetric assay using CM-chitin-RBV as substrate were found to be pH 5 and 50ºC, respectively. The enzyme produced oligomers with degrees of polymerization ranging from DP2 to DP>40 but was unable to hydrolyze chitin oligomers that were shorter than the pentamer, thus, indicating an endo-chitinase activity. Moreover, inhibition of mycelia growth of F. graminearum suggests a role of Wch1 in antifungal resistance both by degrading fungal cell wall as well by releasing chitooligosaccharide elicitors of plant defense mechanisms. From the deduced amino acid sequence, Wch1 enzyme was found to belong to family 19 chitinases. The enzymatic mode of action resolved by TLC, HPLC and 1HNMR analysis of released oligomers following hydrolysis of (GlcNAc)6 showed that Wch1 inverts the anomeric conformation of newly formed reducing ends and possessed the subsite structure (-3)(-2)(-1)(+1)(+2)(+3). However, unexpected data from the physico-chemical analysis of oligomers resulting from the extended depolymerization of highly acetylated chitosan revealed an exclusive preference of Wch1 to acetylated units at the (-1) residues. This substrate preference is a feature exclusively attributed to family 18 chitinases and differed strongly with the mechanism described for family 19 chitinases. The data presented in this study clearly demonstrate that Wch1 is a novel family 19 chitinase in its hydrolytic mechanism. Its stability in a wide range of temperatures and pH as well as its high specificity for GlcNAc residues makes it a promising candidate for upscaled enzymatic depolymerization of chitin and chitosan to provide mixtures of oligosaccharides with predicted chemical composition having defined biological functions."],"dc:identifier":["https://publications.rwth-aachen.de/record/49897","https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-112465%22"],"dc:language":["eng"],"dc:publisher":["Publikationsserver der RWTH Aachen University"],"dc:relation":["info:eu-repo/semantics/altIdentifier/urn/urn:nbn:de:hbz:82-opus-21470"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:source":["Aachen : Publikationsserver der RWTH Aachen University VII, 144 S. : Ill., graph. Darst. (2007). = Aachen, Techn. 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