{"id":{"repo_id":"helsinki","oai_identifier":"oai:helda.helsinki.fi:10138/627069"},"canonical_url":"https://search.dev.ndltd.org/etd/helsinki/oai:helda.helsinki.fi:10138/627069","repository":{"repo_id":"helsinki","name":"University of Helsinki","base_url":"https://helda.helsinki.fi/server/oai/request"},"display":{"title":"Production and characterization of two AA3 enzymes from Arabidopsis thaliana","abstract":"Enzymes involved in the synthesis and degradation of complex carbohydrates are classified in the carbohydrate-active enzyme database (CAZy). One of the classes, auxiliary activities (AA), consists of redox enzymes that assist other carbohydrate-active enzymes. Within this class, the AA3 family, contains FAD-dependent oxidoreductases with diverse catalytic roles in fungi, where they participate in lignocellulose degradation. However, the biological functions of their plant counterparts remain largely unknown, and only a few plant AA3 enzymes have been characterized to date. As current industrial wood-processing methods do not fully utilize the potential of lignocellulosic biomass, there is growing interest in discovering new enzymes that could enhance lignocellulose conversion into valuable products. Plant AA3 oxidoreductases may represent an underexplored source of such biocatalysts, but a deeper understanding of their substrate specificities and catalytic properties is still required. This study presents the production and first characterization of two Arabidopsis thaliana AA3 enzymes, AtAA3C and AtAA3G. The enzymes were heterologously expressed in Pichia pastoris and purified using affinity chromatography. The activities of AtAA3C and AtAA3G were screened against a wide range of substrates using a high-throughput microplate assay. AtAA3C exhibited both oxidase and dehydrogenase activities toward aryl alcohols, with cinnamyl alcohol as its preferred substrate. It also displayed oxidase activity toward primary alcohols. AtAA3G exhibited oxidase activity, with the highest activity observed toward the long-chain alcohols 1-decanol and 1-dodecanol. The catalytic properties of these oxidases were further determined under optimal pH conditions using their preferred substrates. Compared to other characterized plant and fungal AA3 enzymes, AtAA3C and AtAA3G showed relatively low activities and catalytic velocities. This may reflect differences in their biological roles or that their true physiological substrates were not included in the tested substrate sets. Overall, these findings expand the current knowledge of plant AA3 enzymes and provide a basis for future studies.","abstract_html":"Enzymes involved in the synthesis and degradation of complex carbohydrates are classified in the carbohydrate-active enzyme database (CAZy). One of the classes, auxiliary activities (AA), consists of redox enzymes that assist other carbohydrate-active enzymes. Within this class, the AA3 family, contains FAD-dependent oxidoreductases with diverse catalytic roles in fungi, where they participate in lignocellulose degradation. However, the biological functions of their plant counterparts remain largely unknown, and only a few plant AA3 enzymes have been characterized to date. As current industrial wood-processing methods do not fully utilize the potential of lignocellulosic biomass, there is growing interest in discovering new enzymes that could enhance lignocellulose conversion into valuable products. Plant AA3 oxidoreductases may represent an underexplored source of such biocatalysts, but a deeper understanding of their substrate specificities and catalytic properties is still required. This study presents the production and first characterization of two Arabidopsis thaliana AA3 enzymes, AtAA3C and AtAA3G. The enzymes were heterologously expressed in Pichia pastoris and purified using affinity chromatography. The activities of AtAA3C and AtAA3G were screened against a wide range of substrates using a high-throughput microplate assay. AtAA3C exhibited both oxidase and dehydrogenase activities toward aryl alcohols, with cinnamyl alcohol as its preferred substrate. It also displayed oxidase activity toward primary alcohols. AtAA3G exhibited oxidase activity, with the highest activity observed toward the long-chain alcohols 1-decanol and 1-dodecanol. The catalytic properties of these oxidases were further determined under optimal pH conditions using their preferred substrates. Compared to other characterized plant and fungal AA3 enzymes, AtAA3C and AtAA3G showed relatively low activities and catalytic velocities. This may reflect differences in their biological roles or that their true physiological substrates were not included in the tested substrate sets. Overall, these findings expand the current knowledge of plant AA3 enzymes and provide a basis for future studies.","abstract_has_math":false,"creators":["Kiuru, Silja"],"institution":"Helsingin yliopisto","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2026,"date_issued":"2026-01-29","date_published":"2026-01-29","updated_at":"2026-07-27T19:56:25Z","subjects":["Arabidopsis thaliana","AA3","AtAA3C","AtAA3G","oxidoreductase","enzyme characterization","heterologous expression","enzymes"],"languages":["eng"],"rights":["CC BY-NC-ND 4.0"],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10138/627069","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Kiuru, Silja"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2026-01-29T06:21:25Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2026-01-29T06:21:25Z"]},{"key":"dc:date.issued","label":"Date","values":["2026-01-29"]},{"key":"dc:publisher","label":"Institution","values":["Helsingin yliopisto","University of Helsinki","Helsingfors universitet"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Arabidopsis thaliana","AA3","AtAA3C","AtAA3G","oxidoreductase","enzyme characterization","heterologous expression","enzymes"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["CC BY-NC-ND 4.0"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10138/627069"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Enzymes involved in the synthesis and degradation of complex carbohydrates are classified in the carbohydrate-active enzyme database (CAZy). One of the classes, auxiliary activities (AA), consists of redox enzymes that assist other carbohydrate-active enzymes. Within this class, the AA3 family, contains FAD-dependent oxidoreductases with diverse catalytic roles in fungi, where they participate in lignocellulose degradation. However, the biological functions of their plant counterparts remain largely unknown, and only a few plant AA3 enzymes have been characterized to date. As current industrial wood-processing methods do not fully utilize the potential of lignocellulosic biomass, there is growing interest in discovering new enzymes that could enhance lignocellulose conversion into valuable products. Plant AA3 oxidoreductases may represent an underexplored source of such biocatalysts, but a deeper understanding of their substrate specificities and catalytic properties is still required. This study presents the production and first characterization of two Arabidopsis thaliana AA3 enzymes, AtAA3C and AtAA3G. The enzymes were heterologously expressed in Pichia pastoris and purified using affinity chromatography. The activities of AtAA3C and AtAA3G were screened against a wide range of substrates using a high-throughput microplate assay. AtAA3C exhibited both oxidase and dehydrogenase activities toward aryl alcohols, with cinnamyl alcohol as its preferred substrate. It also displayed oxidase activity toward primary alcohols. AtAA3G exhibited oxidase activity, with the highest activity observed toward the long-chain alcohols 1-decanol and 1-dodecanol. The catalytic properties of these oxidases were further determined under optimal pH conditions using their preferred substrates. Compared to other characterized plant and fungal AA3 enzymes, AtAA3C and AtAA3G showed relatively low activities and catalytic velocities. This may reflect differences in their biological roles or that their true physiological substrates were not included in the tested substrate sets. Overall, these findings expand the current knowledge of plant AA3 enzymes and provide a basis for future studies.","CAZy (carbohydrate-active enzymes) on entsyymitietokanta, johon kootut entsyymit osallistuvat monimutkaisten hiilihydraattirakenteiden synteesiin, muokkaukseen ja hajotukseen. Nämä entsyymit jaetaan useaan luokkaan niiden sekvenssien sekä toiminnan mukaan. Yksi luokista on AA (auxiliary activities), jonka entsyymit osallistuvat muiden CAZy-entsyymien avustamiseen. AA3-luokan tunnetut entsyymit hapettavat hiilihydraatteja tai alkoholeja. Sienten AA3-entsyymien tiedetään osallistuvan kasvien lignoselluloosan hajotukseen, mutta kasvien vastaavien AA3-entsyymien tehtäviä ei vielä tunneta hyvin, ja vain joitain kasvien AA3-entsyymeitä on karakterisoitu. Uudenlaisilla AA3- entsyymeillä voisi olla sovelluskohteita esimerkiksi puunjalostusteollisuudessa, ligniinin ja hemiselluloosan hyödyntämisessä ja muokkaamisessa arvokkaiksi tuotteiksi. Kasvien AA3-entsyymien joukosta voi löytyä tähän tarkoitukseen sopivia entsyymejä, mutta niiden substraattispesifisyydestä sekä katalyyttisistä ominaisuuksista tarvitaan vielä lisää tietoa. Tämän tutkimuksen tarkoituksena oli tuottaa ja karakterisoida ensimmäistä kertaa kaksi Arabidopsis thaliana-lituruohon AA3-entsyymiä, AtAA3C ja AtAA3G. Entsyymit tuotettiin Pichia pastoris-hiivassa, minkä jälkeen ne puhdistettiin hyödyntäen affiniteettikromatografiaa. Entsyymien aktiivisuuksien analysoinnissa käytettiin laajaa joukkoa erilaisia substraatteja ja näiden analyysien tulokset osoittivat AtAA3C-entsyymillä olevan sekä oksidaasi- että dehydrogenaasiaktiivisuutta aryylialkoholeja kohtaan. Lisäksi AtAA3C-entsyymin havaittiin hapettavan primaarisia alkoholeja. Paras substraatti aryylialkoholeista oli kanelialkoholi ja primaarisista alkoholeista 1-dodekanoli. Tulokset osoittivat AtAA3G-entsyymillä olevan ainoastaan oksidaasiaktiivisuutta, ja se hapetti parhaiten pitkäketjuisia primaarisia alkoholeja 1-dekanolia ja 1-dodekanolia. Entsyymeille parhaiten sopivien substraattien löydyttyä määritettiin entsyymien katalyyttiset ominaisuudet. AtAA3C- ja AtAA3G-entsyymien aktiivisuudet tutkituilla substraateilla olivat alhaisemmat kuin aikaisemmin julkaistuilla kasvi- ja sienientsyymeillä. Ero voi kertoa entsyymien biologisista tehtävistä tai siitä, että tutkimuksessa käytetyt substraatit eivät vastanneet tutkittujen entsyymien luonnollisia substraatteja. Työn tulokset antavat lisää tietoa kasvien AA3-entsyymeistä ja tarjoavat perustan tuleville tutkimuksille."]},{"key":"dc:title","label":"Title","values":["Production and characterization of two AA3 enzymes from Arabidopsis thaliana"]}]}],"canonical_facts":{"dc:creator":["Kiuru, Silja"],"dc:date.accessioned":["2026-01-29T06:21:25Z"],"dc:date.available":["2026-01-29T06:21:25Z"],"dc:date.issued":["2026-01-29"],"dc:description.abstract":["Enzymes involved in the synthesis and degradation of complex carbohydrates are classified in the carbohydrate-active enzyme database (CAZy). One of the classes, auxiliary activities (AA), consists of redox enzymes that assist other carbohydrate-active enzymes. Within this class, the AA3 family, contains FAD-dependent oxidoreductases with diverse catalytic roles in fungi, where they participate in lignocellulose degradation. However, the biological functions of their plant counterparts remain largely unknown, and only a few plant AA3 enzymes have been characterized to date. As current industrial wood-processing methods do not fully utilize the potential of lignocellulosic biomass, there is growing interest in discovering new enzymes that could enhance lignocellulose conversion into valuable products. Plant AA3 oxidoreductases may represent an underexplored source of such biocatalysts, but a deeper understanding of their substrate specificities and catalytic properties is still required. This study presents the production and first characterization of two Arabidopsis thaliana AA3 enzymes, AtAA3C and AtAA3G. The enzymes were heterologously expressed in Pichia pastoris and purified using affinity chromatography. The activities of AtAA3C and AtAA3G were screened against a wide range of substrates using a high-throughput microplate assay. AtAA3C exhibited both oxidase and dehydrogenase activities toward aryl alcohols, with cinnamyl alcohol as its preferred substrate. It also displayed oxidase activity toward primary alcohols. AtAA3G exhibited oxidase activity, with the highest activity observed toward the long-chain alcohols 1-decanol and 1-dodecanol. The catalytic properties of these oxidases were further determined under optimal pH conditions using their preferred substrates. Compared to other characterized plant and fungal AA3 enzymes, AtAA3C and AtAA3G showed relatively low activities and catalytic velocities. This may reflect differences in their biological roles or that their true physiological substrates were not included in the tested substrate sets. Overall, these findings expand the current knowledge of plant AA3 enzymes and provide a basis for future studies.","CAZy (carbohydrate-active enzymes) on entsyymitietokanta, johon kootut entsyymit osallistuvat monimutkaisten hiilihydraattirakenteiden synteesiin, muokkaukseen ja hajotukseen. Nämä entsyymit jaetaan useaan luokkaan niiden sekvenssien sekä toiminnan mukaan. Yksi luokista on AA (auxiliary activities), jonka entsyymit osallistuvat muiden CAZy-entsyymien avustamiseen. AA3-luokan tunnetut entsyymit hapettavat hiilihydraatteja tai alkoholeja. Sienten AA3-entsyymien tiedetään osallistuvan kasvien lignoselluloosan hajotukseen, mutta kasvien vastaavien AA3-entsyymien tehtäviä ei vielä tunneta hyvin, ja vain joitain kasvien AA3-entsyymeitä on karakterisoitu. Uudenlaisilla AA3- entsyymeillä voisi olla sovelluskohteita esimerkiksi puunjalostusteollisuudessa, ligniinin ja hemiselluloosan hyödyntämisessä ja muokkaamisessa arvokkaiksi tuotteiksi. Kasvien AA3-entsyymien joukosta voi löytyä tähän tarkoitukseen sopivia entsyymejä, mutta niiden substraattispesifisyydestä sekä katalyyttisistä ominaisuuksista tarvitaan vielä lisää tietoa. Tämän tutkimuksen tarkoituksena oli tuottaa ja karakterisoida ensimmäistä kertaa kaksi Arabidopsis thaliana-lituruohon AA3-entsyymiä, AtAA3C ja AtAA3G. Entsyymit tuotettiin Pichia pastoris-hiivassa, minkä jälkeen ne puhdistettiin hyödyntäen affiniteettikromatografiaa. Entsyymien aktiivisuuksien analysoinnissa käytettiin laajaa joukkoa erilaisia substraatteja ja näiden analyysien tulokset osoittivat AtAA3C-entsyymillä olevan sekä oksidaasi- että dehydrogenaasiaktiivisuutta aryylialkoholeja kohtaan. Lisäksi AtAA3C-entsyymin havaittiin hapettavan primaarisia alkoholeja. Paras substraatti aryylialkoholeista oli kanelialkoholi ja primaarisista alkoholeista 1-dodekanoli. Tulokset osoittivat AtAA3G-entsyymillä olevan ainoastaan oksidaasiaktiivisuutta, ja se hapetti parhaiten pitkäketjuisia primaarisia alkoholeja 1-dekanolia ja 1-dodekanolia. Entsyymeille parhaiten sopivien substraattien löydyttyä määritettiin entsyymien katalyyttiset ominaisuudet. AtAA3C- ja AtAA3G-entsyymien aktiivisuudet tutkituilla substraateilla olivat alhaisemmat kuin aikaisemmin julkaistuilla kasvi- ja sienientsyymeillä. Ero voi kertoa entsyymien biologisista tehtävistä tai siitä, että tutkimuksessa käytetyt substraatit eivät vastanneet tutkittujen entsyymien luonnollisia substraatteja. Työn tulokset antavat lisää tietoa kasvien AA3-entsyymeistä ja tarjoavat perustan tuleville tutkimuksille."],"dc:identifier.uri":["http://hdl.handle.net/10138/627069"],"dc:language.iso":["eng"],"dc:publisher":["Helsingin yliopisto","University of Helsinki","Helsingfors universitet"],"dc:rights":["CC BY-NC-ND 4.0"],"dc:subject":["Arabidopsis thaliana","AA3","AtAA3C","AtAA3G","oxidoreductase","enzyme characterization","heterologous expression","enzymes"],"dc:title":["Production and characterization of two AA3 enzymes from Arabidopsis thaliana"]},"updated_at":"2026-07-27T19:56:25Z"}