Volltextdatei(en) vorhanden
DC ElementWertSprache
dc.contributor.advisorStreit, Wolfgang (Prof. Dr.)
dc.contributor.authorRabausch, Ulrich
dc.date.accessioned2020-10-19T12:58:12Z-
dc.date.available2020-10-19T12:58:12Z-
dc.date.issued2013
dc.identifier.urihttps://ediss.sub.uni-hamburg.de/handle/ediss/5796-
dc.description.abstractThe functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside. Further, a novel alpha-L-rhamnosidase was identified. A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-L-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4,095 bp) and encoded for a putative protein of 1,364 amino acids. The C-terminal part of the enzyme revealed an amino acid (AA) sequence identity of 58% to a predicted bacterial α-L-rhamnosidase from Bacteroides nordii. Interestingly, the N-terminal region of the deduced enzyme RhaB contained a GDSL-like lipase motif and an acetyl-xylan esterase (DAP2) motif. While heterologous expression of the complete rhaB failed, subcloning of the gene identified the most active open reading frame (ORF) to be of 3,081 bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in E. coli BL21 (DE3) and was purified to an amount of 75 mg per liter of culture medium. In accordance to the intestinal origin, RhaB1 showed a preference for mesophilic conditions with an optimum activity at a temperature of 40 °C and at pH 6.5, respectively. The recombinant protein had a Km value of 0.79 mM and a specific activity vmax of 18.4 U for pNP-α-L-rhamnose, a calculated Km of 6.36 mM and Vmax of 2.9*10-3 U for naringin, and a Km of 6.75 mM and specific activity Vmax of 8.63*10-2 U for rutin, respectively. Phylogenetic analysis and amino acid domain architecture comparison revealed that RhaB1 belongs to a new subclass of bacterial B type α-L-rhamnosidases of GH 78. To our knowledge RhaB1 is the first biochemically-characterized enzyme of this subclass.en
dc.language.isodede
dc.publisherStaats- und Universitätsbibliothek Hamburg Carl von Ossietzky
dc.relation.isbasedonJournal of Biotechnology 191:38-45, Applied and Environmental Microbiology 79:4551–4563
dc.rightshttp://purl.org/coar/access_right/c_abf2
dc.subjectKohlenhydrat-aktive Enzymede
dc.subjectalpha-L-Rhamnosidasende
dc.subjectfunktionelle Metagenomikde
dc.subject.ddc570 Biowissenschaften, Biologie
dc.titleMetagenome derived carbohydrate active enzymes for directed modification of polyphenolsde
dc.title.alternativeKohlenhydrat-aktive Enzyme aus Metagenomen zur gerichteten Modifizierung von Polyphenolende
dc.typedoctoralThesis
dcterms.dateAccepted2013-06-07
dc.rights.ccNo license
dc.rights.rshttp://rightsstatements.org/vocab/InC/1.0/
dc.subject.bcl42.30 Mikrobiologie
dc.subject.gndGlycosyltransferasen
dc.subject.gndGlykosidasen
dc.subject.gndMetagenom
dc.subject.gndEnzym
dc.subject.gndGlykosylierung
dc.subject.gndBiotransformation
dc.subject.gndBiokonversion
dc.subject.gndBiokatalyse
dc.subject.gndBakterien
dc.subject.gndMikroorganism
dc.type.casraiDissertation-
dc.type.dinidoctoralThesis-
dc.type.driverdoctoralThesis-
dc.type.statusinfo:eu-repo/semantics/publishedVersion
dc.type.thesisdoctoralThesis
tuhh.opus.id7227
tuhh.opus.datecreation2015-03-17
tuhh.type.opusDissertation-
thesis.grantor.departmentBiologie
thesis.grantor.placeHamburg
thesis.grantor.universityOrInstitutionUniversität Hamburg
dcterms.DCMITypeText-
tuhh.gvk.ppn821562819
dc.identifier.urnurn:nbn:de:gbv:18-72276
item.advisorGNDStreit, Wolfgang (Prof. Dr.)-
item.grantfulltextopen-
item.languageiso639-1other-
item.fulltextWith Fulltext-
item.creatorOrcidRabausch, Ulrich-
item.creatorGNDRabausch, Ulrich-
Enthalten in den Sammlungen:Elektronische Dissertationen und Habilitationen
Dateien zu dieser Ressource:
Datei Beschreibung Prüfsumme GrößeFormat  
Dissertation.pdf811b7304762266ece0565de57e8a9fff3.97 MBAdobe PDFÖffnen/Anzeigen
Zur Kurzanzeige

Diese Publikation steht in elektronischer Form im Internet bereit und kann gelesen werden. Über den freien Zugang hinaus wurden durch die Urheberin / den Urheber keine weiteren Rechte eingeräumt. Nutzungshandlungen (wie zum Beispiel der Download, das Bearbeiten, das Weiterverbreiten) sind daher nur im Rahmen der gesetzlichen Erlaubnisse des Urheberrechtsgesetzes (UrhG) erlaubt. Dies gilt für die Publikation sowie für ihre einzelnen Bestandteile, soweit nichts Anderes ausgewiesen ist.

Info

Seitenansichten

478
Letzte Woche
Letzten Monat
geprüft am 27.03.2024

Download(s)

130
Letzte Woche
Letzten Monat
geprüft am 27.03.2024
Werkzeuge

Google ScholarTM

Prüfe