Dokument: Lipase‐specific foldase‐aided folding of Lipase A from Pseudomonas aeruginosa

Titel:	Lipase‐specific foldase‐aided folding of Lipase A from Pseudomonas aeruginosa
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=48826
URN (NBN):	urn:nbn:de:hbz:061-20190312-104304-5
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Dollinger, Peter [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 7,16 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 10.03.2019 / geändert 10.03.2019
Beitragende:	Prof. Dr. Jaeger, Karl-Erich [Gutachter] Prof. Dr. Seidel, Claus A. M. [Gutachter]
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	Secreted bacterial lipases frequently show interesting properties for use in biocatalysis, such as promiscuous reactivity and high stability. Unfortunately, the complexity of the secretion process, which involves many accessory proteins, hampers efficient heterologous and homologous expression, limiting the availability of lipases and therefore their applicability as biocatalysts. One of the major bottlenecks in the production of many secreted lipases is their dependency on lipase-specific foldases, which function as steric chaperones that help lipases overcome an energetic barrier on their folding pathway to the active conformation. In the absence of foldases, lipases aggregate into insoluble inclusion bodies that can be activated by foldases after they have been chemically unfolded and refolded into a so-called preactive state. It is generally accepted that this in vitro-derived preactive conformation is formed on the in vivo folding pathway as well. However, the molecular mechanisms of the in vivo and in vitro foldase-assisted activation of lipases are barely understood. In this thesis, the lipase-specific foldase (LipH) and lipase (LipA) from Pseudomonas aeruginosa PA01 was used as a model system to investigate foldase-assisted lipase activation. After a detailed biochemical and biophysical characterization of the refolding and activation processes of LipA in vitro, the results put into question the hypothesis about the preactive LipA state as a real folding intermediate in vivo. A great aggregation tendency and unstable tertiary structure make the preactive state unlikely to exist as free molecules inside the periplasm. Furthermore, preactive LipA in complex with LipH showed catalytic activity, but comparison of the structural stability and characteristics with the native state of LipA, isolated from P. aeruginosa supernatants, demonstrated clear differences, leading to the assumption that LipH is not able to convert preactive LipA into the native conformation. For the first time, it was possible to demonstrate that the LipAH complex is able to dissociate and that there is a steady exchange of LipA molecules inside the complex during the refolding reaction in vitro. As a consequence, the release of active lipase and multiturnover catalysis of foldases has never been observed, because preactive LipA falls back into an inactive conformation after dissociation from LipH. In vivo, LipH may interact with LipA during the secretion process, before LipA folds into the globular preactive state, to block this pathway and to assist in folding into a highly stable native conformation. The active conformation of preactive LipA in complex with LipH might be an artificial state that does not occur in vivo.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät » WE Biologie » Enzymtechnologie
Dokument erstellt am:	12.03.2019
Dateien geändert am:	12.03.2019
Promotionsantrag am:	29.08.2018
Datum der Promotion:	28.11.2018