Dokument: Analysis of factors influencing enzyme activity and stability in the solid state

Titel:	Analysis of factors influencing enzyme activity and stability in the solid state
URL für Lesezeichen:	https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=15503
URN (NBN):	urn:nbn:de:hbz:061-20100723-080112-8
Kollektion:	Dissertationen
Sprache:	Englisch
Dokumententyp:	Wissenschaftliche Abschlussarbeiten » Dissertation
Medientyp:	Text
Autor:	Kulishova, Liliya [Autor]
Dateien:	[Dateien anzeigen] Adobe PDF [Details] 4,43 MB in einer Datei [ZIP-Datei erzeugen] Dateien vom 02.07.2010 / geändert 02.07.2010
Beitragende:	PD Dr. Pohl, Martina [Gutachter] Prof. Dr. Jaeger, Karl-Erich [Gutachter]
Stichwörter:	Thermostability, alcohol dehydrogenase, gas/solid biocatalysis
Dewey Dezimal-Klassifikation:	500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie
Beschreibung:	The gas/solid biocatalysis represents a developing environmental-friendly technology for the production of volatile compounds like esters and alcohols, in which a dry enzyme catalyzes the reaction of the gaseous substrate to the gaseous product. This approach exhibits significant advantages, such as high conversion rates, higher stability of the immobilized biocatalyst and simplified product recovery. Furthermore, the gas/solid biocatalysis is efficient for investigation of the influence of thermodynamic parameters, such as water activity and substrate activity on activity and stability of a biocatalyst. However, progressive inactivation of the immobilized solid enzymes is usually observed under the operating conditions of a gas/solid reactor. Thus, studies of the temperature-dependent performance of the dry enzymes are important for both fundamental scientific understanding and engineering of novel thermostable biocatalysts. The primary objectives of the current thesis were: (i) Understanding of the key mechanisms responsible for the inactivation of solid enzymes in comparison to enzymes in aqueous solution. (ii) Understanding of the processes leading to inactivation of the solid cofactors NADH and NADPH in comparison to cofactors in aqueous solution. (iii) Generation of novel thermostable enzyme variants for the gas/solid biocatalysis and setup of an appropriate high-throughput screening procedure. (iv) Investigation of the activity and stability of enzymes under operational conditions in the gas/solid reactor. Studies were predominantly performed using the NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbADHwt) and a NADH-dependent variant thereof (LbADH G37D). A comparative study of the biocatalytic activity and protein structure through complementary techniques demonstrated that thermal inactivation of the dissolved and solid enzymes proceeds via different molecular mechanisms. Thermal degradation of NADH differed from NADPH. In the both cases, the main fragmentation routes included the cleavage of the nicotinamide ring as well as oxidative ring opening of the ribose ring. Construction and screening of a mutant library with 6000 enzyme variants resulted in identification of the variant LbADH G37D/V42F/E44C/K48Q with higher stability in the dry state. Comparative studies of LbADHwt and LbADH G37D in the gas/solid reactor demonstrated that thermodynamic activity of water and substrate was decisive for the operational activity and stability of enzymes. Furthermore, already a single amino acid exchange in the enzyme had significant impact on the kinetic characteristics and the stability of the biocatalyst.
Lizenz:	Urheberrechtsschutz
Fachbereich / Einrichtung:	Mathematisch- Naturwissenschaftliche Fakultät
Dokument erstellt am:	23.07.2010
Dateien geändert am:	02.07.2010
Promotionsantrag am:	30.04.2010
Datum der Promotion:	23.06.2010