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One of the most important properties of enzymes is possibly their unwaning reactivity at ambient conditions. Moreover, increasing knowledge successfully counteracts their disadvantages such as limited process stability or costly production by immobilization techniques and modern fermentation process plants. Three different experimental approaches were pursued in order to adapt standard photometric assays for the activity determination of TlL and papain incorporated into polymeric thin films consisting of polyvinylpyrrolidone (PVP) and the tertiary polyamide Ultramid 1C® (U1C). They primarily enable the quantitative measurement probing the suitability of polymer films as supports for enzyme molecules with respect to the activity and long term stability of the composite. All activity measurements of this study are based on the hydrolytic cleavage of p-nitrophenyl fatty acid esters catalyzed by Thermomyces lanuginosus Lipase (TlL) or the amide bond hydrolysis of the model peptide DL-BAPA by papain from Carica papaya. Es wird geschätzt, dass bei der Produktion von mittlerweile der Hälfte aller Feinchemikalien ein biokatalytischer Schritt involviert ist. Dabei stellt die Immobilisierung, die bei ca. 80% aller industriell genutzten Biotransformationsprozesse eine Rolle spielt, einen essentiellen Baustein zur wirtschaftlichen Entwicklung biotechnologischer Prozesse dar. Eine Immobilisierung der Biokatalysatoren ist notwendig, um deren Nachteile bei einer industriellen Anwendung, wie z.B. geringe Stabilität gegenüber hohen Temperaturen, extremen pH-Werten oder organischen Lösungsmitteln zu verringern oder sogar aufzuheben. Hydrolyse- oder Oxidationsreaktionen laufen bei Raumtemperatur ohne Lichteinwirkung in wässrigen Medien nur in Gegenwart von Enzymen katalytisch ab. Um wissenschaftliche Erkenntnisse über die Aktivität und Stabilität von biokatalytisch aktiven Oberflächen zu gewinnen, ist es jedoch nötig, geeignete analytische Werkzeuge zu entwickeln, um darauf aufbauend die Grundlage für die Forschung und Weiterentwicklung dieser Kompositmaterialien unabhängig von deren Anwendungsgebieten zu schaffen. Im Rahmen dieser Arbeit wurden daher verschiedene Analysemethoden entwickelt, um die Aktivität von Enzymen zu bestimmen, die in dünnen Polymerfilmen eingebettet sind. Als grundlegendes Prinzip der Aktivitätsbestimmungen diente dabei die UV/Vis-spektroskopische Aufzeichnung der Konzentration zweier Farbstoffe, p-Nitrophenol und p-Nitroanilin. Ersterer entsteht bei der Hydrolyse des Substrats p-Nitrophenylpalmitat durch die immobilisierte Thermomyces lanuginosus Lipase (TlL) und letzterer durch die hydrolytische Spaltung der Amidbindung des Modellpeptids DL-BAPA durch die Proteinase Papain (Carica papaya). Um die Aktivität von Enzymen, die in dünne Polymerfilme eingebettet wurden, zu bestimmen, sind zum Teil weitreichende Anpassungen des Versuchsaufbaus sowie des Assays selbst nötig. Aufgrund der unterschiedlichen Löslichkeit der verwendeten Trägerpolymere Polyvinylpyrrolidon (PVP) und des tertiären Polyamids Ultramid 1C® (U1C), war die Entwicklung unterschiedlicher Messmethoden erforderlich. Mit Hilfe dieser neu entwickelten oder modifizierten analytischen Werkzeuge kann eine Vielzahl von Fragestellungen betreffend der Stabilität und Aktivität von immobilisierten Enzymen in Abhängigkeit vom verwendeten Trägerpolymer beantwortet werden.

Benzaldehyde lyase from Pseudomonas fluorescens (BAL) and carbonylreductase from Candida parapsilosis (CPCR) are versatile catalysts for the production of various chiral intermediates, which are of interest for the pharmaceutical industry. Due to the mostly hydrophobic reactants only low productivities can be achieved in aqueous reaction systems and thus the use of alternative reaction media is desirable. Especially the use of aqueous-organic biphasic systems is promising, since the extraction of products and the spatial separation from the enzyme facilitate efficient processes. However, both enzymes show only insufficient stability in these systems and therefore a stabilisation is indispensable. Stability of both enzymes was determined in three different biphasic systems as well as the respective solvent-saturated aqueous monophasic systems (BAL: MTBE, 2 octanone, toluene; CPCR: MTBE, cyclohexane, n heptane). It was shown that BAL was already significantly deactivated by the solvent molecules within the aqueous phase. In the respective biphasic systems especially the interfaces towards 2-octanone and toluene led to considerable additional deactivation. On the molecular level CPCR was deactivated mainly by dissolved MTBE-molecules. In contrast, dissolved cyclohexane and n-heptane molecules only had minor effects on CPCR stability, whereas the respective biphasic systems led to significant deactivation of CPCR. The deactivation by the solvents is probably a result of interactions between the solvents and the proteins’ surfaces. Therefore the surface characteristics of both enzymes were altered in order to prevent these interactions. Chemical modification of free amino groups was applied to introduce new chemical functionalities to the enzymes’ surfaces. Acetylation with cyclic anhydrides decreased activity of both enzymes significantly. In contrast, modification with mPEG750 and mPEG2000 resulted in good residual activities of BAL (60-70%), while CPCR retained less than 10% residual activity. Gel electrophoresis showed that apparently only few amino groups of BAL were accessible for mPEG-modification, whereas CPCR was modified to great extent. Storage stability measurements with the mPEG-BAL-variants revealed a considerable increase of stability towards dissolved molecules in the solvent systems with MTBE and toluene. Site-directed mutagenesis was applied to exchange unpolar surface amino acid residues against polar amino acid residues in order to decrease interactions between the enzymes and the solvents. Most of the resulting BAL-variants showed similar activities as the native BAL, whereas many CPCR-variants lost most of their activity. For both enzymes enantioselectivity was unaffected by the introduced mutations. Kinetic constants of BAL-variants differed only marginally from the ones of native BAL. CPCR-variants exhibited higher kcat-values than native CPCR in both reduction- and oxidation reaction. Determination of storage stability in the different mono-and biphasic systems showed that most variants exhibited decreased stability against dissolved solvent molecules compared to native enzymes. However, additional deactivation at the interface towards toluene (BAL) and MTBE, cyclohexane und n-heptane (CPCR) respectively, was decreased. For the technical application enzyme stability under relevant process conditions is of importance. Therefore a reactor set-up was established, that allows stability determination in reactive, aqueous-organic biphasic systems. Respective measurements with BAL indicated that deactivation by the aldehyde substrate superimposed the deactivation by the solvents, thus resulting in similar BAL stability in all tested solvent systems. Neither the mPEG-modification nor the introduced mutations prevented this deactivation by the substrate. CPCR showed a strong stability decrease under process conditions in the MTBE-system, probably due to a combined effect of dissolved MTBE-molecules and isopropanol which was used for substrate-coupled cofactor regeneration. CPCR stability in reactive systems with cyclohexane and n-heptane was comparable to stability under non-reactive conditions. Variant CPCR-I79T,V83S showed increased process stability in all three biphasic systems.