Untersuchungen zum Verständnis der Beziehung von Enzymen mit α/β-Hydrolasefaltung: Generierung von Epoxidhydrolaseaktivität in eine Esterase

Enzyme sind bekannt als Biokatalysatoren, die spezifisch für ein oder wenige Substrate und ihre zu katalysierende Reaktion sind. Die Fähigkeit einiger Enzyme, mehr als nur eine bestimmte chemische Umsetzung zu katalysieren, bezeichnet man als Promiskuität. Einige Enzyme verfügen über ein breites Substratspektrum und können selbst strukturell verschiedene Substrate umsetzen. Man spricht hierbei von der Substratpromiskuität (substrate promiscuity) der Enzyme. Eine weitere Klasse der Promiskuität wird als Konditionspromiskuität (condition promiscuity) bezeichnet. Hierzu zählen Enzyme, die auch bei nicht-natürlichen Reaktionsbedingungen wie hohen Temperaturen, extremen pH-Werten oder in wasserfreiem Medium katalytische Aktivität aufweisen. Die katalytische Promiskuität (catalytic promiscuity) bildet die dritte Gruppe der Enzympromiskuität. Enzyme, die über diese Art der Promiskuität verfügen, zeichnen sich durch eine breite Reaktionsspezifität bei der Katalyse alternativer Reaktionen aus. Zudem lehren uns die Strukturen von über 30.000 Proteinen, dass die Natur nur von einem limitierten Repertoire von Proteingerüsten Gebrauch gemacht hat, um dennoch eine Vielzahl an verschiedensten Reaktionen herbeizuführen. Die Vielfältigkeit der Proteingerüste ist auf einige wenige Vorfahren zurückzuführen, deren Gerüst als Basis zur Generierung von Familien und Superfamilien diente. Die Überreste dieses Prozesses spiegeln sich in den ähnlichen Strukturen und katalytischen Resten der Familienmitglieder wieder. Über die Millionen von Jahren der Evolution haben sich jedoch die Sequenzähnlichkeiten der Mitglieder einer Familie stark verändert. Durch die Untersuchung der Beziehungen von Enzymen mit α/β-Hydrolasefaltung am Beispiel der Generierung von Epoxidhydrolaseaktivität in das Proteingerüst der Pseudomonas fluorescens Esterase (PFE) sollte die verwandtschaftliche Beziehung beider Enzyme näher dargestellt werden. Mit Hilfe der Methoden der positionsgerichteten Mutagenese und der gerichteten Evolution war es möglich eine Vielzahl von Mutanten zu kreieren. Zur Durchmusterung der Mutantenbliotheken kam sowohl ein neu entwickelter Agarplatten-Assay, als auch ein optimiertes Hochdurchsatz-Testsystem zum Einsatz. Mittels dieser Testformate konnten Mutanten der PFE identifiziert werden, die aktiv gegenüber Epoxiden sind. Des Weiteren erfolgte die genaue Charakterisierung der generierten Varianten.The ability of enzymes to catalyse more than one specific reaction is constitute as promiscuity. Some enzymes accept a broad range of substrates. This property of enzymes is called substrate promiscuity. Enzymes, which show catalytic activity under non-natural conditions like high temperatures, extreme pH or non-aqueous medium exhibit condition promiscuity. The catalytic promiscuity defines the last group of enzyme promiscuity. These enzymes are characterised by catalysing alternative reactions. Besides, the structures of more than 30,000 proteins taught us that nature made use of a rather limited repertoire of core structural platforms, or “scaffolds” (on the order of a few thousand), to mediate an amazingly large diversity of functions. This diversity had presumably emerged from a small number of progenitor proteins, each with a different basic scaffold, thus creating enzyme families and superfamilies. The vestiges of this process are the scaffold and active-site architecture or key catalytic residues shared by all family members. Esterases and epoxide hydrolases adopt an a/b-hydrolase fold, but differ in their substrate specificity. The structural relation between these two enzymes should be characterised by converting the Pseudomonas fluorescens esterase (PFE) into an epoxide hydrolase. The generation of mutant libraries is performed by classical method of site-directed mutagenesis followed by directed evolution methods. The libraries were screened with a new developed agar-plate assay and an optimized high-throughput assay system. By using these assay formats it was possible to identify variants of PFE that are active against epoxides

Electrical Wiring of the Aldehyde Oxidoreductase PaoABC with a Polymer Containing Osmium Redox Centers: Biosensors for Benzaldehyde and GABA

Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer

Tunable electric field enhancement and redox chemistry on TiO2 island films via covalent attachment to Ag or Au nanostructures

Abstract Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b5 (Cyt b5) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b 5 were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s -1) of Cyt b5 to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film. © 2013 American Chemical Society

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy

Complementary Surface-Enhanced Resonance Raman Spectroscopic Biodetection of Mixed Protein Solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome <i>b</i>₅, whereas functionalization with SiO₂ amplifies only the spectra of the cationic protein cytochrome <i>c</i>. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy

Bioelectrocatalysis at mesoporous antimony doped tin oxide electrodes—Electrochemical characterization and direct enzyme communication

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