No description available.

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

Detection and quantification of small peptides, such as yeast pheromones, are often challenging. We developed a highly sensitive and robust affinity-assay for the quantification of the α-factor pheromone of Saccharomyces cerevisiae based on recombinant hydrophobins. These small, amphipathic proteins self-assemble into highly stable monolayers at hydrophilic-hydrophobic interfaces. Upon functionalization of solid supports with a combination of hydrophobins either lacking or exposing the α-factor, pheromone-specific antibodies were bound to the surface. Increasing concentrations of the pheromone competitively detached the antibodies, thus allowing for quantification of the pheromone. By adjusting the percentage of pheromone-exposing hydrophobins, the sensitivity of the assay could be precisely predefined. The assay proved to be highly robust against changes in sample matrix composition. Due to the high stability of hydrophobin layers, the functionalized surfaces could be repeatedly used without affecting the sensitivity. Furthermore, by using an inverse setup, the sensitivity was increased by three orders of magnitude, yielding a novel kind of biosensor for the yeast pheromone with the lowest limit of detection reported so far. This assay was applied to study the pheromone secretion of diverse yeast strains including a whole-cell biosensor strain of Schizosaccharomyces pombe modulating α-factor secretion in response to an environmental signal.

Whole-cell sensors represent an emerging branch in biosensor development since they obviate the need for enzyme/antibody purification and provide the unique opportunity to assess global parameters such as genotoxicity and bioavailability. Yeast species such as Saccharomyces cerevisiae are ideal hosts for whole-cell sensor applications. However, current approaches almost exclusively rely on analyte-induced expression of fluorescent proteins or luciferases that imply issues with light scattering and/or require the supply of additional substrates. In this study, the yeast α-factor mating pheromone, a peptide pheromone involved in cell-cell communication in Saccharomyces cerevisiae, was utilized to create the whole-cell sensor read-out signal, in particular by employing engineered sensor cells that couple the response to a user-defined environmental signal to α-factor secretion. Two novel immunoassays - relying on hydrophobin-based surface engineering - were developed to quantify the α-factor. Hydrophobins are amphiphilic fungal proteins that self-assemble into robust monolayers at hydrophobic surfaces. Two recombinant hydrophobins, either lacking (EAS) or exposing the α-factor pheromone (EAS-α) upon self-assembly, were used to functionalize polystyrene supports. In a first approach (competitive immunoassay), pheromone-specific antibodies initially bound to the functionalized surface (due to the α-factor exposed by the hydrophobin layer) were competitively detached by soluble α-factor. In a second approach, the antibodies were first premixed with pheromone-containing samples and subsequently applied to functionalized surfaces, allowing for the attachment of antibodies that still carried available binding sites (inverse immunoassay). Both immunoassays enabled quantitative assessment of the yeast pheromone in a unique but partially overlapping dynamic range and allowed for facile tuning of the assay sensitivity by adjustment of the EAS-α content of the hydrophobin layer. With a limit of detection of 0.1 nM α-factor, the inverse immunoassay proved to be the most sensitive pheromone quantification assay currently available. Due to the high stability of hydrophobin monolayers, functionalized surfaces could be reused for multiple consecutive measurements. Favorably, both immunoassays proved to be largely robust against the changes in the sample matrix composition, allowing for pheromone quantification in complex sample matrices such as yeast culture supernatants. Hence, these immunoassays could also be applied to study the pheromone secretion of wild-type and engineered Saccharomyces cerevisiae strains. Additionally, a proof-of-concept whole-cell sensor for thiamine was developed by combining the hydrophobin-based immunoassays with engineered sensor cells of Schizosaccharomyces pombe modulating the secretion of the α-factor pheromone in response to thiamine. Since this read-out strategy encompasses intrinsic signal amplification and enables flexible choice of the transducer element, it could contribute to the development of miniaturized, portable whole-cell sensors for on-site application.

Glyphosat ist eines der weltweit am häufigsten eingesetzten Herbizide. Sein Einsatz wird u.a. auf Grund einer möglichen karzinogenen Wirkung und eines möglichen negativen Einflusses auf die Biodiversität kritisch diskutiert. Um Aussagen über die Verbreitung von Glyphosat in der Umwelt treffen zu können, werden verlässliche Nachweissysteme benötigt. Das Ziel der vorliegenden Arbeit bestand darin, ein einfaches optisches System zum schnellen Nachweis von Glyphosat in wässrigen Proben, basierend auf einer Hydrophobin-funktionalisierten Oberfläche, die das Glyphosat Zielprotein präsentiert, zu entwickeln. Hierfür wurden verschiedene Fusionsproteine aus dem Glyphosat Zielprotein, der 5-Enolpyruvylshikimat-3-phosphatsynthase (EPSPS, hier aus dem Bakterium Escherichia coli (EcEPSPS)) und dem zur Selbstassemblierung an hydrophilen/hydrophoben Grenzflächen befähigten Hydrophobin Ccg2 aus Neurospora crassa erzeugt, welche für die Oberflächenfunktionalisierung eingesetzt wurden. Die Expression und Reinigung der Fusionsproteine und von Ccg2 in E. coli verlief erfolgreich. Nach initialen Kontaktwinkelmessungen zur Untersuchung der Funktionalität des Hydrophobins und Enzymaktivitätsmessungen für die Fusionsproteine, konnte deren Aktivität auch nach der Reinigung nachgewiesen werden. Dabei erwies sich das Fusionsprotein Ccg2_GS_EcEPSPS, aufgrund einer hohen enzymatischen Aktivität nach Immobilisierung, als am besten geeignet. Es wurden verschiedene Belegverhältnisse zwischen Hydrophobin und Fusionsprotein untersucht, um etwaige sterische Behinderungen zu minimieren. Hierbei erwies sich ein Belegverhältnis von 1 µM Ccg2_GS_EcEPSPS und 5 µM Ccg2 für die künftigen Messungen als gut geeignet. Auf Basis der so funktionalisierten Oberfläche wurden zwei Verfahren zum optischen Nachweis von Glyphosat entwickelt. Eines der Verfahren, der Malachitgrün-Assay, weist die enzymatische Aktivität der EPSPS auf der Oberfläche nach, genauer das entstehende anorganische Phosphat (Pi). Durch Glyphosathemmung entsteht weniger Pi, dies kann mittels Malachitgrün-Assay nachgewiesen werden. Unter Laborbedingungen konnte ein Detektionslimit von 50 nM erreicht werden. Des Weiteren zeigte der Assay keine nennenswerte Querempfindlichkeit und erwies sich damit als sehr spezifisch. Zusätzlich wurde der Einfluss unterschiedlicher Temperaturen und pH-Werte untersucht. Es zeigte sich, dass Schwankungen dieser Parameter den Assay beeinflussen. Auch ein Einfluss der Ionenstärke konnte festgestellt werden. Deshalb sind entsprechende Kontrollen unerlässlich. Der Einfluss nicht-reaktionsbedingten Phosphates konnte durch Vorinkubation der Oberfläche mit der Glyphosat-haltigen Analyselösung mit anschließender Entfernung der Selbigen und Durchführung des Malachitgrün-Assays minimiert werden. Das zweite Verfahren, der Hydrogelsonden (HGS)-Assay, weist direkt die Interaktion von Glyphosat und der immobilisierten EcEPSPS nach. Hierfür wurden verformbare, Glyphosat-dekorierte HGS aus Polyethylenglykol benötigt. Bei Abwesenheit von freiem Glyphosat liegen die Bindestellen der immobilisierten EPSPS frei vor, sodass sie für die Bindung des immobilisierten Glyphosats an den HGS zur Verfügung stehen. Zwischen den HGS und der Oberfläche entsteht auf diese Weise eine große Kontaktfläche, welche mittels Reflektionsinterferenzkontrastmikroksopie messbar ist. Freies Glyphosat in der Analyselösung reduziert die verfügbaren Bindestellen an der Oberfläche. Dies resultiert in einer kleineren Kontaktfläche. Auf diese Weise kann durch Ermittlung der Größe der Kontaktfläche zwischen HGS und funktionalisierter Oberfläche und der daraus berechneten Adhäsionsenergie, auf das Vorhandensein von Glyphosat in der Analyselösung geschlossen werden. Im Rahmen dieser Arbeit konnte nach Optimierung der Oberflächenbeschichtung, ein positiver Machbarkeitsbeweis für dieses Verfahren erbracht werden.

This work was performed in the framework of an interdisciplinary graduate program that focuses on the establishment and extension of innovative compounds for the packaging of electronic systems. Such chemically or biotechnologically tailored compounds can be used for the direct patterning of optically, magnetically or biologically functional structures in nano- and biotechnical products. In order to organize matter at the nanometer scale, imprinting litho-graphy techniques or self-organization processes are appropriate. Fine-tuning of numerous engineering processes requires continuous and high precision monitoring as well as control of diverse parameters. These demands are only partially met by physical or chemical components since they use surrogate parameters, measure off-line, or provide insufficient performances. Biological compounds, in particular protein-based feedback systems, fulfill certain system requirements to a considerable degree. Hydrophobins and S-layer proteins are surface active proteins, produced by filamentous fungi or bacteria. In nature, these (self )assembly proteins form highly ordered and robust structures. In addition, their tolerance for different sequence manipulations and chemical modifications allows extensive functionalization of these nanometer-sized proteins. Hence, these surface active proteins can also be fused with other protein domains to create chimera, which retain function of both original proteins. In conclusion, both hydrophobins and S-layer proteins represent a versatile tool in numerous fields of applied biotechnology, medicine or diagnostics. But until now, efficient in vitro operation in molecular designed protein coatings is strongly restricted due to their complex assembly mechanism. In the first phase of this work, it was demonstrated, that representatives of class I and class II hydrophobins tend to form multilayered structures on solid surfaces. It was found that only two protein orientations seems to be preferentially formed. In the process of assembly, the orientation of the first hydrophobin layer strictly depends on the substrate wettability. Consequently, each of the following hydrophobin layers is inverse oriented to the layer before. This alternating assembly mechanism has to be taken into account, when working with functionalized hydrophobins, because a hydrophobin-fused functional protein domain is exclusively located on one side of the protein. Due to the densely packed structure of surface active proteins, a fused functional domain, embedded between two hydrophobins is barely available for external reagents. Basically, the simultaneous existence of a broad spectrum of ordered and disordered assembly structures, demonstrated the need of an uniform protein film assembly for applications in fine-diagnostics or biomedicine. With regard to molecular designed protein coatings, this work further aimed at establishing conditions to develop a method for a ‘layer-by-layer’ assembly of protein chimeras. Based on their amphiphilic character, self-assembly behavior of surface active proteins can be influenced by conventional ionic surfactants. In order to study the effect of surfactants on the composition and morphology of adsorbed protein films, contact angle measurements, nulling ellipsometry, SEM, AFM and AFAM were performed. It was found that the layer thickness of assembled protein films is strictly dependent on the amount of added surfactant. At certain threshold surfactant concentrations, hydrophobins and S-layer proteins assemble in uniform layers, which are as thick as expected for a protein monolayer or a bilayer. Assembled protein films are covered by a smooth surfactant layer, which prevents further protein assembly. AFAM measurements reveal the formation of well defined lattice structures under the coverage of surfactants. Even the removal of the surfactant layer is possible without inter-fering with protein specific secondary structures. Solvent accessibility and functionality of protein-fused domains was successfully demonstrated. As compared to conventional assembly techniques, this novel protein deposition method offers a possibility for a ‘directed’ protein coating on solid surfaces. In addition, it guarantees broadly ranged homogeneous assembly of protein chimeras on non-planar or even porous surfaces independent of their position. Finally, a prototype for an interfacial FRET was developed in a close collaboration with the Institute of Physical Chemistry (TUD). This innovative FRET between semiconducting nano-particles and illuminating protein chimeras takes place across an oil/water interface. Hydro-phobins were used to stabilize artificial oil droplets in aqueous solution. These small proteins possess the ability to attach fused functional domains very close to an oil/water interface. When, in addition to this, an optically active nanostructure directly docks to the hydrophobin, the distance of a protein-fused domain and the nanostructure are in the range of the FÖRSTER radius. It was successfully demonstrated that quantum dots and fluorescent proteins fulfill the spectroscopic requirements of such a donor/acceptor pair. The FRET performance of these excitable oil droplets was examined as a ‘proof of concept’. Due to its modular design, this signal amplification setup could be exploited in numerous fields of technical application ranging from quantification of micronutrient to photothermal cancer therapy.

Biosurfactants are naturally occurring amphiphiles with potential for use as alternatives to traditional petrochemical and oleochemical surfactants. The unique properties of biosurfactants, including their biodegradability and tolerance of a wide range of temperature and pH, make their use in a range of novel applications attractive. Currently the wider ultilisation of biosurfactants is hindered by a lack of economically viable production routes, with downstream processing presenting a significant challenge. This thesis presents an investigation into the production of HFBII, a hydrophobin protein, using an adsorptive bubble separation technique called foam fractionation for in situ recovery of the biosurfactant. The effects of foaming on the production of HFBII by fermentation were investigated at two different scales. Foaming behaviour was characterised in standard terms of the product enrichment and recovery achieved. Additional specific attention was given to the rate at which foam, product and biomass overflowed from the fermentation system in order to assess the utility of foam fractionation for HFBII recovery. HFBII was expressed as an extracellular product during fed batch fermentations with a genetically modified strain of Saccharomyces cerevisiae, which were carried out with and without antifoam. In the presence of antifoam HFBII production is shown to be largely unaffected by process scale, with similar yields of HFBII on dry matter obtained. More variation in HFBII yield was observed between fermentations without antifoam. In fermentations without antifoam a maximum HFBII enrichment in the foam phase of 94.7 was measured with an overall enrichment of 54.6 at a recovery of 98.1%, leaving a residual HFBII concentration of 5.3 mg L-1 in the fermenter. It is also shown that uncontrolled foaming reduced the concentration of biomass in the fermenter vessel, affecting total production. This series of fermentation experiments illustrates the potential for the application of foam fractionation for efficient in situ recovery of HFBII, through simultaneous high enrichment and recovery which are greater than those reported for similar systems. After the suitability of foam fractionation was demonstrated a novel apparatus design was developed for continuously recovering extracellular biosurfactants from fermenters. The design allows for the operating conditions of the foam fractionation process, feed rate and airflow rate, to be chosen independently of the fermentation parameters. Optimal conditions can then be established for each process, such as the aeration rate required to meet the biological oxygen demand of the cell population. The recirculating foam fractionation process was tested on HFBII producing fermentations. It is shown that by using foam fractionation to strip HFBII from fermentation broth in situ the amount of uncontrolled overflowing from the fermenter was greatly reduced from 770.0 g to 44.8 g, compared to fermentations without foam fractionation. Through optimisation of the foam column operating conditions the proportion of dry matter retained in the fermenter was increased from 88% to 95%, in contrast to a dry matter retention of 66% for fermentation without the new design. With the integrated foam fractionation process a HFBII recovery of 70% was achieved at an enrichment of 6.6. This work demonstrates the utility of integrated foam fractionation in minimising uncontrolled foaming in fermenters whilst recovering an enriched product. This integrated production and separation process has the potential to facilitate improved biosurfactant production, currently a major barrier to their wider use.

This work was performed in the framework of an interdisciplinary graduate program that focuses on the establishment and extension of innovative compounds for the packaging of electronic systems. Such chemically or biotechnologically tailored compounds can be used for the direct patterning of optically, magnetically or biologically functional structures in nano- and biotechnical products. In order to organize matter at the nanometer scale, imprinting litho-graphy techniques or self-organization processes are appropriate. Fine-tuning of numerous engineering processes requires continuous and high precision monitoring as well as control of diverse parameters. These demands are only partially met by physical or chemical components since they use surrogate parameters, measure off-line, or provide insufficient performances. Biological compounds, in particular protein-based feedback systems, fulfill certain system requirements to a considerable degree. Hydrophobins and S-layer proteins are surface active proteins, produced by filamentous fungi or bacteria. In nature, these (self )assembly proteins form highly ordered and robust structures. In addition, their tolerance for different sequence manipulations and chemical modifications allows extensive functionalization of these nanometer-sized proteins. Hence, these surface active proteins can also be fused with other protein domains to create chimera, which retain function of both original proteins. In conclusion, both hydrophobins and S-layer proteins represent a versatile tool in numerous fields of applied biotechnology, medicine or diagnostics. But until now, efficient in vitro operation in molecular designed protein coatings is strongly restricted due to their complex assembly mechanism. In the first phase of this work, it was demonstrated, that representatives of class I and class II hydrophobins tend to form multilayered structures on solid surfaces. It was found that only two protein orientations seems to be preferentially formed. In the process of assembly, the orientation of the first hydrophobin layer strictly depends on the substrate wettability. Consequently, each of the following hydrophobin layers is inverse oriented to the layer before. This alternating assembly mechanism has to be taken into account, when working with functionalized hydrophobins, because a hydrophobin-fused functional protein domain is exclusively located on one side of the protein. Due to the densely packed structure of surface active proteins, a fused functional domain, embedded between two hydrophobins is barely available for external reagents. Basically, the simultaneous existence of a broad spectrum of ordered and disordered assembly structures, demonstrated the need of an uniform protein film assembly for applications in fine-diagnostics or biomedicine. With regard to molecular designed protein coatings, this work further aimed at establishing conditions to develop a method for a ‘layer-by-layer’ assembly of protein chimeras. Based on their amphiphilic character, self-assembly behavior of surface active proteins can be influenced by conventional ionic surfactants. In order to study the effect of surfactants on the composition and morphology of adsorbed protein films, contact angle measurements, nulling ellipsometry, SEM, AFM and AFAM were performed. It was found that the layer thickness of assembled protein films is strictly dependent on the amount of added surfactant. At certain threshold surfactant concentrations, hydrophobins and S-layer proteins assemble in uniform layers, which are as thick as expected for a protein monolayer or a bilayer. Assembled protein films are covered by a smooth surfactant layer, which prevents further protein assembly. AFAM measurements reveal the formation of well defined lattice structures under the coverage of surfactants. Even the removal of the surfactant layer is possible without inter-fering with protein specific secondary structures. Solvent accessibility and functionality of protein-fused domains was successfully demonstrated. As compared to conventional assembly techniques, this novel protein deposition method offers a possibility for a ‘directed’ protein coating on solid surfaces. In addition, it guarantees broadly ranged homogeneous assembly of protein chimeras on non-planar or even porous surfaces independent of their position. Finally, a prototype for an interfacial FRET was developed in a close collaboration with the Institute of Physical Chemistry (TUD). This innovative FRET between semiconducting nano-particles and illuminating protein chimeras takes place across an oil/water interface. Hydro-phobins were used to stabilize artificial oil droplets in aqueous solution. These small proteins possess the ability to attach fused functional domains very close to an oil/water interface. When, in addition to this, an optically active nanostructure directly docks to the hydrophobin, the distance of a protein-fused domain and the nanostructure are in the range of the FÖRSTER radius. It was successfully demonstrated that quantum dots and fluorescent proteins fulfill the spectroscopic requirements of such a donor/acceptor pair. The FRET performance of these excitable oil droplets was examined as a ‘proof of concept’. Due to its modular design, this signal amplification setup could be exploited in numerous fields of technical application ranging from quantification of micronutrient to photothermal cancer therapy.

No description available.