Calixto Mancipe, Natalia2019-03-132019-03-132019-01https://hdl.handle.net/11299/202101University of Minnesota M.S. thesis.January 2019. Major: Bioproducts/Biosystems Science Engineering and Management. Advisor: Ping Wang. 1 computer file (PDF); xiii, 91 pages.Biosurfactants are amphipathic molecules required for vital processes in all life forms. They help to reduce surface tension facilitating interfacial processes such as breathing and evaporative cooling, modulate environmental conditions, control surface wettability, interact with substrates, form protective layers, etc. Thus, nature has evolved a wide variety of biosurfactants combining hydrophilic building blocks such as acid groups, sugars or polar amino acids with hydrophobic ones such as lipids, creating an enormous pallet of possibilities. Among them, hydrophobins (hereafter HFBs) are a family of self-assembling surfactant proteins with the highest surface activity known to date and an intrinsic amphipathic structure that does not require additional functional groups such as lipids or sugars. These characteristics give them a great potential for their industrial use as interfacial stabilizers, dispersal agents, surface modifiers and molecular anchors for protein immobilization on solids. The unique sequences and folding structures of HFBs particularly promote interfacial and intermolecular interactions, along with robust self-assembling mechanisms and surface activity. Unfortunately, a lack of knowledge on their sequence-structure-function relationships hinders the optimal selection of proteins for specific applications as well as manipulation of their properties. For a specific consideration, the use of HFBs for standard coating processes usually results in irregular products; at the same time, such an application requires a large-scale production that has been difficult to achieve limited by the productivity of native organisms, the HFB toxicity for heterologous hosts and the challenges of their purification. These obstacles suggest the need to leverage HFBs’ interfacial behaviors to better fit a broader range of applications. Therefore, an HFB from the fungus Trichoderma reesei is taken as a model surfactant protein in this work to explore the variation of its characteristics by a modular fusion strategy, as an alternative approach to protein directed mutagenesis and engineering. We aim to combine its high surfactant activity with the functionalities of different fusion partners to enhance its productivity and interfacial properties. Our results show that the fusion with a small metal binding protein from Nitrosomonas eurepaea (SMBP) achieves much improved product solubility and easier purification without compromising the hydrophobin properties. SMBP-HFBII shows a critical micelle concentration (CMC) < 0.5 mg/l and is prone to form stable nanobubbles of 50-60 nm radius and thin films at liquid-air interfaces. The adsorption of SMBP-HFBII on hydrophilic substrates (mica and glass) generates homogeneous coatings that reverse their wettability increasing the water contact angle (WCA) 460% and 70%, respectively. Adsorbed SMBP-HFBII also demonstrated a slight increase of Botrytis cinerea spores’ adhesion to coated glass, dependent on pH and spore concentration. This work shows that fusion HFBs can expand the application potentials of their native parent proteins as the fusion provides a powerful avenue to manipulate their functionalities. The lack of knowledge on their structure-function relationships and uncontrolled self-assembling can be subsidized by the addition of domains that influence the overall protein performance. This fusion strategy also enhances HFB productivity, facilitating their use in research and industry.enHydrophobinInterfacialSurfactantThesis or Dissertation