Browsing by Subject "Hydrophobicity"

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Toward effects of hydrophobicity on biosurfactant production by Bacillus subtilis isolates from crude-oil-exposed environments
(2024) Hashemi, Seyedeh Zahra; Fooladi, Jamshid; Vahidinasab, Maliheh; Hubel, Philipp; Pfannstiel, Jens; Pillai, Evelina; Hrenn, Holger; Hausmann, Rudolf; Lilge, Lars
Background: Due to their structural features, biosurfactants reveal promising physicochemical properties, making them interesting for various applications in different fields, such as the food, cosmetics, agriculture, and bioremediation sectors. In particular, the bioproduction of surfactin, one of the most potent microbially synthesized biosurfactant molecules, is of great interest. However, since the wild-type productivities are comparably low, stimulatory environmental conditions have to be identified for improved bioproduction This study aims to find a correlation between the hydrophobicity and production of the biosurfactant surfactin by B. subtilis isolates from crude-oil-contaminated soil and water. Methods: The surfactin production yield was characterized in adapted batch cultivations using high-performance thin-layer liquid chromatography (HPTLC). Defined hydrophobic environmental conditions were achieved by supplementation with hexadecane or polystyrene beads, and the effects on biosurfactant production were measured. Adaptations at the protein level were analyzed using mass spectrometry measurements. Results: The correlation between hydrophobicity and surfactin production was characterized using Bacillus subtilis strains ZH1 and P7 isolated from crude-oil-contaminated soil and water. Since these isolates show the biodegradation of crude oil and hexadecane as hydrophobic substrates, respectively, a first-time approach, using polystyrene beads, was applied to provide a hydrophobic environment. Interestingly, contrary to popular opinion, reduced biosurfactant production was determined. Using mass spectrometric approaches, the physiological effects of co-cultivation and the cellular response at the protein level were investigated, resulting in altered quantities of stress proteins and proteins involved in the carbon metabolism counter to polystyrene beads. Conclusions: Contrary to common opinion, increasing hydrophobicity does not have a stimulating effect, and even reduces the effect on the bioproduction of surfactin as the main biosurfactant using selected B. subtilis strains.
Untersuchungen zur autonomen und YidC-vermittelten Membraninsertion von Pf3 coat-Protein mit Hilfe Fluoreszenz-spektroskopischer Einzelmolekülmessungen
(2011) Schönbauer, Anne-Kathrin; Kuhn, Andreas
Pf3 coat is the capsid protein of the bacteriophage Pf3. The phage leaves the host cell by continuous extrusion without damaging the cell. The protein itself consists of 44 amino acid residues and has a rod-like shape. Because of its simple structure, the protein needs only the help of the insertase YidC to insert into the bacterial inner membrane. 3L-Pf3 coat, a protein mutant with three additional leucine residues in the center of the transmembrane region (TMD), has an increased hydrophobicity. It is independent of YidC and inserts into the membrane autonomously (Serek et al., 2004). In this work, a newly developed physical method was used to find out whether the elongation or the increased hydrophobicity accounts for the autonomous insertion of the protein. For this reason, two new protein mutants were constructed. Each mutant has only one of the changed properties of the 3L-Pf3 coat protein: GAT-Pf3 coat has an elongated TMD with three additional residues (glycine, alanin and threonine). The second mutant, 2M-Pf3 coat, shows an increased hydrophobicity due to the substitution of two alanine residues by two methionine residues at the positions 30 and 31. So it had an increased hydrophobicity like 3L-Pf3 coat. The above mentioned proteins, wt-Pf3 coat and its mutants, were modified with a fluorescent label to follow the proteins with optical methods. The Proteins were first modified with a single cysteine and then labeled by a fluorescent marker, Atto520 maleimid. Proteins with a labeled N-terminal tail were called NC-Pf3 coat, whereas CC-Pf3 coat had a labeled C-terminal tail. In addition, the orientation of the protein in the membrane was identified by quenching the fluorescence of the NC- and CC- labeled proteins. A new method employing single molecules was developed using fluorescence correlation spectroscopy. This method allows real time observations of binding and insertion of the protein into semisynthetical liposomes. By using fluorescent quenching the membrane insertion and binding were distinguished. It became clear that both the elongation of the TMD as well as an increased hydrophobicity play a crucial role in the autonomous insertion of the protein into the membrane. Therefore, the interaction between the hydrophobic region of the protein and the hydrophobic core region of the membrane is important for the binding of the protein and its insertion into the membrane.