Hydrophobin Research Articles

Simple Method to Assess Foam Structure and Stability using Hydrophobin and BSA as Model Systems.

The properties and arrangement of surface-active molecules at air-water interfaces influence foam stability and bubble shape. Such multiscale-relationships necessitate a well-conducted analysis of mesoscopic foam properties. We introduce a novel automated and precise method to characterize bubble growth, size distribution and shape based on image analysis and using the machine learning algorithm Cellpose. Studying the temporal evolution of bubble size and shape facilitates conclusions on foam stability. The addition of two sets of masks, for tiny bubbles and large bubbles, provides for a high precision of analysis. A python script for analysis of the evolution of bubble diameter, circularity and dispersity is provided in the Supporting Information. Using foams stabilized by bovine serum albumin (BSA), hydrophobin (HP), and blends thereof, we show how this technique can be used to precisely characterize foam structures. Foams stabilized by HP show a significantly increased foam stability and rounder bubble shape than BSA-stabilized foams. These differences are induced by the different molecular structure of the two proteins. Our study shows that the proposed method provides an efficient way to analyze relevant foam properties in detail and at low cost, with higher precision than conventional methods of image analysis.

Identification and characterization of a hydrophobin Vmh3 from Pleurotus ostreatus

Hydrophobins (HPs) are relatively small surface-active proteins of fungal origin. Being an industrially important protein, isolation of new molecules from GRAS (Generally Regarded as Safe) strains like mushrooms is the need of the time. In the present work, hydrophobin Vmh3-1 is isolated, purified, and identified from a culture broth and vegetative mycelia of Pleurotus ostreatus grown in a Potato dextrose broth (PDB) in static culture conditions. Purified proteins from the broth and the cell wall showed bands of 11 kDa and 17 kDa when analyzed on SDS-PAGE. Hydrophobin Vmh3-1 was identified in purified protein samples by the Orbitrap-HR-LC-MS/MS analysis with a maximum of 66% sequence coverage. The amphipathic nature of the protein was revealed by an increase in the water contact angle (WCA) of the hydrophilic surface of glass by 87% as well as a decrease in the WCA of the hydrophobic surface of Teflon by 19%. The emulsification property was tested with food-grade oils and Hexane. A maximum activity (EI 24) of 87.64% was recorded for Sunflower oil. In CD (Circular dichroism) spectra, Vmh3-1 showed the typical spectra of hydrophobin with a dominance of β-sheets (51%) in the secondary structure and a minimum percentage of the α-helix (2%). The protein did not show a self-aggregating property on vigorous shaking making it suitable for numerous industrial applications. The identification of Vmh3-1 with detailed amino acid sequencing and the characterization of the protein to evaluate its potential in surface modifications for various industrial applications is demonstrated herein for the first time.

Molecular Dynamics Simulations of Nanostructures Formed by Hydrophobins and Oil in Seawater.

Classical molecular dynamics simulations using the Martini coarse-grained force field were performed to study oil nanodroplets surrounded by fungal hydrophobin (HP) proteins in seawater. The class I EAS and the class II HFBII HPs were studied along with two model oils, namely, benzene and n-decane. Both HPs exhibit free energy minima at the oil-seawater interface, which is deeper in benzene compared to the n-decane systems. Larger constraint forces are required to keep both HPs within the n-decane phase compared to inside benzene, with HFBII being more affine to benzene compared to EAS. Smaller surface tensions are observed at benzene-seawater interfaces coated with HPs compared to their n-decane counterparts. In the latter the surface tension remains unchanged upon increases in the concentration of HPs, whereas in benzene systems adding more HPs lead to decreases in surface tension. EAS has a larger tendency to cluster together in the interface compared to HFBII, with both HPs having larger coordination numbers when surrounding benzene droplets compared to when they are around n-decane nanoblobs. The HP-oil nanostructures in seawater examined have radii of gyration ranging between 2 and 12 nm, where the n-decane structures are larger and have more irregular shapes compared to the benzene systems. The n-decane molecules within the nanostructures form a compact spherical core, with the HPs partially covering its surface and clustering together, conferring irregular shapes to the nanostructures. The EAS with n-decane structures are larger and have more irregular shapes compared to their HFBII counterparts. In contrast, in the HP-benzene structures both HPs tend to penetrate the oil part of the droplet. The HFBII-benzene structures having the larger oil/HP ratios examined tend to be more compact and spherical compared to their EAS counterparts; however, some of the HFBII-benzene systems that have smaller oil/HP ratios have a more elongated structure compared to their EAS counterparts. This simulation study provides insights into HP-oil nanostructures that are smaller than the oil droplets and gas bubbles recently studied in experiments and, thus, might be challenging to examine with experimental techniques.

Different Hydrophobins of Fusarium graminearum Are Involved in Hyphal Growth, Attachment, Water-Air Interface Penetration and Plant Infection.

Hydrophobins (HPs) are small secreted fungal proteins possibly involved in several processes such as formation of fungal aerial structures, attachment to hydrophobic surfaces, interaction with the environment and protection against the host defense system. The genome of the necrotrophic plant pathogen Fusarium graminearum contains five genes encoding for HPs (FgHyd1-5). Single and triple FgHyd mutants were produced and characterized. A reduced growth was observed when the ΔFghyd2 and the three triple mutants including the deletion of FgHyd2 were grown in complete or minimal medium. Surprisingly, the growth of these mutants was similar to wild-type when grown under ionic, osmotic or oxidative stress conditions. All the mutant strains confirmed the ability to develop conidia and perithecia, suggesting that the FgHyds are not involved in normal development of asexual and sexual structures. A reduction in the ability of hyphae to penetrate through the water-air interface was observed for the single mutants ΔFghyd2 and ΔFghyd3 as well as for the triple mutants including the deletion of FgHyd2 and FgHyd3. Besides, ΔFghyd3 and the triple mutant ΔFghyd234 were also affected in the attachment to hydrophobic surface. Indeed, wheat infection experiments showed a reduction of symptomatic spikelets for ΔFghyd2 and ΔFghyd3 and the triple mutants only when spray inoculation was performed. This result could be ascribed to the affected ability of mutants deleted of FgHyd2 and FgHyd3 to penetrate through the water-air interface and to attach to hydrophobic surfaces such as the spike tissue. This hypothesis is strengthened by a histological analysis, performed by fluorescence microscopy, showing no defects in the morphology of infection structures produced by mutant strains. Interestingly, triple hydrophobin mutants were significantly more inhibited than wild-type by the treatment with a systemic triazole fungicide, while no defects at the cell wall level were observed.

Chemical shifts assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus

Chemical shifts assignments of the RodA hydrophobin from the opportunistic pathogen Aspergillus fumigatus

Production of Hydrophobins from fungi

Abstract Hydrophobins (HPs) are industrially important surface active, amphipathic proteins produced by fungi. There are many applications reported for HPs in the literature notably as, agents for enhancing bioavailability of water insoluble drugs, food stabilizers, antifouling agents for biomedical devices like catheters, fusion partner for recombinant proteins for purification, low friction coatings on biomaterials, immobilizing enzymes in biosensors, etc. However, there are limitations for industrial scale production of HPs. Various methods have been reported for their production e.g. use of wild fungi from natural hydrophobic environments, use of modified bioreactors for submerged and solid state fermentation and recombinant homologous as well as heterologous microbes. Knowing the industrial importance of HPs many reviews have been published focusing on technical and medical applications of these proteins; however there is no comprehensive overview of HP production in the literature. This review summarizes the efforts made to improve yields of HPs by various bioprocesses and also highlights the strategies designed to overcome problems of low yield of HPs.