Evidence of Small Fungal Cysteine-Rich Proteins Acting as Biosurfactants and Self-Assembling into Large Fibers.

Fungi produce surface-active proteins, among which hydrophobins are the most characterized and attractive also for their ability to form functional amyloids. Our most recent findings show that these abilities are shared with other classes of fungal proteins. Indeed, in this paper, we compared the characteristics of a class I hydrophobin (Vmh2 from Pleurotus ostreatus) and an unknown protein (named PAC3), extracted from the marine fungal strain Acremonium sclerotigenum, which does not belong to the same protein family based on its sequence features. They both proved to be good biosurfactants, stabilizing emulsions in several conditions (concentration, pH, and salinity) and decreasing surface tension to a comparable value to that of some synthetic surfactants. After that, we observed for both Vmh2 and PAC3 the formation of giant fibers without the need for harsh conditions or long incubation time, a remarkable ability herein reported for the first time.

Sorafenib-Loaded PLGA Carriers for Enhanced Drug Delivery and Cellular Uptake in Liver Cancer Cells.

Introduction: Currently, conventional treatments of hepatocellular carcinoma (HCC) are not selective enough for tumor tissue and lead to multidrug resistance and drug toxicity. Although sorafenib (SOR) is the standard first-line systemic therapy approved for the clinical treatment of HCC, its poor aqueous solubility and rapid clearance result in low absorption efficiency and severely limit its use for local treatment. Methods: Herein, we present the synthesis of biodegradable polymeric Poly (D, L-Lactide-co-glycolide) (PLGA) particles loaded with SOR (PS) by emulsion-solvent evaporation process. The particles are carefully characterized focusing on particle size, surface charge, morphology, drug loading content, encapsulation efficiency, in vitro stability, drug release behaviour and tested on HepG2 cells. Additionally, PLGA particles have been coupled on side emitting optical fibers (seOF) integrated in a microfluidic device for light-triggered local release. Results: PS have a size of 248 nm, tunable surface charge and a uniform and spherical shape without aggregation. PS shows encapsulation efficiency of 89.7% and the highest drug loading (8.9%) between the SOR-loaded PLGA formulations. Treating HepG2 cells with PS containing SOR at 7.5 µM their viability is dampened to 40%, 30% and 17% after 48, 129 and 168 hours of incubation, respectively. Conclusion: The high PS stability, their sustained release profile and the rapid cellular uptake corroborate the enhanced cytotoxicity effect on HepG2. With the prospect of developing biomedical tools to control the spatial and temporal release of drugs, we successfully demonstrated the potentiality of seOF for light-triggered local release of the carriers. Our prototypical system paves the way to new devices integrating microfluidics, optical fibers, and advanced carriers capable to deliver minimally invasive locoregional cancer treatments.

Innovative surface bio-functionalization by fungal hydrophobins and their engineered variants.

Research on innovative surface functionalization strategies to develop materials with high added value is particularly challenging since this process is a crucial step in a wide range of fields (i.e., biomedical, biosensing, and food packaging). Up to now, the main applied derivatization methods require hazardous and poorly biocompatible reagents, harsh conditions of temperature and pressure, and are time consuming and cost effective. The discovery of biomolecules able to adhere by non-covalent bonds on several surfaces paves the way for their employment as a replacement of chemical processes. A simple, fast, and environment-friendly method of achieving modification of chemically inert surfaces is offered by hydrophobins, small amphiphilic proteins produced by filamentous fungi. Due to their structural characteristics, they form stable protein layers at interfaces, serving as anchoring points that can strongly bind molecules of interest. In addition, genetic engineering techniques allow the production of hydrophobins fused to a wide spectrum of relevant proteins, providing further benefits in term of time and ease of the process. In fact, it is possible to bio-functionalize materials by simply dip-casting, or by direct deposition, rendering them exploitable, for example, in the development of biomedical and biosensing platforms.

The growth of marine fungi on seaweed polysaccharides produces cerato-platanin and hydrophobin self-assembling proteins.

The marine fungi Paradendryphiela salina and Talaromyces pinophilus degrade and assimilate complex substrates from plants and seaweed. Additionally, these fungi secrete surface-active proteins, identified as cerato-platanins and hydrophobins. These hydrophobic proteins have the ability to self-assemble forming amyloid-like aggregates and play an essential role in the growth and development of the filamentous fungi. It is the first time that one cerato-platanin (CP) is identified and isolated from P. salina (PsCP) and two Class I hydrophobins (HFBs) from T. pinophilus (TpHYD1 and TpHYD2). Furthermore, it is possible to extract cerato-platanins and hydrophobins using marine fungi that can feed on seaweed biomass, and through a submerged liquid fermentation process. The propensity to aggregate of these proteins has been analyzed using different techniques such as Thioflavin T fluorescence assay, Fourier-transform Infrared Spectroscopy, and Atomic Force Microscopy. Here, we show that the formation of aggregates of PsCP and TpHYD, was influenced by the carbon source from seaweed. This study highlighted the potential of these self-assembling proteins generated from a fermentation process with marine fungi and with promising properties such as conformational plasticity with extensive applications in biotechnology, pharmacy, nanotechnology, and biomedicine.

A versatile and user-friendly approach for the analysis of proteins in ancient and historical objects.

Identification and characterization of ancient proteins still require technical developments towards non-invasiveness, sensitivity, versatility and ease of use of the analyses. We report that the enzyme functionalized films, described in Cicatiello et al. (2018), can be used efficiently on the surface of different objects ranging from fixative-coated paper to canvas to the coating on an albumen photograph, as well as the much harder surfaces of ivory objects and the proteinaceous binders in the decoration of a wooden Egyptian coffin. The mixture of digested peptides that are efficiently captured on the functionalized surface are also amenable to LC-MS/MS analysis, which is necessary to confidently identify chemical modifications induced upon degradation, in order to characterize the conservation state of proteins. Moreover, in a two-step procedure, we have combined the trypsin functionalized film with a PNGaseF functionalized film, which adds a deglycosylation pretreatment allowing improved detection of glycosylated proteins. SIGNIFICANCE: User friendly trypsin functionalized films were implemented to expand their potential as versatile, modular tools that can be widely exploited in the world of diagnosis of cultural heritage objects, ancient proteins, and palaeoproteomics: a procedure that could be carried out by conservators or archaeologists first on-site and later analysed with standard MS techniques.

Cerato-Platanins from Marine Fungi as Effective Protein Biosurfactants and Bioemulsifiers.

Two fungal strains, Aspergillus terreus MUT 271 and Trichoderma harzianum MUT 290, isolated from a Mediterranean marine site chronically pervaded by oil spills, can use crude oil as sole carbon source. Herein, these strains were investigated as producers of biosurfactants, apt to solubilize organic molecules as a preliminary step to metabolize them. Both fungi secreted low molecular weight proteins identified as cerato-platanins, small, conserved, hydrophobic proteins, included among the fungal surface-active proteins. Both proteins were able to stabilize emulsions, and their capacity was comparable to that of other biosurfactant proteins and to commercially available surfactants. Moreover, the cerato-platanin from T. harzianum was able to lower the surface tension value to a larger extent than the similar protein from A. terreus and other amphiphilic proteins from fungi. Both cerato-platanins were able to make hydrophilic a hydrophobic surface, such as hydrophobins, and to form a stable layer, not removable even after surface washing. To the best of our knowledge, the ability of cerato-platanins to work both as biosurfactant and bioemulsifier is herein demonstrated for the first time.

Characterization of a Surface-Active Protein Extracted from a Marine Strain of Penicillium chrysogenum.

Marine microorganisms represent a reservoir of new promising secondary metabolites. Surface-active proteins with good emulsification activity can be isolated from fungal species that inhabit the marine environment and can be promising candidates for different biotechnological applications. In this study a novel surface-active protein, named Sap-Pc, was purified from a marine strain of Penicillium chrysogenum. The effect of salt concentration and temperature on protein production was analyzed, and a purification method was set up. The purified protein, identified as Pc13g06930, was annotated as a hypothetical protein. It was able to form emulsions, which were stable for at least one month, with an emulsification index comparable to that of other known surface-active proteins. The surface tension reduction was analyzed as function of protein concentration and a critical micellar concentration of 2 µM was determined. At neutral or alkaline pH, secondary structure changes were monitored over time, concurrently with the appearance of protein precipitation. Formation of amyloid-like fibrils of SAP-Pc was demonstrated by spectroscopic and microscopic analyses. Moreover, the effect of protein concentration, a parameter affecting kinetics of fibril formation, was investigated and an on-pathway involvement of micellar aggregates during the fibril formation process was suggested.

Minimally Invasive and Portable Method for the Identification of Proteins in Ancient Paintings.

A novel method for the analysis of proteinaceous materials present on painted surfaces was developed by taking advantage of the adhesive ability of some fungal proteins which can form a stable and homogeneous layer on flexible transparency sheets able to capture trypsin in a fully active form. We demonstrated that the bioactive sheets were able to efficiently digest proteins, present as such, on surfaces of painted tests and historical samples, releasing peptides that can allow an easy and confident identification of the proteinaceous binders by standard bottom-up proteomic approach. By this method there is no need: (i) to transport the artifacts and (ii) to remove, even at micro level, a sample from the object. The ingenuity of the method lies in the easily accommodated sampling coupled with a minimal invasiveness.

New clues into the self-assembly of Vmh2, a basidiomycota class I hydrophobin.

Hydrophobins are fungal proteins that can self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces. Class I hydrophobin aggregates resemble amyloid fibrils, sharing some features with them. Here, five site-directed mutants of Vmh2, a member of basidiomycota class I hydrophobins, were designed and characterized to elucidate the molecular determinants playing a key role in class I hydrophobin self-assembly. The mechanism of fibril formation proposed for Vmh2 foresees that the triggering event is the destabilization of a specific loop (L1), leading to the formation of a ß-hairpin, which in turn generates the ß-spine of the amyloid fibril.

A step forward in laccase exploitation: Recombinant production and evaluation of techno-economic feasibility of the process.

Protein heterologous production offers viable opportunities to tailor laccase properties to specific industrial needs. The high redox potential laccase POXA1b from Pleurotus ostreatus was chosen as case study of marketable enzyme, due to its desirable properties in terms of activity/stability profile, and already assessed applicability. POXA1b was heterologously produced in Pichia pastoris by investigating the effect of inducible and constitutive expression systems on both the yield and the cost of its production. System performances were first assessed in shaken-flasks and then scaled-up in bioreactor. The production level obtained in the inducible system is 42U/mL, while the activity value achieved with the constitutive one is 60U/mL, the highest obtained in constitutive systems so far. The economic feasibility of recombinant laccase production was simulated, describing the case of an Italian small-medium enterprise. Two scenarios were evaluated: Scenario (I) production based on methanol inducible system; Scenario (II) production based on the constitutive system, fed with glycerol. At all the scales the glycerol-based fermentation is more economic than the methanol-based one. The price forecast for rPOXA1b production is 0.34€kU-1 for glycerol-based process, and is very competitive with the current price of commercial laccase.

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins.

Class I hydrophobins produced from fungi are amongst the first proteins recognized as functional amyloids. They are amphiphilic proteins involved in the formation of aerial structures such as spores or fruiting bodies. They form chemically robust layers which can only be dissolved in strong acids. These layers adhere to different surfaces, changing their wettability, and allow the binding of other proteins. Herein, the modification of diverse types of surfaces with Class I hydrophobins is reported, highlighting the applications of the coated surfaces. Indeed, these coatings can be exploited in several fields, spanning from biomedical to industrial applications, which include biosensing and textile manufacturing.

Hydrophobin coating prevents Staphylococcus epidermidis biofilm formation on different surfaces.

Staphylococcus epidermidis is a significant nosocomial pathogen in predisposed hosts because of its capability of forming a biofilm on indwelling medical devices. The initial stage of biofilm formation has a key role in S. epidermidis abiotic surface colonization. Recently, many strategies have been developed to create new anti-biofilm surfaces able to control bacterial adhesion mechanisms. In this work, the self-assembled amphiphilic layers formed by two fungal hydrophobins (Vmh2 and Pac3) have proven to be able to reduce the biofilm formed by different strains of S. epidermidis on polystyrene surfaces. The reduction in the biofilm thickness on the coated surfaces and the preservation of cell vitality have been demonstrated through confocal laser scanning microscope analysis. Moreover, the anti-biofilm efficiency of the self-assembled layers on different medically relevant materials has also been demonstrated using a CDC biofilm reactor.

Self-assembly of two hydrophobins from marine fungi affected by interaction with surfaces.

Hydrophobins are amphiphilic fungal proteins endowed with peculiar characteristics, such as a high surface activity and an interface triggered self-assembly. Several applications of these proteins have been proposed in the food, cosmetics and biomedical fields. Moreover, their use as proteinaceous coatings can be effective for materials and nanomaterials applications. The discovery of novel hydrophobins with diverse properties may be advantageous from both the scientific and industrial points of view. Stressful environmental conditions of fungal growth may induce the production of proteins with peculiar features. Two Class I hydrophobins from fungi isolated from marine environment have been recently purified. Herein, their propensity to aggregate forming nanometric fibrillar structures has been compared, using different techniques, such as circular dichroism, dynamic light scattering and Thioflavin T fluorescence assay. Furthermore, TEM and AFM images indicate that the interaction of these proteins with specific surfaces, are crucial in the formation of amyloid fibrils and in the assembly morphologies. These self-assembling proteins show promising properties as bio-coating for different materials via a green process. Biotechnol. Bioeng. 2017;114: 2173-2186. © 2017 Wiley Periodicals, Inc.

Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein.

A fusion protein designed in order to combine the fluorescence emission of the Green Fluorescent Protein (GFP) with the adhesion ability of the class I hydrophobin Vmh2 was heterologously produced in the yeast Pichia pastoris. The Vmh2-GFP fusion protein has proven to be a smart and effective tool for the study of Vmh2 self-assembling. Since the two proteins were linked by the specific cutting site of the thrombin, the fusion protein was used as the active biological element in the realization of a thrombin biosensor. When the thrombin present in the target solution specifically hydrolyzed its cleavage sequence, a consequent decrease in the fluorescence intensity of the sample could be observed. The Vmh2-GFP based assay allowed quantification of thrombin in solution with a detection limit of 2.27aM. The specificity of the assay with respect to other proteases and proteins granted the measurement of thrombin added to healthy human plasma with same high sensitivity and a limit of detection of 2.3aM. Further advantages of the developed biosensor are the simplicity of its design and preparation, and the low requirements in terms of samples, reagents and time.

Marine fungi as source of new hydrophobins.

Hydrophobins have been described as the most powerful surface-active proteins known. They are produced by filamentous fungi and exhibit a distinct amphiphilic structure determining their self-assembly at hydrophilic-hydrophobic interfaces and surfactant properties which have been demonstrated to be useful for several biotechnological applications. The marine environment represents a vast natural resource of new molecules produced by organisms growing in various stressful conditions. This study was focused on the screening of 100 marine fungi from Mycoteca Universitatis Taurinensis (MUT) for the identification of new hydrophobins. Four different methods were set up to extract hydrophobins of class I and II, from the mycelium or the culture broth of fungi. Six fungi were selected as the best producers of hydrophobins endowed with different characteristics. Their ability to form stable amphiphilic films and their emulsification capacity in the presence of olive oil was evaluated.

Efficient immobilization of a fungal laccase and its exploitation in fruit juice clarification.

The clarification step represents, in fruit juices industries, a bottleneck process because residual phenols cause severe haze formation affecting juice quality and impairing customers acceptance. An enzymatic step can be efficiently integrated in the process, and use of immobilized enzymes entails an economical advantage. In this work, covalent immobilization of recombinant POXA1b laccase from Pleurotus ostreatus on epoxy activated poly(methacrylate) beads was optimized thanks to a Response Surface Methodologies approach. Through regression analysis the process was well fitted by a quadratic polynomial equation (R(2)=0.9367, adjusted R(2)=0.8226) under which laccase activity reached 2000 ± 100 Ug(-1) of beads, with an immobilization efficiency of 98%. The immobilized biocatalyst was characterized and then tested in fruit juice clarification reaching up to 45% phenol reduction, without affecting health-effective flavanones content. Furthermore, laccase treated juice displays an improved sensory profile, due to the reduction of vinyl guaiacol, a potent off-flavor possessing a peppery/spicy aroma.

Fungal laccases degradation of endocrine disrupting compounds.

Over the past decades, water pollution by trace organic compounds (ng/L) has become one of the key environmental issues in developed countries. This is the case of the emerging contaminants called endocrine disrupting compounds (EDCs). EDCs are a new class of environmental pollutants able to mimic or antagonize the effects of endogenous hormones, and are recently drawing scientific and public attention. Their widespread presence in the environment solicits the need of their removal from the contaminated sites. One promising approach to face this challenge consists in the use of enzymatic systems able to react with these molecules. Among the possible enzymes, oxidative enzymes are attracting increasing attention because of their versatility, the possibility to produce them on large scale, and to modify their properties. In this study five different EDCs were treated with four different fungal laccases, also in the presence of both synthetic and natural mediators. Mediators significantly increased the efficiency of the enzymatic treatment, promoting the degradation of substrates recalcitrant to laccase oxidation. The laccase showing the best performances was chosen to further investigate its oxidative capabilities against micropollutant mixtures. Improvement of enzyme performances in nonylphenol degradation rate was achieved through immobilization on glass beads.