The essential role of aggregation for the emulsifying ability of a fungal CYS-rich protein.

Biosurfactants are in demand by the global market as natural commodities suitable for incorporation into commercial products or utilization in environmental applications. Fungi are promising producers of these molecules and have garnered interest also for their metabolic capabilities in efficiently utilizing recalcitrant and complex substrates, like hydrocarbons, plastic, etc. Within this framework, biosurfactants produced by two Fusarium solani fungal strains, isolated from plastic waste-contaminated landfill soils, were analyzed. Mycelia of these fungi were grown in the presence of 5% olive oil to drive biosurfactant production. The characterization of the emulsifying and surfactant capacity of these extracts highlighted that two different components are involved. A protein was purified and identified as a CFEM (common in fungal extracellular membrane) containing domain, revealing a good propensity to stabilize emulsions only in its aggregate form. On the other hand, an unidentified cationic smaller molecule exhibits the ability to reduce surface tension. Based on the 3D structural model of the protein, a plausible mechanism for the formation of very stable aggregates, endowed with the emulsifying ability, is proposed. KEY POINTS: • Two Fusarium solani strains are analyzed for their surfactant production. • A cationic surfactant is produced, exhibiting the ability to remarkably reduce surface tension. • An identified protein reveals a good propensity to stabilize emulsions only in its aggregate form.

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.

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 laccase mediator system at carbon nanotubes for anthracene oxidation and femtomolar electrochemical biosensing.

We investigated the use of POXA1b laccase from Pleurotus ostreatus for the oxidation of anthracene into anthraquinone. We show that different pathways can occur depending on the nature of the redox mediator combined to laccase, leading to different structural isomers. The laccase combined with 2,2'-azine-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) leads to the formation of 1,4-anthraquinone and/or 1,2-anthraquinone. The unprecedented role of carbon nanotubes (CNTs) as redox mediators for oxidation of anthracene into 9,10-anthraquinone is shown and corroborated by density-functional theory (DFT) calculations. Owing to the efficient adsorption of anthraquinones at CNT electrodes, anthracene can be detected with low limit-of-detection using either laccase in solution, CNT-supported laccase or laccase immobilized at magnetic beads exploiting the adhesive property of a chimeric hydrophobin-laccase.

A machine learning-enhanced biosensor for mercury detection based on an hydrophobin chimera.

Marine waters are becoming contaminated by diverse pollutants at a fast rate, and detection of these water pollutants has become a major concern in recent years. Among these, mercury is considered the most toxic element for human health. At present, despite the commonly used methods for its detection are accurate, they often require sophisticated equipments, have relatively high costs, are demanding and time-consuming. Herein a novel solution to detect mercury (II) pollution in sea water is proposed, and an easy and portable detection method has been developed. Indeed, a hydrophobin based chimera able to both adhere to polystyrene multiwell plates and bind mercury (II) with a consequent fluorescent decrease was designed. The chimera was the recognition element in a fluorescence-based biosensor able to detect mercury (II) in the nM range. Indeed, this biosensor specifically measure Hg2+ concentration also in the presence of other metals, reaching a limit of detection of 0.4 nM in tap water and 0.3 nM in sea water. Moreover, the developed biosensor was coupled to machine learning methodologies with the big advantage of predicting mercury concentration levels without the use of classical reader devices, thus allowing in situ monitoring of sea pollution by non-skilled personnel.

Immobilization of Antibodies by Genetic Fusion to a Fungal Self-Assembling Adhesive Protein.

Although antibody immobilization on solid surfaces is extensively used in several applications, including immunoassays, biosensors, and affinity chromatography, some issues are still challenging. Self-assembling protein layers can be used to coat easily different surfaces by direct deposition. A specific biofunctional layer can be formed using genetic engineering techniques to express fused proteins acting as self-immobilizing antibodies. In this study, fusion proteins combining the self-assembling adhesive properties of a fungal hydrophobin and the functionality of the single chain fragment variables (ScFvs) of two antibodies were produced. The chosen ScFvs are able to recognize marine toxins associated with algal blooms, saxitoxin, and domoic acid, which can bioaccumulate in shellfish and herbivorous fish causing food poisoning. ScFvs fused to hydrophobin Vmh2 from Pleurotus ostreatus were produced in Escherichia coli and recovered from the inclusion bodies. The two fusion proteins retained the functionality of both moieties, being able to adhere on magnetic beads and to recognize and bind the two neurotoxins, even with different performances. Our immobilization procedure is innovative and very easy to implement because it allows the direct functionalization of magnetic beads with ScFvs, without any surface modification. Two different detection principles, electrochemical and optical, were adopted, thus achieving a versatile platform suitable for different antigen detection methods. The sensitivity of the saxitoxin optical biosensor [limit of detection (LOD) 1.7 pg/ml] is comparable to the most sensitive saxitoxin immunosensors developed until now.

Trichoderma harzianum cerato-platanin enhances hydrolysis of lignocellulosic materials.

Considering its worldwide abundance, cellulose can be a suitable candidate to replace the fossil oil-based materials, even if its potential is still untapped, due to some scientific and technical gaps. This work offers new possibilities demonstrating for the first time the ability of a cerato-platanin, a small fungal protein, to valorize lignocellulosic Agri-food Wastes. Indeed, cerato-platanins can loosen cellulose rendering it more accessible to hydrolytic attack. The cerato-platanin ThCP from a marine strain of Trichoderma harzianum, characterized as an efficient biosurfactant protein, has proven able to efficiently pre-treat apple pomace, obtaining a sugar conversion yield of 65%. Moreover, when used in combination with a laccase enzyme, a notable increase in the sugar conversion yield was measured. Similar results were also obtained when other wastes, coffee silverskin and potato peel, were pre-treated. With respect to the widespread laccase pre-treatments, this new pre-treatment approach minimizes process time, increasing energy efficiency.

Self-assembling thermostable chimeras as new platform for arsenic biosensing.

The correct immobilization and orientation of enzymes on nanosurfaces is a crucial step either for the realization of biosensors, as well as to guarantee the efficacy of the developed biomaterials. In this work we produced two versions of a chimeric protein, namely ArsC-Vmh2 and Vmh2-ArsC, which combined the self-assembling properties of Vmh2, a hydrophobin from Pleurotus ostreatus, with that of TtArsC, a thermophilic arsenate reductase from Thermus thermophilus; both chimeras were heterologously expressed in Escherichia coli and purified from inclusion bodies. They were characterized for their enzymatic capability to reduce As(V) into As(III), as well as for their immobilization properties on polystyrene and gold in comparison to the native TtArsC. The chimeric proteins immobilized on polystyrene can be reused up to three times and stored for 15 days with 50% of activity loss. Immobilization on gold electrodes showed that both chimeras follow a classic Langmuir isotherm model towards As(III) recognition, with an association constant (KAsIII) between As(III) and the immobilized enzyme, equal to 650 (± 100) L mol-1 for ArsC-Vmh2 and to 1200 (± 300) L mol-1 for Vmh2-ArsC. The results demonstrate that gold-immobilized ArsC-Vmh2 and Vmh2-ArsC can be exploited as electrochemical biosensors to detect As(III).

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.

Hydrolyzable vs. Condensed Wood Tannins for Bio-based Antioxidant Coatings: Superior Properties of Quebracho Tannins.

Tannins have always been the subject of great interest for their countless properties, first of all their ability to produce functional coatings on a variety of materials. We report herein a comparative evaluation of the antioxidant properties of wood tannin-based coated substrates. In particular, nylon membrane filters were functionalized with chestnut (hydrolyzable) or quebracho (condensed) tannins by dip coating under different conditions. The efficiency of functionalization was evaluated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) assays, which invariably highlighted the superior ability of condensed tannins to induce the formation of a functional and robust coating. The results of the antioxidant assays revealed also the deleterious effects of aerial or enzymatic oxidation conditions on substrate functionalization, being more significant in the case of hydrolyzable tannins. On the other hand, the use of oxidizing conditions allowed to obtain more stable coatings, still exhibiting good antioxidant properties, in the case of condensed tannins. The presence of iron ions did not lead to a significant improvement of the coating efficiency for either tannins. The systematic approach used in this work provides novel and useful information for the optimal exploitation of tannins in antioxidant functional coatings.

Development of anti-bacterial surfaces using a hydrophobin chimeric protein.

The search for new approaches for developing antimicrobial surfaces is a challenge of great urgency to prevent and control microbial growth on surfaces. The strategy herein proposed relies on the design of a new, simple and general tool for creating antimicrobial surfaces, based on a hydrophobin chimeric protein which fuses the adhesive self-assembling class I hydrophobin Vmh2 from Pleurotus ostreatus to the human antimicrobial peptide LL-37. The recombinant LL37-Vmh2 protein displayed both the adhesive and the antimicrobic properties of its members, and when deposited on polystyrene surface, a positive effect due to the fusion was observed in terms of both efficacy and versatility of the coating. Indeed, the chimeric protein significantly enlarges the range of pathogens affected by Vmh2 layer rendering it able to inhibit three Gram-positive and two Gram-negative pathogens, selected among the renowned biofilm producer bacteria. Confocal Laser Scanning Microscopy analysis performed on Staphylococcus epidermidis biofilms formed on coated surfaces proved that, besides inhibiting biofilm formation, the LL37-Vmh2 coating also displayed biocidal activity, since dead cells were present in the biofilm layer. The reported results open new perspectives in various fields of application of LL37, and of antimicrobial peptides in general. LL37-Vmh2 increases the inventory of chimeric hydrophobins, further proving their effectiveness and versatility in surface functionalization.

From Graphite to Laccase Biofunctionalized Few-Layer Graphene: A "One Pot" Approach Using a Chimeric Enzyme.

A chimeric enzyme based on the genetic fusion of a laccase with a hydrophobin domain was employed to functionalize few-layer graphene, previously exfoliated from graphite in the presence of the hydrophobin. The as-produced, biofunctionalized few-layer graphene was characterized by electrochemistry and Raman spectroscopy, and finally employed in the biosensing of phenols such as catechol and dopamine. This strategy paves the way for the functionalization of nanomaterials by hydrophobin domains of chimeric enzymes and their use in a variety of electrochemical applications.

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.

Beyond natural laccases: extension of their potential applications by protein engineering.

Laccases bring exciting promises into the green industries, and the development of enzymes with improved properties is further raising their exploitation potential. Molecular engineering methods to build highly efficient catalysts both through rational and random mutagenesis were extensively applied. Moreover, computational approaches are becoming always more reliable in aiding proper design of efficient and tailored catalyst for specific applications. In this review, the results of the last 10 years about industrial application of engineered laccases in different fields are analyzed. Tailoring laccase towards a target substrate and defining a proper screening strategy for the selection of the "jackpot mutant" represent the keys of a winning mutagenesis pathway. Likewise, laccase chimerae, built by the fusion of laccases with relevant proteins, emerged as an added value in the designing of flexible and well-rounded biocatalysts. Despite being promising in most of the reported examples, evolved laccases are currently tested at a laboratory scale and a feedback from the industry world is continuously required to strengthen the biotechnological exploitation of these improved enzymes.

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.

Development of a biosensing platform based on a laccase-hydrophobin chimera.

A simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform.

Biological interactions of biocompatible and water-dispersed MoS2 nanosheets with bacteria and human cells.

Two dimensional materials beyond graphene such as MoS2 and WS2 are novel and interesting class of materials whose unique physico-chemical properties can be exploited in applications ranging from leading edge nanoelectronics to the frontiers between biomedicine and biotechnology. To unravel the potential of TMD crystals in biomedicine, control over their production through green and scalable routes in biocompatible solvents is critically important. Furthermore, considering multiple applications of eco-friendly 2D dispersions and their potential impact onto live matter, their toxicity and antimicrobial activity still remain an open issue. Herein, we focus on the current demands of 2D TMDs and produce high-quality, few-layered and defect-free MoS2 nanosheets, exfoliated and dispersed in pure water, stabilized up to three weeks. Hence, we studied the impact of this material on human cells by investigating its interactions with three cell lines: two tumoral, MCF7 (breast cancer) and U937 (leukemia), and one normal, HaCaT (epithelium). We observed novel and intriguing results, exhibiting evident cytotoxic effect induced in the tumor cell lines, absent in the normal cells in the tested conditions. The antibacterial action of MoS2 nanosheets is then investigated against a very dangerous gram negative bacterium, such as two types of Salmonellas: ATCC 14028 and wild-type Salmonella typhimurium. Additionally, concentration and layer-dependent modulation of cytotoxic effect is found both on human cells and Salmonellas.

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.

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.