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.

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.

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.

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.

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.

A new and efficient procedure to load bioactive molecules within the human heavy-chain ferritin nanocage.

For their easy and high-yield recombinant production, their high stability in a wide range of physico-chemical conditions and their characteristic hollow structure, ferritins (Fts) are considered useful scaffolds to encapsulate bioactive molecules. Notably, for the absence of immunogenicity and the selective interaction with tumor cells, the nanocages constituted by the heavy chain of the human variant of ferritin (hHFt) are optimal candidates for the delivery of anti-cancer drugs. hHFt nanocages can be disassembled and reassembled in vitro to allow the loading of cargo molecules, however the currently available protocols present some relevant drawbacks. Indeed, protein disassembly is achieved by exposure to extreme pH (either acidic or alkaline), followed by incubation at neutral pH to allow reassembly, but the final protein recovery and homogeneity are not satisfactory. Moreover, the exposure to extreme pH may affect the structure of the molecule to be loaded. In this paper, we report an alternative, efficient and reproducible procedure to reversibly disassemble hHFt under mild pH conditions. We demonstrate that a small amount of sodium dodecyl sulfate (SDS) is sufficient to disassemble the nanocage, which quantitatively reassembles upon SDS removal. Electron microscopy and X-ray crystallography show that the reassembled protein is identical to the untreated one. The newly developed procedure was used to encapsulate two small molecules. When compared to the existing disassembly/reassembly procedures, our approach can be applied in a wide range of pH values and temperatures, is compatible with a larger number of cargos and allows a higher protein recovery.

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.

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.

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.