Surface-active Proteins Research Articles

Antimicrobial Functionalization of Surfaces by a Chimeric Adhesive Protein.

The main aim of this work is to account for the prevention and control of microbial growth on surfaces of interest in medical technology. Surface modification is often achieved by physiotherapy or chemical treatments that can involve time-consuming steps, hazardous reagents, and harsh conditions. One of the ways to overcome these drawbacks is the use of surface-active proteins such as hydrophobins. They can form stable protein layers on different surfaces, serving as anchoring points for other molecules of interest. The fungal hydrophobin Vmh2, already exploited for its adhesive ability, has been fused with the antimicrobial peptide GKY20, forming the chimeric protein used herein for functionalizing polystyrene (PS) and bacterial cellulose (BC). As a natural biomass, BC has multiple advantages, including biodegradability, low cost, renewability, high purity, and excellent mechanical properties. The chimeric protein has been proven to successfully adhere to both surfaces. A strong decrease in biofilm formation on PS and a good bactericidal effect of BC have been demonstrated. These findings provide evidence of an alternative strategy to obtain functional composites using a green, easy process.

Hydrophobins: multitask proteins

Hydrophobins are small amphiphilic extracellular proteins produced by filamentous fungi; they are surface-active proteins, and their functions are mainly related to their ability to self-assemble into amphipathic monolayers at hydrophobic–hydrophilic interfaces. Depending on their hydropathy patterns and purification requirements, they are classified into class I and class II; both present eight conserved cysteines throughout their sequence, forming four disulfide bridges, which generate four loops that give stability to the protein in its monomeric and folded forms. Class I hydrophobin loops are more extended than class II hydrophobin loops, resulting in differences in assembly on divergent surfaces, additionally accompanied by conformational changes in the protein structure. In the monomer hydrophobin glycosylated form, hydrophobins are rich in β-sheet structure, while being assembled at the water–air interface increases the content of the β-sheet in their structure and is at the interface with water, and a hydrophobic solid such as Teflon also induces the formation of an α-helix structure. The monolayers generated by class I are stable structures called fibrils or rodlets, and class II only produces aggregates. Class I presents a glycosylated chain in its sequence; this causes the formation of the α-helix structure, promoting ordered assemblies, which entails their stability and high insolubility. Fibrils could be dissociated with trifluoroacetic acid and formic acid, which unfolds the protein, while 60% ethanol and 2% sodium dodecyl sulfate solutions dissociate class II aggregates.

Biosensing with Oleosin-Stabilized Liquid Crystal Droplets.

Liquid crystals (LCs) are emerging as novel platforms for chemical, physical, and biological sensing. They can be used to detect biological amphiphiles such as lipids, fatty acids, digestive surfactants, and bacterial endotoxins. However, designing LC-based sensors in a manner that preserves their sensitivity and responsiveness to these stimuli, and possibly improves biocompatibility, remains challenging. In this work, the stabilization of LC droplets by oleosins, plant-sourced and highly surface active proteins due to their extended amphipathic helix, is investigated. Purified oleosins, at sub-micromolar concentrations, are shown to readily stabilize nematic LC droplets without switching their alignment, allowing them to detect surfactants at micromolar concentrations. Direct evidence of localization of oleosins at the LC-water interface is provided with fluorescent labeling, and the stabilized droplets remain stable over months. Interestingly, chiral LC droplets readily switch in the presence of nanomolar oleosin concentrations, an unexpected behavior that is explained by accounting for the energy barriers required for switching the alignment between the two cases. This leads thus to a twofold conclusion: oleosin-stabilized nematic LC droplets present a biocompatible alternative for bioanalyte detection, while chiral LCs can be further investigated for use as highly sensitive sensors for detecting amphipathic helices in biologicalsystems.

Molecular Modeling of the Adsorption of an Egg Yolk Protein on a Water-Oil Interface.

Egg yolk contains several molecular species with emulsifying properties, such as proteins and phospholipids. In particular, these molecules have both polar and nonpolar parts and thus can act as surfactants. One of the most surface-active proteins from egg yolk low-density lipoproteins is the so-called Apovitellenin-1. Experimental studies have been hindered by difficulties in isolating individual species from egg yolk lipoproteins. The purpose of this work was to assess the emulsifying properties of Apovitellenin-1 and any potential cooperative or competitive behavior in the presence of phospholipids. To do so, molecular simulations were carried out in a liquid-liquid interfacial system consisting of water and soybean oil, with varying concentrations of phospholipids and for different spatial configurations. To evaluate the conformational stability of the protein at the water-oil interface, the Gibbs free energy was computed from Metadynamics simulations as a function of the distance from the interface and of the radius of gyration. Moreover, a detailed analysis was also performed to determine which peptide residues were responsible for the protein adsorption at the oil-water interface as well as the lowering of the interfacial tension. Lastly, we combined the simulation results with a thermodynamic model to predict the interfacial tension behavior at increasing protein bulk concentration, which cannot be measured experimentally.

An efficient method of improving essential oil retention and sustained release of chitosan films: Ultrasound-assisted preparation of chitosan composites with surface active chickpea proteins

This work aimed at preparing chitosan (CHI) composites with surface active chickpea protein (CP) showing better eugenol (EUG) retention and sustained release capacity than pristine CHI films. For this purpose, ionic complexation of CHI with CP (CHI:CP ratio = 2:1, w/w) in the presence of EUG at pH 5.0 was achieved using mechanical homogenization alone (HM) or in combination with ultrasonic homogenization (HM-HUS). The HM-HUS treatment provided better solubility of CP (4.4-fold), increased emulsified EUG in film-forming solutions, and denser films than HM treatment. The composite films obtained using HM-HUS (FLMCHI-CP-EUG/HM-HUS) retained 1.2–1.4-fold higher EUG after drying, and showed almost 2-fold slower EUG release in air at room temperature than composite films prepared by HM, and control CHI films prepared by HM (FLMCHI-EUG/HM) or HM-HUS (FLMCHI-EUG/HM-HUS). The FLMCHI-CP-EUG/HM-HUS films also showed better moisture barrier and mechanical properties than other films. The developed films were proved in a challenging coating application with onions. Escherichia coli and Listeria innocua counts of inoculated and FLMCHI-CP-EUG/HM-HUS (average coating thickness = 4.5 ± 1.3 μm) coated onions were significantly lower than those of uncoated (2.8 and 3.8 log) and FLMCHI/HM-HUS (1.4 and 1.3 log) coated onions after 5-days at room temperature. FLMCHI-CP-EUG/HM-HUS coating also reduced percentage of sprouted onions from 30 to 10% during storage. EUG odor of coated onions could not have been detected by 80% of panelists after 4 weeks. Compositing with CP boosts the performance of essential oil loaded CHI films by enabling use of film matrix as an encapsulant.

The emulsifying performance of brewers' spent grains treated by colloid milling

The sustainable production of food ingredients can be achieved by utilising waste streams, but it is difficult to isolate pure components from a heterogeneous matrix. In this work, brewers' spent grains (BSG), which is a by-product from the brewing industry, was mildly treated with a colloid mill and its emulsifying ability was evaluated. The milled samples contained 26–32% protein and 52–62% fibre and were generally smaller than 10 μm. In its soluble fraction, a small amount of surface-active proteins was present and reduced oil-water interfacial tension by 35%. Emulsions prepared with the milled BSG and its insoluble particles were stable against coalescence for 10 days, with respective droplet sizes of 40 μm and 25 μm. The presence of fibres caused droplet flocculation but also helped to promote network formation. Our results suggest that obtaining pure components may not be necessary for emulsification purposes, especially for the case of particle-stabilised emulsions. Industrial relevanceIndustrial by-products from food processing are generated in large quantities and typically discarded due to their recalcitrant nature. Yet, there is more that can be exploited from this waste stream including proteins and fibres. Separation of these components in their purified form would require harsh methods that are not environmentally sustainable. This study provided insights into the use of a mild and scalable process that can produce functionally active dispersions that are of interest in the preparation of emulsions.

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.

Characterization of biosurfactant-producing bacterial strains isolated from agro-industrial wastes in southwestern, Nigeria

Introduction. The difficulty of managing trash and cleaning up the environment prompted interest in biosurfactants and surface-active proteins made by microbes. The study aims to augment bacterial isolates from agro-industrial wastes targeted for possible mass production of biosurfactants. Methods. Six agro-industrial wastes from Cassava, Palm kernel, and Sawdust from six agro-industrial sites within Ijebu area in Ogun State were collected for standard laboratory analyses in the Biotechnology Unit of the Federal Industrial Institute for Research, Oshodi (FIIRO). Five screening methods; blood hemolysis, lipase activity, blue agar hydrolysis, oil spreading, and emulsification index (EI24) were carried out to confirm biosurfactant production. Isolates with the highest hyper-production were subjected to 16rRNA molecular identification. Results. The study justified efficient biosurfactant production from 4 bacterial isolates out of 26 screened bacterial isolates from hydrocarbon degraders and 29 heterotrophic screened bacterial isolates, making a total of 55 screened bacterial isolates. Screening results reveal the emulsification capacities of identified Pseudomonas putida strain SG1, Acinetobacter baumanii strain MS14413, Bacillus zhangzhouensis strain cdsV18, and Burkholderia cepacia strain 717. Conclusion. Biosurfactant bacteria produced in all agricultural and industrial wastes considered in this study are capable of mass production.

Compressibility and porosity modulate the mechanical properties of giant gas vesicles

Gas vesicles used as contrast agents for noninvasive ultrasound imaging must be formulated to be stable, and their mechanical properties must be assessed. We report here the formation of perfluoro-n-butane microbubbles coated with surface-active proteins that are produced by filamentous fungi (hydrophobin HFBI from Trichoderma reesei). Using pendant drop and pipette aspiration techniques, we show that these giant gas vesicles behave like glassy polymersomes, and we discover novel gas extraction regimes. We develop a model to analyze the micropipette aspiration of these compressible gas vesicles and compare them to incompressible liquid-filled vesicles. We introduce a sealing parameter to characterize the leakage of gas under aspiration through the pores of the protein coating. Utilizing this model, we can determine the elastic dilatation modulus, surface viscosity, and porosity of the membrane. These results demonstrate the engineering potential of protein-coated bubbles for echogenic and therapeutic applications and extend the use of the pipette aspiration technique to compressible and porous systems.

Entrapment of air microbubbles by ice crystals during freezing exacerbates freeze-induced denaturation of proteins

Freezing techniques are an essential part of biologics manufacturing processes, yet the formation of ice/water interfaces can impart detrimental effects on proteins. However, the absence of chemical and structural differences between ice and liquid water poses the question as to why ice can destabilize proteins. We hypothesize that the destabilizing stress of the ice-liquid water interface does not originate from the ice-water system itself but rather from the air microbubbles present during the freezing process. As the temperature decreases, the dissolved air is expelled from the ice crystal lattices in the form of microbubbles and is subsequently trapped by the advancing ice front. This newly formed air–water interface represents an additional interfacial area for the proteins to be adsorbed onto and denatured. The result showed that freezing at ∼ 1 K/s led to the formation of small circular microbubbles with diameters ranging from 100 µm to 500 µm. In contrast, slower freezing resulted in the formation of larger, elongated millimeter-size bubbles. The reduction of the number of microbubbles was carried out by the deaeration process using agitation under reduced pressure at 20 kPa. The resulting deaerated (i.e., low dissolved air) protein samples were frozen and monitored for the formation of subvisible aggregates using micro-flow imaging (MFI). The results demonstrated that deaerating the samples prior to intermediate freezing (i.e., TFF) reduced the number of aggregates for both highly surface-active and low surface-active proteins (lactoferrin and bovine IgG, respectively). This reduction was more pronounced in spray freeze drying (SFD) than thin-film freezing (TFF), and less apparent in conventional lyophilization.

Non-Invasive Rheo-MRI Study of Egg Yolk-Stabilized Emulsions: Yield Stress Decay and Protein Release.

A comprehensive understanding of the time-dependent flow behavior of concentrated oil-in-water emulsions is of considerable industrial importance. Along with conventional rheology measurements, localized flow and structural information are key to gaining insight into the underlying mechanisms causing time variations upon constant shear. In this work, we study the time-dependent flow behavior of concentrated egg-yolk emulsions with (MEY) or without (EY) enzymatic modification and unravel the effects caused by viscous friction during shear. We observe that prolonged shear leads to irreversible and significant loss of apparent viscosity in both emulsion formulations at a mild shear rate. The latter effect is in fact related to a yield stress decay during constant shearing experiments, as indicated by the local flow curve measurements obtained by rheo-MRI. Concurrently, two-dimensional D-T2 NMR measurements revealed a decrease in the T2 NMR relaxation time of the aqueous phase, indicating the release of surface-active proteins from the droplet interface towards the continuous water phase. The combination of an increase in droplet diameter and the concomitant loss of proteins aggregates from the droplet interface leads to a slow decrease in yield stress.

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 modeling of the interface of an egg yolk protein-based emulsion

Many food emulsions are stabilized by functional egg yolk biomolecules, which act as surfactants at the oil/water interface. Detailed experimental studies on egg yolk emulsifying properties have been largely hindered due to the difficulty in isolating individual chemical species. Therefore, this work presents a molecular model of an oil/water interfacial system where the emulsifier is one of the most surface-active proteins from the egg yolk low-density lipoproteins (LDL), the so-called Apovitellenin I. Dissipative particle dynamics (DPD) was here adopted in order to simulate large systems over long time scales, when compared with full-atom molecular dynamics (MD). Instead of a manual assignment of the DPD simulation parameters, a fully automated coarse-graining procedure was employed. The molecular interactions used in the DPD system were determined by means of a parameter calibration based on matching structural data from atomistic MD simulations. Despite the little availability of experimental data, the model was designed to test the most relevant physical properties of the protein investigated. Protein structural and dynamics properties obtained via MD and DPD were compared highlighting advantages and limits of each molecular technique. Promising results were achieved from DPD simulations of the oil/water interface. The proposed model was able to properly describe the protein surfactant behavior in terms of interfacial tension decrease at increasing protein surface concentration. Moreover, the adsorption time of a free protein molecule was estimated and, finally, an LDL-like particle adsorption mechanism was qualitatively reproduced.

The pleiotropic functions of intracellular hydrophobins in aerial hyphae and fungal spores.

Higher fungi can rapidly produce large numbers of spores suitable for aerial dispersal. The efficiency of the dispersal and spore resilience to abiotic stresses correlate with their hydrophobicity provided by the unique amphiphilic and superior surface-active proteins–hydrophobins (HFBs)–that self-assemble at hydrophobic/hydrophilic interfaces and thus modulate surface properties. Using the HFB-enriched mold Trichoderma (Hypocreales, Ascomycota) and the HFB-free yeast Pichia pastoris (Saccharomycetales, Ascomycota), we revealed that the rapid release of HFBs by aerial hyphae shortly prior to conidiation is associated with their intracellular accumulation in vacuoles and/or lipid-enriched organelles. The occasional internalization of the latter organelles in vacuoles can provide the hydrophobic/hydrophilic interface for the assembly of HFB layers and thus result in the formation of HFB-enriched vesicles and vacuolar multicisternal structures (VMSs) putatively lined up by HFBs. These HFB-enriched vesicles and VMSs can become fused in large tonoplast-like organelles or move to the periplasm for secretion. The tonoplast-like structures can contribute to the maintenance of turgor pressure in aerial hyphae supporting the erection of sporogenic structures (e.g., conidiophores) and provide intracellular force to squeeze out HFB-enriched vesicles and VMSs from the periplasm through the cell wall. We also show that the secretion of HFBs occurs prior to the conidiation and reveal that the even spore coating of HFBs deposited in the extracellular matrix requires microscopic water droplets that can be either guttated by the hyphae or obtained from the environment. Furthermore, we demonstrate that at least one HFB, HFB4 in T. guizhouense, is produced and secreted by wetted spores. We show that this protein possibly controls spore dormancy and contributes to the water sensing mechanism required for the detection of germination conditions. Thus, intracellular HFBs have a range of pleiotropic functions in aerial hyphae and spores and are essential for fungal development and fitness.

Effective drug delivery system based on hydrophobin and halloysite clay nanotubes for sustained release of doxorubicin

Halloysite clay nanotubes (HNTs), which are natural nano-clays with a hollow tubular structure, have been widely studied for drug delivery and sustained release while largely limited by the complexity of surface modification. Hydrophobins are small surface-active proteins produced by filamentous fungi that self-assemble into nanolayers at hydrophobic/hydrophilic interfaces. In this study, hydrophobins including HGFI-his and a mutant MGF6-his were used as unique biomaterials to modify HNTs to construct drug delivery systems. Water contact angle and HNT dispersion assays demonstrated that MGF6-his performed better surface activity and rendered HNT more lasting stability than HGFI-his. MGF6-his-coated HNTs loaded with the anticancer drug doxorubicin hydrochloride ([email protected]) were further prepared and characterized by dynamic light scattering, fluorescence microscopy and transmission electron microscopy. This composite was found to form nanoparticles in size of 375.62 ± 17.85 nm and possess better dispersion stability owing to the coating of the MGF6-his. Excellent sustained release was observed at pH 3.3 and 5.0 but not at pH 7.4 in in vitro release assay. Moreover, an in vitro cytotoxicity assay showed that this system exhibited good bioactivity, killing about 70% of HeLa cells at a concentration of 150 μg/mL. Herein, the hydrophobin MGF6-his was designed as a novel approach for HNT surface modification, facilitating the dispersion and prolonging drug release simultaneously. These findings suggested that coating with hydrophobins has potential as a novel approach to develop HNT-based drug delivery systems.

Class I hydrophobin fusion with cellulose binding domain for its soluble expression and facile purification

Hydrophobins, highly surface-active proteins, have the ability to reverse surface hydrophobicity through self-assembly at the hydrophilic-hydrophobic interfaces. Their unique structure and interfacial activity lead hydrophobins to have potential applications on surface functional modifications. However, class I hydrophobins are prone to self-assemble into highly insoluble amyloid-like rodlets structure. Recombinant hydrophobins could be produced by Escherichia coli but generally as an insoluble inclusion body. To overcome this insoluble expression limitation, cellulose-binding domain (CBD) from Clostridium thermocellum was fused to the N-terminal of class I hydrophobin HGFI to enhance its soluble expression in E. coli. Approximately, 94% of expressed CBD fused HGFI (CBD-HGFI) was found as soluble protein. The fused CBD could also bind specifically onto bacterial cellulose (BC) nanofibrils produced by Komagataeibacter xylinus to facilitate rapid isolation and purification of HGFI from crude extract. Lysostaphin (Lst), known as GlyGly endopeptidase could successfully cleave the flexible linker (GGGGS)2 between CBD and HGFI to recover HGFI from BC-bound CBD-HGFI. CBD-HGFI purified by immobilized metal-chelated affinity chromatography (IMAC) and Lst cleaved BC-CBD-HGFI still retained interfacial activity of hydrophobin and its effect on accelerating PETase hydrolysis against poly(ethylene terephthalate) (PET) fiber.

The post-translational modification landscape of commercial beers

Beer is one of the most popular beverages worldwide. As a product of variable agricultural ingredients and processes, beer has high molecular complexity. We used DIA/SWATH-MS to investigate the proteomic complexity and diversity of 23 commercial Australian beers. While the overall complexity of the beer proteome was modest, with contributions from barley and yeast proteins, we uncovered a very high diversity of post-translational modifications (PTMs), especially proteolysis, glycation, and glycosylation. Proteolysis was widespread throughout barley proteins, but showed clear site-specificity. Oligohexose modifications were common on lysines in barley proteins, consistent with glycation by maltooligosaccharides released from starch during malting or mashing. O-glycosylation consistent with oligomannose was abundant on secreted yeast glycoproteins. We developed and used data analysis pipelines to efficiently extract and quantify site-specific PTMs from SWATH-MS data, and showed incorporating these features into proteomic analyses extended analytical precision. We found that the key differentiator of the beer glyco/proteome was the brewery, with beer from independent breweries having a distinct profile to beer from multinational breweries. Within a given brewery, beer styles also had distinct glyco/proteomes. Targeting our analyses to beers from a single brewery, Newstead Brewing Co., allowed us to identify beer style-specific features of the glyco/proteome. Specifically, we found that proteins in darker beers tended to have low glycation and high proteolysis. Finally, we objectively quantified features of foam formation and stability, and showed that these quality properties correlated with the concentration of abundant surface-active proteins from barley and yeast.

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.

Engineered Hydrophobin as a Crystallization Inhibitor for Flufenamic Acid.

Hydrophobins are multifunctional, highly surface-active proteins produced in filamentous fungi. Due to their surface-active properties, resistance to degradation, and potential immunological inertness, hydrophobins have been used in many applications such as protein purification, increasing implant biocompatibility, increasing water solubility of insoluble drugs, and foam stabilizers for food products. To further explore surface-active and self-assembly properties of hydrophobins, we evaluated an engineered, recombinant hydrophobin (class II type 1, HFB1) as a potential crystallization inhibitor for maintaining drug supersaturation for an amorphous drug delivery system. A supersaturation-precipitation method was employed utilizing an ultraviolet (UV) fiber optic system for tracking precipitation kinetics of a model drug, flufenamic acid (FA), that was selected due to its low aqueous solubility in its crystalline form. The effectiveness of HFB1 as a crystallization inhibitor was compared with commonly used pharmaceutical grade polymeric crystallization inhibitors. The following polymers were selected to compare with HFB1: methocel (A4C grade), methocel (K15M grade), Kollidon vinylpyrrolidone-vinyl acetate (VA64), and hydroxypropyl methylcellulose acetate succinate (HPMCAS) (MF grade). The supersaturation-precipitation experiments concluded that HFB1 outperformed all polymers tested in this study and can potentially be used as a crystallization inhibitor at significantly lower concentrations in amorphous drug delivery systems. Dynamic light scattering (DLS) and circular dichroism (CD) results suggest a crystallization inhibition mechanism in which HFB1 functions differently depending on whether flufenamic acid is molecularly dispersed but supersaturated relative to its crystalline solubility or it has exceeded its amorphous solubility limit and exists as a phase-separated drug-rich colloid.

Improvement Thermal Stability of D-Lactate Dehydrogenase by Hydrophobin-1 and in Silico Prediction of Protein-Protein Interactions.

Hydrophobins are small surface-active proteins. They can connect to hydrophobic or hydrophilic regions and oligomerize in solution to form massive construction. In nature, these proteins are produced by filamentous fungi at different stages of growth. So far, researchers have used them in various fields of biotechnology. In this study, recombinant hydrophobin-1 (rHFB1, 7.5kDa) was used to stabilize recombinant D-lactate dehydrogenase (rD-LDH, 35kDa). rD-LDH is a sensitive enzyme deactivated and oxidized by external agents such as O2 and lights. So, its stabilization with rHFB1 can be the best index to demonstrate the positive effect of rHFB1 on preserving and improving enzyme's activity. The unique ability of rHFB1 for interacting with hydrophobic regions of rD-LDH was predicted by protein-protein docking study with ClusPro and PIC servers and confirmed by fluorescence experiments, and Colorless Native-PAGE. Measurement of thermodynamic parameters allows for authenticating the role of rHFB1 as a thermal stabilizer in the protein-protein complex (rD-LDH@rHFB1). Interaction between rHFB1 and rD-LDH improved half-life of enzyme 2.25-fold at 40°C. Investigation of the kinetic parameters proved that the presence of rHFB1 along with the rD-LDH enhancement strongly the affinity of the enzyme for pyruvate. Furthermore, an increase of Kcat/Km for complex displayed the effect of rHFB1 for improving the enzyme's catalytic efficiency.