Aug 2026· International Journal of Biological Macromolecules· pp.
153896
· 0 citations· 39 references
Medicine
TL;DR
A new class of fungal biosurfactant proteins is introduced and a simplified downstream process based on methanol/chloroform extraction is developed, reducing costs while preserving functionality.
Abstract
Protein-based biosurfactants remain underexplored compared to glycolipids and lipopeptides, despite their unique interfacial properties and self-assembly behavior. PAC3, a surface-active protein produced by the marine fungus Acremonium sclerotigenum, exhibits dual behavior as both a biosurfactant and bioemulsifier. For this reason, it can be seen as a high molecular weight proteinaceous compound, able to efficiently reduce surface tension. Here, we identify PAC3 as the first member of a previously unrecognized family of fungal protein biosurfactants. The complete amino acid sequence of PAC3 was determined through a combined de novo transcriptomic and mass spectrometry approach, revealing an 83-residue protein that lacks the canonical eight-cysteine motif typical of hydrophobins, the most surface-active proteins known. Sequence, phylogenetic, and structural analyses revealed a distinct fold and amphipathic architecture, with a negatively charged surface and a hydrophobic planar region, providing a molecular basis for its strong interfacial activity. The identification of homologous sequences across fungi supports the existence of a novel protein family. Notably, we show through spectroscopy and confocal microscopy that PAC3 fibrils exhibit deep-blue intrinsic fluorescence, a property recently associated with amyloid architecture. To support industrial application, we developed a simplified downstream process based on methanol/chloroform extraction, reducing costs while preserving functionality. In parallel, the use of waste frying oil enhanced fungal biomass production and supported efficient PAC3 synthesis, demonstrating a sustainable production strategy. Overall, this study introduces a new class of fungal biosurfactant proteins and provides a foundation for their biotechnological exploitation.
Biosurfactants from lactic acid bacteria are attractive as safe, multifunctional ingredients for food and cosmetic applications, yet many remain only partially characterized. An olive-derived strain was identified as Lactiplantibacillus plantarum J2K-1229 by 16S rRNA gene and genome-based phylogeny. Grown in rapeseed oil-containing medium, it secreted extracellular material with surface/interfacial activity, and its cell-free supernatant emulsified diverse plant oils (emulsification index after 24 h, 26.67−41.33%). A purified biosurfactant fraction gave carbohydrate- and lipid-positive, ninhydrin-negative thin-layer chromatography and Fourier transform infrared bands of a hydroxylated, ester- and carboxylic acid-containing lipid. Gas chromatography, NMR, and HPAEC–PAD indicated a complex, lipid-dominant mixture with major oleic and palmitic acids, NMR signals consistent with co-extracted phenyllactic acid-type metabolites, and fructose as the only detected monosaccharide. Genome mining revealed glucosyltransferases for glucose-based glycolipids but no fructosyltransferase, suggesting that the detected fructose may represent co-extracted free fructose, possibly released as a glucansucrase by-product. The preparation inhibited Candida albicans in disk diffusion assays and was non-cytotoxic to RAW 264.7 macrophages and HaCaT keratinocytes up to 200 ppm. In lipopolysaccharide-stimulated macrophages it suppressed nitric oxide production (88.07% at 200 ppm) and inducible nitric oxide synthase expression, and in cytokine-stimulated keratinocytes it reduced TARC, MDC, and RANTES expression. Overall, this olive-derived L. plantarum produces a multicomponent, glycolipid-containing biosurfactant preparation with emulsifying, antifungal, and anti-inflammatory activities suited to cosmetic and personal-care applications.
Jong Woo Hyeon, Min Hui Jeon, Jun-Tae Bae et al.· Journal of Microbiology and...· 0 citations
Traditional enzyme immobilization strategies often rely on chemical crosslinkers or solid carriers, thereby increasing processing complexity and potentially compromising catalytic efficiency. Here, we present a carrier-free approach for generating stable biocatalytic particles by exploiting the intrinsic aggregation behavior of four phylogenetically distinct AA10 LPMO homologues (Kpapp40, Karip40, Alipp40, and Psufp40) as scaffolds for catalytically active inclusion bodies (CatIBs) in Escherichia coli. Each AA10 variant was genetically fused to either mCherry or a thermostable Bacillus α-amylase (BacAmy) and expressed in E. coli BL21(DE3), resulting in the predominant formation of insoluble protein inclusion bodies (IBs). Protein partitioning was quantified by SDS–PAGE densitometry, intracellular localization by confocal microscopy, particle size and morphology by dynamic light scattering and FESEM, and secondary structure by FTIR spectroscopy. All variants assembled into submicron, structured aggregates with hydrodynamic diameters ranging from 620 to 824 nm and were enriched in α-helical and β-sheet secondary structure, consistent with the formation of structured aggregates rather than extensive amorphous misfolding. mCherry IBs retained fluorescence and displayed polar localization in vivo, while BacAmy CatIBs exhibited maximal catalytic activity at 80 °C, maintained substantial activity up to 95 °C, and demonstrated broad pH tolerance with pronounced pH stability from a slightly acidic to a mild alkaline range. FTIR analysis showed that BacAmy CatIBs contained 47–54% α-helical structure, while mCherry IBs contained 42–45% α-helical structure, indicating the preservation of partially native protein conformations within the aggregated state. Differences among variants influenced particle size, dispersity, and aggregate morphology. These findings demonstrate the potential of AA10 LPMO domains as versatile structural modules for engineering thermostable, carrier-free biocatalysts and provide a foundation for expanding their application beyond oxidative polysaccharide cleavage toward sustainable enzyme material design.
Biosurfactants present strong potential to replace petroleum‐based surfactants due to their amphiphilic structure, high surface activity, and broad applicability. In this sense, this study reports the physicochemical characterization of novel biosurfactants produced by the underexplored strain
Arthrobacter crystallopoietes
B14903. GC–MS analyses and FT‐IR spectroscopy suggested the presence of cyclic lipopeptides, structurally related to arthrofactin, syringopeptin, and syringomycin. These biosurfactants exhibited high emulsifying efficiency, with EI
24
values up to 65% against hydrophobic substrates such as almond, olive, and motor oils. Purified biosurfactants effectively reduced water surface tension to 28 mN/m, showing a critical micelle concentration of approximately 900 mg/L and a C20 value of 125 mg/mL. Notably, surface tension remained stable at approximately 38–40 mN/m after exposure to temperatures ranging from 20°C to 100°C, pH values between 7 and 11, and salinity levels up to 20% (w/w) NaCl, with no statistically significant differences detected. Antimicrobial assays demonstrated no inhibitory effects on pathogenic microorganisms. Overall, these findings position
A. crystallopoietes
B14903 lipopeptides as efficient, stable, and environmentally compatible biosurfactants suitable for applications requiring strong surface activity without disrupting microbial communities.
Georgina Martini, Juan Ignacio D'Alessandro, C. Osorio-González et al.· Journal of Surfactants and D...· 0 citations
BACKGROUND
The discovery of novel biocatalysts for the sustainable valorization of complex biomass feedstocks remains a significant challenge. Domain-centric exploration of characterized CAZyme families offers a promising but underexplored strategy for identifying enzymes with unusual architectures and potentially expanded substrate specificities.
RESULTS
Systematic analysis of archaeal glycoside hydrolase family 18 (GH18) chitinases using the CANDy domain annotation pipeline led to the identification of TcChi from Thermococcus chitonophagus, a multidomain enzyme combining a GH12 and a GH18 catalytic domain alongside two carbohydrate-binding modules. Given that T. chitonophagus also encodes dedicated standalone cellulases and chitinases, we hypothesized that this multidomain assembly may have evolved a broader functional range than either composing domain alone. Biochemical assays of truncated constructs confirmed this hypothesis: the GH18 domain hydrolyzed chitin, chitosan, and β-1,3-glucan, marking the first report of β-1,3-glucanase activity (EC 3.2.1.58) in a GH18 chitinase, while the GH12 domain exhibited strong cellulase activity alongside unexpected chitosanase activity (EC 3.2.1.132), extending the known functional range of this family. Both domains demonstrated high thermostability consistent with the hyperthermophilic origin of T. chitonophagus.
CONCLUSIONS
TcChi is a thermostable, multifunctional biocatalyst capable of degrading chitin, chitosan, cellulose, and β-1,3-glucan from a single protein scaffold, making it a promising candidate for consolidated biomass deconstruction and waste valorization. These findings also demonstrate that domain-centric analysis of CAZyme families is an effective strategy for uncovering hidden functional diversity in well-characterized enzyme families.
Alex Windels, S. Dhaene, Tom Desmet· Biotechnology for Biofuels a...· 0 citations
Hydrophobins are small, cysteine-rich amphipathic proteins predominantly produced by filamentous fungi, known for their ability to self-assemble at hydrophobic–hydrophilic interfaces. These proteins are essential for fungal development, surface interactions, pathogenicity, and environmental adaptation, and they have attracted growing interest for biotechnological applications. In this work, we provide a narrative review of fungal hydrophobins, based on a systematic literature search and manual curation of eligible studies integrated with protein database records from Ascomycota and Basidiomycota. Information was compiled from peer-reviewed publications selected according to predefined eligibility criteria and complemented with UniProt records, covering taxonomic distribution, functional and biophysical properties, and physiological and pathogenic roles. Significant diversity in molecular features and physicochemical profiles was observed, indicating functional specialization across different ecological niches and lifestyles. Additionally, the compiled data highlight various biotechnological applications, such as surface modification, enzyme immobilization, drug delivery systems, biomaterial development, and environmentally sustainable technologies. By consolidating molecular, functional, and applied information across a wide range of fungal species, this review provides a comprehensive reference framework for hydrophobin research and biotechnological innovation.
Sandra de Camargo Lameu, Matheus Henrique Galvão, Isabelle Teixeira Mello et al.· Journal of Fungi· 0 citations
The present study is the first to report the antifungal activity of bacillopeptins against Sclerotinia sclerotiorum, a fungus responsible for white mold.
Maria Luiza A. Jesus-Nicoletto, J. P. Baptista, S. Noriler et al.· Scientific Reports· 1 citation