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Herein, a new method was demonstrated for effective immobilization of the antibacterial peptide nisin on Grifola frondosa hydrophobin (HGFI), without the need of any additional complex reaction. Hydrophobin can self-assemble as a monolayer to form continuous negative-charged surfaces with enhanced wettability and biocompatibility. Adding nisin solution to such hydrophobin surface created antibacterial surfaces. The quantification analysis revealed that more nisin could be adsorbed on the HGFI-coated than to control polystyrene surfaces at different pH values. This suggested that electronic attraction and wettability may play important roles in this process. The transmission electron microscopy, atomic force microscopy and fourier transform infrared (FTIR) analysis indicated the adsorption mode of nisin on the HGFI film, i.e., hydrophobins served as an adhesive layer for binding charged peptides to interfaces. The antibacterial activity of the treated surface was investigated via counting, a nucleic acid release test, scanning electron microscopy, and biofilm detection. These results indicated the excellent antibacterial activity of nisin adsorbed on the HGFI-coated surfaces. The activity retention of adsorbed nisin was demonstrated by immersing the modified substrates in a flowed liquid condition. • Hydrophobin HGFI was employed to immobilize AMP (Nisin) on material surfaces. • Nisin on the HGFI-coated surfaces exhibited excellent bioactivity. • The coating formation was easier without complex and rigorous conditional limits. [ABSTRACT FROM AUTHOR]

Hydrophobins are a series of low molecular weight proteins produced by filamentous fungi that play an important role in fungal growth. They have a globular structure and possess a unique hydrophobic patch on their surface that makes them amphiphilic, making them among the most surface-active proteins. Herein, the surface charge properties of HGFI, a class I hydrophobin from Grifola frondosa, were altered by replacing the negatively charged Glu24 with a positively charged Lys to generate the ME24 mutant. Pichia pastoris GS115 was used for recombinant expression of the ME24 mutant, which was purified by a two-step procedure. The function of the mutated residue in HGFI self-assembly was investigated. Reverse-phase high-performance liquid chromatography analysis revealed that the polarity of ME24 was enhanced compared with HGFI. Circular dichroism, thioflavin T assay, water contact angle and atomic force microscopy indicated that Glu24 participates in rodlet formation. Water solubility detection and dynamic light scattering showed that Glu24 affects the assembled state of HGFI in aqueous solution. The behaviour of the mutant in an emulsion, in the dispersion of insoluble materials and in large-scaled protein production suggests the functions of hydrophobins can be tuned for new applications. [ABSTRACT FROM AUTHOR]

Class IIa bacteriocin pediocin PA-1 has broad-spectrum activity and is a well-characterized candidate food biopreservative. Here, a simple approach is designed to extend the application of pediocin PA-1 in improving the antibacterial activity of electrospun poly(caprolactone) (PCL) grafts through combining PA-1 with HGFI, which is a self-assembled protein with characteristics allowing the modulation of surface properties of other materials originated from Grifola frondosa . Saccharomyces cerevisiae was used as the host for expression of fusion protein PA-1-linker-HGFI (pH) and his-tag purification was used to purify recombinant protein pH. An antibacterial activity assay showed the fusion protein pH retained the biological property of native PA-1. Water contact angle, X-ray photoelectron spectroscopy, immunofluorescence assay and atomic force microscopy indicated the surface properties of HGFI were greatly preserved by the fusion protein pH. Finally, antibacterial activity of pH-modified PCL substrate measurements implied the fusion protein significantly improved the bacterial-resistance of the PCL film through dressing the PCL fibers with the recombinant pH protein. This work presents a new perspective on the application of hydrophobin and pediocin PA-1 in antibacterial medical devices. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • The alteration of charged bases influenced the pI value of HGFI. • MGF3 and MGF6 exhibited better surface activity than the wild type HGFI. • Neither MGF3 nor MGF6 have the ability to form rodlets. • Both MGF3 and MGF6 enhanced the stability of IND. • The mutant hydrophobins extended the release time of IND in simulated GI system. Abstract HGFI (hydrophobin found in Grifola frondosa) is a small surface-active protein, belonging to class I hydrophobins, and can form amphipathic protein films at hydrophobic- hydrophilic material interfaces. HGFI has a low isoelectric point (pI) because no basic amino acids are present in its sequence. Here we constructed two HGFI mutants using a site-directed mutation method. The two mutants were named MGF3 (E24K/D35K/D37K) and MGF6 (E12K/D19K/E24K/D35K/D37K/D66K). MGF3 was generated by replacing three acidic amino acids, namely Glu24, Asp35 and Asp37, with the basic amino acid Lys. MGF6 was obtained by substituting Glu12, Asp19, Glu24, Asp35, Asp37 and Asp66 with six Lyses. Zeta-potential analysis showed that the pI values of MGF3 and MGF6 were between 5 and 6. The water contact angle (WCA) and quartz-crystal microbalance (QCM) analyses revealed enhanced adsorption capacity of these two mutants. Circular dichroism measurements (CD), Thioflavin T (THT) assays and atomic force microscopy (AFM) tests indicated that neither MGF3 nor MGF6 have the ability to form rodlets. An in vitro drug release assay showed the water solubility of the indometacin (IND) nano-particles (NPs) was improved by both MGF3 and MGF6 modification. Furthermore, the mutant hydrophobin-modified NPs were more stable and exhibited a lower release rate in solutions at pH 1.2 and 7 at 37 °C. The MGF6-IND NPs showed a particularly improved drug release behavior. These results suggest new applications of the mutated hydrophobins in sustained delivery of oral drugs in the gastrointestinal tract. [ABSTRACT FROM AUTHOR]