The enhancement of surface activity and nanoparticle stability through the alteration of charged amino acids of HGFI.

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.