Structure and antimicrobial activity relationship of royalisin, an antimicrobial peptide from royal jelly of Apis mellifera

•The secondary structure that might affect the antimicrobial activity of royalisin was evaluated.•Intramolecular disulfide linkages of royalisin play a very important role in the antimicrobial activity.•The 11-amino-acid-long peptide at the C-terminus of royalisin would stabilize the royalisin structure. Royalisin is a 5.5-kDa antibacterial peptide isolated from the royal jelly of the honeybee (Apis mellifera). The antimicrobial activity of royalisin against fungi, Gram-positive and Gram-negative bacteria has been revealed. Compared with another insect antibacterial peptide, there is an extra stretch of 11 amino acid residues at the C-terminus of royalisin. In this study, a recombinant shortened form of royalisin named as royalisin-D, was constructed without the 11 amino acid residues at the C-terminal of royalisin and linked to the C-terminal of oleosin by an inteinS fragment. The recombinant protein was overexpressed in Escherichia coli, purified by artificial oil body system and subsequently released through self-splicing of inteinS induced by the changes of temperature. The antibacterial activity of royalisin-D was compared with royalisin via minimal inhibitory concentration (MIC) assay, minimal bactericidal concentration (MBC) assay, microbial adhesion to solvents (MATS) methods, and cell membrane permeability. Furthermore, the recombinant royalisin and royalisin-D have also been treated with the reducing agent of disulfide bonds, dithiothreitol (DTT), to investigate the importance of the intra-disulfide bond in royalisin. In our results, royalisin-D exhibited similar antimicrobial activity to royalisin. Royalisin and royalisin D lost their antimicrobial activities when the intra-disulfide bonds were reduced by DDT. The intra-disulfide bond plays a more important role than the extra stretch of 11 amino acid residues at the C-terminus of royalisin in terms of the antimicrobial properties of the native royalisin.