Abstract
The antibacterial activity of royalisin, an antimicrobial peptide from the royal jelly produced by honeybees, has been addressed extensively. However, its mechanism of action remains unclear. In this study, a recombinant royalisin, RAcc-royalisin from the royal jelly of Asian honeybee Apis cerana cerana, was expressed by fusing with glutathione S-transferase (GST) in Escherichia coli BL21, isolated and purified. The agar dilution assays with inhibition zone showed that RAcc-royalisin, similar to nisin, inhibits the growth of Gram-positive bacteria. The antibacterial activity of RAcc-royalisin was associated with its concentration, and was weakened by heat treatment ranging from 55°C to 85°C for 15 min. Both RAcc-royalisin and nisin exhibited the minimum inhibitory concentrations (MIC) of 62.5 µg/ml, 125 µg/ml, and 250 µg/ml against Gram-positive bacterial strains, Bacillus subtilis and Micrococcus flavus and Staphyloccocus aureus in the microplate assay, respectively. However, RAcc-royalisin did not show antimicrobial activity against tested Gram-negative bacterial and fungal strains. The antibacterial activity of RAcc-royalisin agrees well with the decrease in bacterial cell hydrophobicity, the leakage of 260-nm absorbing materials, and the observation by transmission electron microscopy, all indicating that RAcc-royalisin induced the disruption and dysfunction of cell walls and membranes. This is the first report detailing the antibacterial mechanism of royalisin against Gram-positive bacteria, and provides insight into the application of recombinant royalisin in food and pharmaceutical industries as an antimicrobial agent.
Highlights
Royalisin, a potent antimicrobial peptide (AMP), was first isolated from royal jelly of the Western honeybee Apis mellifera [1]
Expression and Purification of RAcc-royalisin To obtain a large quantity of RAcc-roylisin, we have developed a new method to express and purify proteins using GSTrap FF column connected to GE A KTA Explorer 100 FPLC-Fast Protein Liquid Chromatography System
Antibacterial Activity of RAcc-royalisin The lyophilized soluble RAcc-royalisin diluted in sterilized ddH2O was used to assay inhibitory activity against Gram-positive bacterial strains, B. subtilis and M. luteus
Summary
A potent antimicrobial peptide (AMP), was first isolated from royal jelly of the Western honeybee Apis mellifera [1]. The mature royalisin peptide from the Western honeybee consists of 51 amino acid residues, with a molecular weight of 5.52 kDa, belonging to the family of defensins, the most common group of AMPs in insects [2,3]. Classical defensins and b-defensins that are found in mammalian neutrophils and play important roles in defending against Gram-positive and Gramnegative bacteria, mycobacteria, fungi, and some enveloped viruses. Protein sequences of insect defensins share only limited sequence similarity with mammalian defensins, but do share a common structure comprising an amino-terminal loop, R-helix and two anti-parallel b-strands stabilized by disulfide bridges [5]
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