Detection of secreted antimicrobial peptides isolated from cell-free culture supernatant of Paenibacillus alvei AN5

Abstract

An antimicrobial substance produced by the Paenibacillus alvei strain AN5 was detected in fermentation broth. Subsequently, cell-free culture supernatant (CFCS) was obtained by medium centrifugation and filtration, and its antimicrobial activity was tested. This showed a broad inhibitory spectrum against both Gram-positive and -negative bacterial strains. The CFCS was then purified and subjected to SDS-PAGE and infrared spectroscopy, which indicated the proteinaceous nature of the antimicrobial compound. Some de novo sequencing using an automatic Q-TOF premier system determined the amino acid sequence of the purified antimicrobial peptide as Y-S-K-S-L-P-L-S-V-L-N-P (1,316 Da). The novel peptide was designated as peptide AN5-1. Its mode of action was bactericidal, inducing cell lysis in E. coli ATCC 29522 and S. aureus, and non-cell lysis in both S. marcescens and B. cereus ATCC 14579. Peptide AN5-1 displayed stability at a wide range of pH values (2-12) and remained active after exposure to high temperatures (100 °C). It also maintained its antimicrobial activity after incubation with chemicals such as SDS, urea and EDTA.

Fig. 1

Profile of Paenibacillus alvei AN5 growth and antimicrobial peptide production in Modified Landy Medium at 30 °C, 150 rpm in 2 h intervals, absorbance at 600 nm (filled circle), and antimicrobial peptide production (AU ml⁻¹) determined by micro-dilution assay (filled square)

Fig. 2

Tricine SDS-PAGE supplemented with glycerol for purified antimicrobial peptide AN5-1 produced by Paenibacillus alvei AN5. Lane 1 color marker ultra-low range (M.W. 1.06–26.6) KDa (Sigma, USA), lane 2 purified AN5-1 stained with Coomassie blue, lane 3 bacteriocin assay overlying on soft agar shows inhibitory activity of the active peptide against E. coli ATCC 29522

Fig. 3

Fourier transform infrared (FT-IR) of purified antimicrobial peptide AN5-1

Fig. 4

Mass spectroscopy of purified antimicrobial peptide AN5-1, a ESI spectra of purified antimicrobial peptide AN5-1 obtained from UPLC shows the single band has three charges of molecular mass 439.9275m/z ionized by ESI with overall molecular mass 1,316.7340 Da. b MS/MS spectra of purified antimicrobial peptide AN5-1 performed automatically using ProteinLynx Global SERVER v 2.4 software based on Q-TOF premier system

Fig. 5

Effects of 80 AU ml⁻¹ of purified antimicrobial peptide AN5-1 produced by P. alvei AN5 on early exponential growth phase against target strains: a E. coli ATCC 29522, b S. marcescens, c S. aureus and d B. cereus ATCC 14579, in the absence (filled circle) and presence (filled triangle) of antimicrobial peptide AN5-1. The bacterial growth was measured by means of optical density at 600 nm (OD₆₀₀)