Isolation and identification of a new intracellular antimicrobial peptide produced by Paenibacillus alvei AN5

Abstract

A wild-type, Gram-positive, rod-shaped, endospore-forming and motile bacteria has been isolated from palm oil mill sludge in Malaysia. Molecular identification using 16S rRNA gene sequence analysis indicated that the bacteria belonged to genus Paenibacillus. With 97% similarity to P. alvei (AUG6), the isolate was designated as P. alvei AN5. An antimicrobial compound was extracted from P. alvei AN5-pelleted cells using 95% methanol and was then lyophilized. Precipitates were re-suspended in phosphate buffered saline (PBS), producing an antimicrobial crude extract (ACE). The ACE showed antimicrobial activity against Salmonella enteritidis ATCC 13076, Escherichia coli ATCC 29522, Bacillus cereus ATCC 14579 and Lactobacillus plantarum ATCC 8014. By using SP-Sepharose cation exchange chromatography, Sephadex G-25 gel filtration and Tricine SDS-PAGE, the ACE was purified, which produced a ~2-kDa active band. SDS-PAGE and infrared (IR) spectroscopy indicated the proteinaceous nature of the antimicrobial compound in the ACE, and liquid chromatography electrospray ionization mass spectroscopy and de novo sequencing using an automatic, Q-TOF premier system detected a peptide with the amino acid sequence F-C-K-S-L-P-L-P-L-S-V-K (1,330.7789Da). This novel peptide was designated as AN5-2. The antimicrobial peptide exhibited stability from pH 3 to 12 and maintained its activity after being heated to 90°C. It also remained active after incubation with denaturants (urea, SDS and EDTA).