World Journal of Microbiology and Biotechnology

, Volume 30, Issue 4, pp 1377–1385 | Cite as

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

  • Bassam Alkotaini
  • Nurina Anuar
  • Abdul Amir Hassan Kadhum
  • Asmahani Azira Abdu Sani
Original Paper
First Online:
Received:
Accepted:

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 (1,330.7789 Da). 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).

Keywords

Paenibacillus alvei AN5 Antimicrobial peptide AN5-2 LC/ESI–MS Characterization Mode of action 
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Notes

Acknowledgments

This study was supported by Research Grants UKM-DPP-2013-044 and GUP-2012-002 from the Universiti Kebangsaan Malaysia. The author would also like to thank the Proteomic Laboratory of the Malaysian Genome Institute for LC–ESI/TOF–MS service.

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Bassam Alkotaini
    • 1
  • Nurina Anuar
    • 1
  • Abdul Amir Hassan Kadhum
    • 1
  • Asmahani Azira Abdu Sani
    • 2
  1. 1.Department of Chemical and Process Engineering, Faculty of Engineering and Built EnvironmentUniversiti Kebangsaan MalaysiaBangiMalaysia
  2. 2.Proteomic LaboratoryMalaysian Genome InstituteBangiMalaysia

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