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Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112

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Abstract

The antibacterial activity of antimicrobial peptides produced by strain LFB 112 was determined using the cup-plate method and a standard curve of penicillin. An effective method to evaluate the antimicrobial activity of antibacterial peptides was established. Three active fractions, Fraction A (5.0–5.5 kDa), Fraction B (3.0–3.5 kDa) and Fraction C (400–800 Da), were separated by anion-exchange, reversed phase liquid chromatography (RPLC) and gel filtration chromatography. Fraction A contained an antimicrobial peptide; its partial sequence (LVQSPNGNFAASFVLDGTK), determined by liquid chromatography-mass spectrometry (LC-MS), was homologous with antimicrobial peptide LCI. In addition, there were three major ingredients in fraction B (3,371.811, 3,442.379 and 3,486.596 Da, respectively), and several small peptides (400–800 Da) in fraction C. After purification, the potencies of fractions A, B and C were 188.6, 11,217.26 and 10,290.63 U/mg, respectively. Furthermore, antimicrobial peptides in fraction B, apparently different from known antimicrobial peptides produced by Bacillus subtilis (based on molecular weight), and small (molecular weight 400–800 Da) antimicrobial peptides produced by B. subtilis LFB112 were also identified. Antimicrobial peptides produced by strain LFB112 with high potency against several undesirable bacteria were identified. These compounds have potential as alternatives to known antibiotics.

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References

  • Aunpad R, Na-Bangchang K (2007) Pumilicin 4, a novel bacteriocin with anti-MRSA and anti-VRE activity produced by newly isolated bacteria Bacillus pumilus strain WAPB4. Curr Microbiol 55:308–313

    Article  PubMed  CAS  Google Scholar 

  • Babasaki K, Takao T, Shimonishi Y, Kurahashi K (1985) Subtilosin A, a new antibiotic peptide produced by Bacillus subtilis 168: isolation, structural analysis, and biogenesis. J Biochem 98:585–603

    PubMed  CAS  Google Scholar 

  • Barefoot SF, Klaenhammer TR (1983) Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus. Appl Environ Microbiol 45:1808–1815

    PubMed  CAS  Google Scholar 

  • Bierbaum G, Brotz H, Koller KP, Sahl HG (1995) Cloning, sequencing and production of the lantibiotic mersacidin. FEMS Microbiol Lett 127:121–126

    Article  PubMed  CAS  Google Scholar 

  • Cherif A, Chehimi S, Limem F, Hansen BM, Hendriksen NB, Daffonchio D, Boudabous A (2003) Detection and characterization of the novel bacteriocin entomocin 9, and safety evaluation of its producer, Bacillus thuringiensis ssp. entomocidus HD9. J Appl Microbiol 95:990–1000

    Article  PubMed  CAS  Google Scholar 

  • Cherif A, Rezgui W, Raddadi N, Daffonchio D, Boudabous A (2008) Characterization and partial purification of entomocin 110, a newly identified bacteriocin from Bacillus thuringiensis subsp. Entomocidus HD110. Microbiol Res 163:684–692

    Article  PubMed  CAS  Google Scholar 

  • Duitman EH, Hamoen LW, Rembold M, Venema G, Seitz H, Saenger W, Bernhard F, Reinhardt R, Schmidt M, Ullrich C, Stein T, Leenders F, Vater J (1999) The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc Natl Acad Sci USA 96:13294–13299

    Article  PubMed  CAS  Google Scholar 

  • Green G, Dicks LM, Bruggeman G, Vandamme EJ, Chikindas ML (1997) Pediocin PD-1, a bactericidal antimicrobial peptide from Pediococcus damnosus NCFB 1832. J Appl Microbiol 83:127–132

    Article  PubMed  CAS  Google Scholar 

  • Gross E, Kiltz HH, Nebelin E (1973) Subtilin, VI. Die Struktur des Subtilins. Hoppe-Seyler Z Physiol Chem 354:810–812

    PubMed  CAS  Google Scholar 

  • Holo H, Nilssen O, Nes IF (1991) Lactococcin A, a new bacteriocin from Lactococcus lactis subsp. cremoris: isolation and characterization of the protein and its gene. J Bacteriol 173:3879–3887

    PubMed  CAS  Google Scholar 

  • Inaoka T, Takahashi K, Yada H, Yoshida M, Ochi K (2004) RNA polymerase mutation activates the production of a dormant antibiotic 3,3′-neotrehalosadiamine via an autoinduction mechanism in Bacillus subtilis. J Biol Chem 279:3885–3892

    Article  PubMed  CAS  Google Scholar 

  • Joerger RD (2003) Alternatives to antibiotics: bacteriocins, antimicrobial peptides and bacteriophages. Poult Sci 82:640–647

    PubMed  CAS  Google Scholar 

  • Kimura H, Matsusaki H, Sashihara T, Sonomoto K, Ishizaki A (1998) Purification and partial indentification of bacteriocin ISK-1, a new lantibiotic produced by Pediococcus sp. ISK-1. Biosci Biotechnol Biochem 62:2341–2345

    Article  CAS  Google Scholar 

  • Lightbown JW, Mussett MV, Sulitzeanu D (1958) A method for the blood-serum assay of the blood-level duration test for oily injection of procaine penicillin. Bull World Health Organ 19:1085–1091

    PubMed  CAS  Google Scholar 

  • Liu JY, Pan NS, Chen ZL (1990) Characterization of an anti-rice bacterial blight polypeptide LCI. Rice Genet Newsl 7:151–154

    Google Scholar 

  • Moyne AL, Cleveland TE, Tuzun S (2004) Molecular characterization and analysis of the operon encoding the antifungal lipopeptide bacillomycin D. FEMS Microbiol Lett 234:43–49

    Article  PubMed  CAS  Google Scholar 

  • Nagao J, Asaduzzaman SM, Aso Y, Okuda K, Nakayama J, Sonomoto K (2006) Lantibiotics: insight and foresight for new paradigm. J Biosci Bioeng 102:139–149

    Article  PubMed  CAS  Google Scholar 

  • Paik SH, Chakicherla A, Hansen JN (1998) Identification and characterization of the structural and transporter genes for, and the chemical and biological properties of, sublancin 168, a novel lantibiotic produced by Bacillus subtilis 168. J Biol Chem 273:23134–23142

    Article  PubMed  CAS  Google Scholar 

  • Peypoux F, Bonmatin JM, Wallach J (1999) Recent trends in the biochemistry of surfactin. Appl Microbiol Biotechnol 51:553–563

    Article  PubMed  CAS  Google Scholar 

  • Pieterse R, Todorov SD (2010) Bacteriocins—exploring alternatives to antibiotics in mastitis treatment. Braz J Microbiol 41:542–562

    Article  Google Scholar 

  • Pinchuk IV, Bressollier P, Sorokulova IB, Verneuil B, Urdaci MC (2002) Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats. Res Microbiol 153:269–276

    Article  PubMed  CAS  Google Scholar 

  • Riley MA, Wertz JE (2002) Bacteriocins: evolution, ecology, and application. Annu Rev Microbiol 56:117–137

    Article  PubMed  CAS  Google Scholar 

  • Rose SB, Miller RE (1939) Studies with the agar cup-plate method: I. A standardized agar cup-plate technique. J Bacteriol 38:525–537

    PubMed  CAS  Google Scholar 

  • Schägger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22

    Article  PubMed  Google Scholar 

  • Sharma N, Kapoor G, Neopaney B (2006) Characterization of a new bacteriocin produced from a novel isolated strain of Bacillus lentus NG121. Antonie Van Leeuwenhoek 89:337–343

    Article  PubMed  CAS  Google Scholar 

  • Stein T, Borchert S, Conrad B, Feesche J, Hofemeister B, Hofemeister J, Entian KD (2002) Two different lantibiotic-like peptides originate from the ericin gene cluster of Bacillus subtilis A1/3. J Bacteriol 184:1703–1711

    Article  PubMed  CAS  Google Scholar 

  • Steller S, Vollenbroich D, Leenders F, Stein T, Conrad B, Hofemeister J, Jacques P, Thonart P, Vater J (1999) Structural and functional organization of the fengycin synthetase multienzyme system from Bacillus subtilis b213 and A1/3. Chem Biol 6:31–41

    Article  PubMed  CAS  Google Scholar 

  • Tamehiro N, Okamoto-Hosoya Y, Okamoto S, Ubukata M, Hamada M, Naganawa H, Ochi K (2002) Bacilysocin, a novel phospholipid antibiotic produced by Bacillus subtilis 168. Antimicrob Agents Chemother 46:315–320

    Article  PubMed  CAS  Google Scholar 

  • Xie J, Zhang R, Shang C, Guo Y (2009) Isolation and characterization of a bacteriocin produced by an isolated Bacillus subtilis LFB112 that exhibits antimicrobial activity against domestic animal pathogens. Afr J Biotechnol 8:5611–5619

    Google Scholar 

  • Xiraphi N, Georgalaki M, Rantsios K, Cocolin L, Tsakalidou E, Drosinos EH (2008) Purification and characterization of a bacteriocin produced by Leuconostoc mesenteroides E131. Meat Sci 80:194–203

    Article  PubMed  CAS  Google Scholar 

  • Yamazaki K, Suzuki M, Kawai Y, Inoue N, Montville TJ (2005) Purification and characterization of a novel class IIa bacteriocin, piscicocin CS526, from surimi-associated Carnobacterium piscicola CS526. Appl Environ Microbiol 71:554–557

    Article  PubMed  CAS  Google Scholar 

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Acknowledgment

This work was supported by National High-Tech Research and Development Projects (863) 2006AA10A208.

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Authors and Affiliations

  1. Laboratory of Feed Biotechnology, State Key Laboratry of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, No. 2 West Road Yuanmingyuan, Beijing, 100193, People’s Republic of China

    Guiguan Li, Baosheng Liu, Yajing Shang, Zhanqiao Yu & Rijun Zhang

  2. College of Animal Science and Technology, Jiangxi Agricultural University, Nanchang, 330045, People’s Republic of China

    Baosheng Liu

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  1. Guiguan Li
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  2. Baosheng Liu
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  3. Yajing Shang
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  4. Zhanqiao Yu
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  5. Rijun Zhang
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Correspondence to Rijun Zhang.

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Guiguan Li and Baosheng Liu contributed equally to this article.

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Li, G., Liu, B., Shang, Y. et al. Novel activity evaluation and subsequent partial purification of antimicrobial peptides produced by Bacillus subtilis LFB112. Ann Microbiol 62, 667–674 (2012). https://doi.org/10.1007/s13213-011-0303-9

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  • DOI: https://doi.org/10.1007/s13213-011-0303-9

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