Characterization and Quantitative Determination of a Diverse Group of Bacillus subtilis subsp. subtilis NCIB 3610 Antiba
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Characterization and Quantitative Determination of a Diverse Group of Bacillus subtilis subsp. subtilis NCIB 3610 Antibacterial Peptides Angeliki Karagiota 1 & Hara Tsitsopoulou 2 & Rafail Nikolaos Tasakis 3 & Varvara Zoumpourtikoudi 2 & Maria Touraki 2 Accepted: 31 August 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract Five antibacterial peptides produced by Bacillus subtilis NCIB 3610 were purified, quantified, characterized, and identified in the present study. Cell-free extracts were subjected to three purification protocols employing ammonium sulfate or organic solvent precipitation and their combination, followed by ion-exchange chromatography, solid-phase extraction, and preparative highperformance liquid chromatography (HPLC). The combined ammonium sulfate and organic solvent precipitation extraction protocol presented the best results for peptide purification. In the five fractions that presented antimicrobial activity, antibacterial peptides were quantified by the turbidometric method and by HPLC using nisin for external calibration, with the second providing more accurate results. All peptides were pH- and temperature-resistant and their sensitivity to proteases treatment indicated their proteinic nature. The five peptides were subjected to microwave-assisted acid hydrolysis (MAAH) and following derivatization were analyzed using norleucine as the internal standard, to determine their amino acid content. The identification of the isolated peptides using the UniProt and PubChem databases indicated that the four peptides correspond to UniProt entries of the bacteriocins Subtilosin-A (Q1W152) Subtilosin-SbOX (H6D9P4), Ericin B (Q93GH3), Subtilin (P10946), and the fifth to the non-ribosomal antibacterial lipopeptide surfactin (CID:443592). The amino acid content determination and computational analyses, applied in the present work on the antimicrobial peptides of B. subtilis, proved an efficient screening and quantification method of bacteriocins that could potentially be applied in other bacterial strains. The constructed phylogenetic trees heterogeneity observed across the five peptides investigated might be indicative of competitive advantage of the strain. Keywords Bacillus subtilis . Bacteriocins . Antibacterial peptides . HPLC quantitation . Amino acid content analysis
Introduction Bacterial infections are treated with antibiotics, once considered as the panacea that could target any bacterial cell without affecting host cells [1]. However, the effectiveness of antibiotics is Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12602-020-09706-y) contains supplementary material, which is available to authorized users. * Maria Touraki [email protected] 1
Laboratory of Biochemistry, Faculty of Medicine, University of Thessaly, Biopolis, 41110 Larissa, Greece
2
Laboratory of General Biology, Division of Genetics, Development and Molecular Biology, Department of Biology, School of Sciences, Aristotle University of Thessalon
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