Considering ambiguities in de novo sequencing, search was also made by replacing Leu residue with Ile as well as other de novo sequences obtained with low score values, however, no ORF was found in genome sequence. As no ORF detected in genome, it is anticipated that antimicrobial peptide might be produced

from medium components by the strain IE-3. Nevertheless, synthesis of peptide from the medium components is ruled out as the peptide production was observed in minimal medium containing an inorganic nitrogen source. Though the de novo sequence similarity search using APD2 p38 protein kinase [26] revealed low similarity (37% similarity) with the eukaryotic antimicrobial peptide, temporin LTb, it did not show any conserved motifs observed for temporins [27]. Antimicrobial peptide prediction analysis [26] of the de novo sequence suggested that the peptide could be a potential antimicrobial peptide with the presence of cationic, aromatic and hydrophobic

amino acids, along with two cysteine residues. Moreover, this LMW peptide shares an amino acid arrangement with N-terminal sequence of other pediocin-like bacteriocins such as pediocin PA-1 [9,28]. Remarkably, the five amino acids (CTRGC) of the peptide showed amino acids pattern similarity with β-hairpin composition (CTKSGC) of pediocin-like bacteriocins [29] where the positively charged amino acid play crucial role in antimicrobial activity [10]. In fact, addition of a positively charged see more amino acid within this patch showed significant increase in antimicrobial activity of pediocin PA-1 [30]. Additionally, structure prediction analysis for the LMW peptide showed antiparallel β-sheet confirmation (Figure 4) where hydrophobic and positively charged amino acids are enclosed by two cysteine residues (Cyt7-Cyt11). Figure 3 De novo sequence derived for the antimicrobial peptide generated by de – novo explorer of AB Sciex with highest score value (b ion values shown at bottom and y ion Reverse transcriptase values at top). Figure

4 Predicted 3-dimensional structure of de novo sequence obtained for low molecular weight antimicrobial peptide showing the presence of antiparallel β-sheets. Effect of pH, temperature, proteolytic enzymes, reducing agent and H2O2 on antimicrobial activity The LMW antimicrobial peptide was found to be thermo-stable as there was no reduction observed in its antimicrobial activity even after 30 min of incubation at 100°C. However, it displayed sensitivity towards the pH as the maximum activity was observed at pH 5 and significant loss was found at pH 8 and above (Table 2). Unlike pediocin-like bacteriocins, the low molecular weight peptide in this study was found to be resistant to proteolytic cleavage as an antimicrobial assay performed upon incubation with proteolytic enzymes showed no reduction in activity.