The culture was incubated
NVP-BSK805 mw at 30°C with shaking at 120 rpm for optimal growth. The CFB obtained by removing the cells present in the medium by centrifugation (6,000 g for 10 min, 4°C) and subsequent filtration of the supernatant through 0.22 μm filter (Millipore, USA). The CFB was used to test the growth inhibition activity by agar well diffusion assay using actively growing test strains (between 0.2-0.4 OD). A growth curve verses antimicrobial production graph up to 48 h was constructed for strain IE-3 to examine the bacteriocin production at regular time intervals using anaerobic broth. Bacterial growth was measured as absorbance at 600 nm after constant time intervals of 2 h and antimicrobial activity at same time point was estimated
by zone inhibition assay against L. monocytogenes test strain. Purification of low molecular weight antimicrobial peptide Strain IE-3 was grown anaerobically in serum vials at 30°C for 48 h for the maximum production of a LMW peptide. Antimicrobial compound was extracted from CFB using 2% activated Diaion HP20 (Sigma, USA) hydrophobic resin. The crude HIF inhibitor extract obtained was further purified through cation exchange (Capto S, GE Healthcare, USA) chromatography column linked to an AKTA prime plus (GE healthcare, USA), in 20 mM sodium acetate buffer (pH 4.6) and eluted with NaCl gradient (50 learn more to 1000 mM) in binding buffer. The peptide was desalted using dialysis tube (molecular cutoff 0.5 kDa, Spectrum, USA). Approximate molecular mass of peptide was determined by gel filtration column (Sodex KW-802.5) using standard molecular weight markers as described earlier [31]. Purity was confirmed by reversed phase HPLC (10 mm × 250 mm × 150 Å) C-18 column (venusil, Agela Technologies) under isocratic flow (1.5 ml/min) of acetonitrile (20%) along with 0.1% TFA. Elution
was monitored at 200–340 nm wavelength range on PDA detector and peaks were collected by fraction collector (1260 Infinity, Agilent technology, USA). In-gel activity assay The partially purified antimicrobial peptide (50 μg/lane) was electrophoresed in duplicate on 18.0% tricine SDS-PAGE [32]. One set of the gel lane along with protein ladder (multi-color low range protein ladder, Thermo Spectra™) was stained with Coomassie brilliant blue to confirm the PRKACG location of the antimicrobial peptide and the other lane of the gel was used to test antimicrobial activity as described earlier [33] by overlaying with 5 ml of log-phase culture of L. monocytogenes (106 cells/ml) and was incubated at 30°C overnight. Intact mass analysis and de novo sequencing To analyze the molecular mass of peptide, purified peptide was electrophoresed, eluted from tricine SDS-PAGE by 75% acetonitrile with 0.1% TFA and used only for mass analysis and sequencing. Eluted peptide was mixed with equal ratios (1:1) of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.1% (v/v) TFA.