Purification, HR-LC-ESI-MS-MS Identification, and Peptide Prediction of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from Chanos chanos

Abstract

Consumption of fresh and minimally processed food is closely related to foodborne diseases. To minimize the adverse effects, bacteriocin-like inhibitory substance (BLIS) as a natural preservative can be used. One of the bacteriocins with promising activity was produced by Streptomyces sp. Using gel filtration chromatography, the bacteriocin purification process succeeded in obtaining semi-purified fractions with broad-spectrum inhibitory activity to foodborne pathogen bacteria. These fractions are also stable up to 100 °C and pH 2.0–7.0. High-Resolution Liquid Chromatography Electrospray Ionization-Tandem Mass Spectrometry analysis followed by orthogonal projection to latent structure showed that each fraction had eight peaks with the highest positive correlation to BLIS-specific activity. Peptide identification based on MS spectrum found 597 predictive peptides, of which 42 predictive peptides with antimicrobial peptide characteristics and the highest iAMPpred antimicrobial peptide probability (>0.5) were selected. The selected predictive peptides have molecular mass of 247.13-615.37 Da and consist of at least 20% hydrophobic amino acids with a hydrophobicity value of 14.72 Kcal mol-1. The results of this study indicate the effectiveness of BLIS purification by gel filtration chromatography and the promising potential of semi-purified BLIS as a natural preservative. Besides, the active peptides in semi-purified BLIS can also be identified quickly so that the isolation process to obtain purified-BLIS can be carried out more efficiently.

Bioinformatics analysis of purified and identified novel bacteriocin‐like inhibitory substances produced by Streptomyces sp.

“…Bacteria Staphylococcus aureus ATCC 25922, Listeria monocytogenes, Salmonella enterica serovar Typhimurium ATCC 14028 and Escherichia coli ATCC 25922 in TSA (tryptic soy agar; Oxoid, UK) slant agar were picked up using an ose needle and inoculated on TSB (tryptic soy broth; Oxoid, UK) medium. Bacterial cultures were incubated at 37 °C for 24 h (Kurnianto et al ., 2020, 2022).…”

“…BLIS fractions that have been partially purified using gel filtration chromatography (BLIS‐GF) with potential activity (AS53, AS82, AS85, AS112, AA122, BS116 and BA122) were purified using RP‐HPLC to obtain pure BLIS fractions (Pei et al ., 2020; Kurnianto et al ., 2022). First, BLIS‐GF was dissolved with distilled water (LC–MS grade) and injected into a C18 column (Shim‐pack PREP(H)‐Kit 250 × 4.6 mm; 5 μm, Shimadzu, Japan) placed in an RP‐HPLC (reversed‐phase high‐performance liquid chromatography) system (Shimadzu, Japan) with a photodiode array (PDA) detector at a flow rate of 0.5 mL min −1 with mobile phase A (99.5% water, 0.05% trifluoroacetic acid/TFA) and mobile phase B (100% acetonitrile).…”

“…This analysis uses bovine serum albumin (BSA; Sigma Aldrich, US) as a standard solution and distilled water as a blank. The BLIS‐specific activity was calculated using the protein concentration (Jamaluddin et al ., 2019; Kurnianto et al ., 2022).…”

“…Our previous study reported that the partially purified bacteriocin fraction produced by Streptomyces sp. associated with milkfish ( Chanos chanos ) has promising antimicrobial activity and stability (Kurnianto et al ., 2020, 2021, 2022).…”

“…In previous studies, two‐stage partial purification of BLIS was carried out using ultrafiltration membranes and chromatography gel filtration (Sephadex G‐25). The analysis showed that seven BLIS fractions resulting from partial purification had good antibacterial bioactivity and stability (Kurnianto et al ., 2021, 2022). Based on these results, this study aims to purify the potential BLIS fraction using RP‐HPLC to obtain a purified BLIS fraction, identify the purified BLIS peptide sequence using HR‐LC‐ESI‐MS/MS, characterise toxicity (LC 50 ) and stability against various pH levels and high temperatures and analyse the molecular interactions between the purified BLIS fraction and the target penicillin‐binding protein 3 (PBP‐3) by methicillin‐resistant Staphylococcus aureus (MRSA) using a bioinformatics approach.…”