Purification of a Bacteriocin‐Like Inhibitory Substance Derived from Pediococcus acidilactici Kp10 by an Aqueous Micellar Two‐Phase System

Abstract

Aqueous micellar two‐phase system (AMTPS) is an extractive technique of biomolecule, where it is based on the differential partitioning behavior of biomolecule between a micelle‐rich and a micelle‐poor phase. In this study, an AMTPS composed of a nonionic surfactant, Triton X‐100 (TX‐100) was used for purifying a bacteriocin‐like inhibitory substance (BLIS) derived from Pediococcus acidilactici Kp10. The influences of the surfactant concentration and the effect of additives on the partitioning behavior and activity yield of the BLIS were investigated. The obtained coexistence curves showed that the mixtures of solutions composed of different surfactant concentrations (5–30% w/w) and 50% w/w crude load were able to separate into two phases at temperatures of above 60 °C. The optimum conditions for BLIS partitioning using the TX‐100‐based AMTPS were: TX‐100 concentration of 22.5% w/w, CFCS load of 50% w/w, incubation time of 30 min at 75 °C, and back‐extraction using acetone precipitation. This optimal partitioning resulted in an activity yield of 64.3% and a purification factor of 5.8. Moreover, the addition of several additives, such as sorbitol, KCl, dioctyl sulfosuccinate sodium salt, and Coomassie® Brilliant Blue, demonstrated no improvement in the BLIS separation, except for Amberlite® resin XAD‐4, where the activity yield was improved to 70.3% but the purification factor was reduced to 2.3. Results from this study have demonstrated the potential and applicability of TX‐100‐based AMTPS as a primary recovery method for the BLIS from a complex fermentation broth of P. acidilactici Kp10. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2719, 2019

Partial Purification and Characterization of Bacteriocin-Like Inhibitory Substances Produced by Streptomyces sp. Isolated from the Gut of Chanos chanos

“…The total protein concentration of each sample was determined on the basis of the Bradford method (Bradford 1976), which refers to Jamaluddin et al [ 27 ]. A total of 160 μ L of each sample was reacted with 40 μ L of Bradford reagent in a 96-well microtiter plate and then incubated at 37°C in an orbital shaker for 10 min.…”

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

“…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).…”

“…The antibacterial activity of purified BLIS fractions was stated as BLIS activity (AU mL −1 ) and BLIS‐specific activity (AU μL −1 ) (Jamaluddin et al ., 2019; Kurnianto et al ., 2022). BLIS activity was obtained by calculating the ratio of inhibition zone area (mm 2 ) and sample volume (mL) (eq.…”

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

“…Protein concentration was calculated as μ g mL −1 . The protein concentration was then used for the calculation of BLIS specific activity [ 21 ].…”

“…The diameter of the inhibition zone (mm) was measured. Based on the inhibition zone and protein concentration, the value of BLIS activity and the specific activity of BLIS can be determined [ 21 ]. BLIS activity is calculated using Eq.…”