1887

Abstract

Pediocin PA −1 is class IIa bacteriocin that displays efficient antimicrobial activity against pathogenic Listeriaspp. This bacteriocin is known to lose activity during long periods of storage especially at non optimal pH, thus reducing its usefulness for the pharmaceutical and food industries. Loss of activity has been attributed to oxidation of the methionine residue at position 31, however, replacement of this residue by leucine results in a peptide with activity equivalent to that of the native peptide. In this work, the heterologous expression of the structural (with Met31 to Leu substitution), accessory and transport genes from pediocin PA-1 operon was carried out in Escherichia coli TunerTM (DE3) cells. The sequences of all genes were redesigned using codon bias for the host and were cloned into an expression vector that allows control of plasmid copy number. The heterologous expression of pediocin Met31Leu was optimized for temperature, induction time, IPTG concentration and plasmid copy number and was evaluated via antimicrobial activity assays against Listeria innocua DPC3572. Maximum activity (2560 AU mL) was achieved using low plasmid copy number and 6 h of induction at 37 °C with 1 mM of IPTG. Recombinant pediocin PA-1M31L was successfully purified in 5 steps (>95 % purity) as confirmed by mass spectrometry (4606.27 Da) with a yield of 0.725 mg per liter of culture. This variant showed a similar spectrum of activity to the native pediocin PA-1 and is an interesting alternative for industrial applications due to its greater stability.

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/content/journal/acmi/10.1099/acmi.ac2019.po0471
2019-04-08
2019-12-13
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http://instance.metastore.ingenta.com/content/journal/acmi/10.1099/acmi.ac2019.po0471
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