Production of enterocin P by a bioengineered Pichia pastoris to control of pathogen bacteria

Document Type : Original Article

Authors

1 Ph.D. of Agricultural Biotechnology, Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Shahrood University of Technology, Shahrood, Iran.

2 Assistant Professor in Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

3 Ph.D. student of Animal Sciences, Department of Animal Sciences, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran.

Abstract

Entrocin p has been characterized in class IIa bacteriocins and showed a broad spectrum of antimicrobial activity. In this study, the nucleotide sequence of enterocin P transformed and expressed in Pichia pastoris genomically, and antimicrobial activity of the extracted protein was evaluated on pathogenic bacteria. The synthetic sequence of entrocin p was ligated into pPICZαA, an integrated vector, in EcoRI - XbaI site. The recombinant plasmid was amplified in Escherichia coli strain DH5α and proved for ligation and probably mutation by sequencing. The linearized plasmid was electroporated into Pichia pastoris. The presence of recombinant protein in yeast cell extraction was verified by SDS-PAGE and antimicrobial activity of the purified protein. Recombinant entrocin p tested for the inhibition of 8 avian pathogen bacteria via MIC test. Electrophoresis of purified protein and cell culture supernatant showed a protein band about 5.4 kDa. The mass spectrometry of the sample exhibit an Amino acid sequence corresponds to enterocin P in Enterococcus faecium with an accession number of OTN91526.1. Recombinant bacteriocin MIC test shows the most inhibition on Salmonella typhimurium and E. coli O157. Recombinant Pichia pastoris exhibited a potential for producing enterocin P in high concentrations; therefore, this antimicrobial peptide can be studied for pharmacological use or industrial applications.

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History

  • Receive Date: 30 October 2022
  • Revise Date: 29 January 2024
  • Accept Date: 17 February 2024

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