Genome-assisted Identification, Purification, and Characterization of Bacteriocins

Kirill V Ovchinnikov; Thomas F Oftedal; Sebastian J Reich; Nadav S Bar; Helge Holo; Morten Skaugen; Christian U Riedel; Dzung B Diep

doi:10.21769/BioProtoc.4477

Genome-assisted Identification, Purification, and Characterization of Bacteriocins

Bio Protoc. 2022 Jul 20;12(14):e4477. doi: 10.21769/BioProtoc.4477.

Authors

Kirill V Ovchinnikov¹, Thomas F Oftedal¹, Sebastian J Reich², Nadav S Bar³, Helge Holo¹, Morten Skaugen¹, Christian U Riedel², Dzung B Diep¹

Affiliations

¹ Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, Ås, Norway.
² Institute of Microbiology and Biotechnology, Ulm University, Ulm, Germany.
³ Department of Chemical Engineering, Norwegian University of Science and Technology, Trondheim, Norway.

Abstract

Bacteriocins are antimicrobial peptides with activity against antibiotic resistant bacterial pathogens. Here, we describe a set of methods aimed at purifying, identifying, and characterizing new bacteriocins. The purification consists of ammonium sulphate precipitation, cation-exchange chromatography, and reversed-phase chromatography. The yield of the bacteriocin is quantified by bacteriocin antimicrobial activity in a microtiter plate assay after each purification step. The mass of the purified bacteriocin is assessed by MALDI TOF MS analysis of the active fractions after reversed-phase chromatography. The mass is compared with the theoretical mass based on genetic information from the whole genome sequencing of the bacteriocin producer strain. Physicochemical characterization is performed by assessing antimicrobial activity following heat and protease treatments. Fluorescent techniques are used to examine the capacity of the bacteriocin to disrupt membrane integrity. Herein a set of protocols for purification and characterization of the bacteriocin nisin Z is used as a typical example in this paper.

Keywords: Antibiotic resistance; Antimicrobial peptide; Bacteriocin; Biosensor; Chromatography; MALDI; MALDI TOF; Peptides; Pore formation; Propidium iodide; Purification; pHluorin.