Recombinant Bacteriophage LysKB317 Endolysin Mitigates Lactobacillus Infection of Corn Mash Fermentations

Abstract

Background Commercial ethanol fermentation facilities traditionally rely on antibiotics for bacterial contamination control. Here we demonstrate an alternative approach to treat contamination using a novel peptidoglycan hydrolase (LysKB317) isolated from a bacteriophage, EcoSau. This endolysin was specially selected against Lactobacillus strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in Escherichia coli as a 33 kDa purified enzyme. Results In turbidity reduction assays, the recombinant enzyme was subjected to a panel of 32 bacterial strains and was active against 28 bacterial strains representing one species of Acetobacter, eight species of Lactobacillus, one species of Pediococcus, three species of Streptococcus, and one species of Weissella. The activity of LysKB317 was optimal around pH 6, but it has broad activity and stability from pH 4.5 – 7.5 up to at least 48 h. Maximum activity was observed at 50°C up to at least 72 hrs. In addition, LysKB317 was stable in 30% ethanol up to at least 72 hrs. In experimentally infected corn mash fermentations, 1 µM endolysin reduced bacterial load by 3-log fold change, while 0.01 µM reduced bacteria by 2-log fold change. Concentration of fermentation products (ethanol, residual glucose, lactic acid, and acetic acids) for infected cultures treated with ≥ 0.01 µM LysKB317 were similar to uncontaminated controls. Conclusion Exogenously added LysKB317 endolysin is functional in conditions typically found in fuel ethanol fermentations tanks and may be developed as an alternative to antibiotics for contamination control during fuel ethanol fermentations.