Recombinant Bacteriophage LysKB317 Endolysin Mitigates Infection of Corn Mash Fermentations

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 strains that were isolated as contaminants from a fuel ethanol plant. The LysKB317 gene was recombinantly expressed in 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 1 species of , 8 species of , 1 species of , 3 species of , and 1 species of . 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 h. In addition, LysKB317 was stable in 30% ethanol up to at least 72 h. 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 was 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.