Lu Shao-Yeh, Bischoff Kenneth M, Rich Joseph O, Liu Siqing, Skory Christopher D
Renewable Product Technology Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604-3902 USA.
Agricultural Research Service, U.S. Department of Agriculture, Fort Collins, CO 80526 USA.
Biotechnol Biofuels. 2020 Sep 8;13:157. doi: 10.1186/s13068-020-01795-9. eCollection 2020.
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.
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.
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.
传统上，商业乙醇发酵设施依靠抗生素来控制细菌污染。在此，我们展示了一种使用从噬菌体EcoSau中分离出的新型肽聚糖水解酶（LysKB317）来处理污染的替代方法。这种内溶素是专门针对从燃料乙醇工厂分离出的作为污染物的菌株筛选出来的。LysKB317基因在大肠杆菌中重组表达为一种33 kDa的纯化酶。
在浊度降低试验中，该重组酶对一组32种细菌菌株进行了测试，对代表1种芽孢杆菌属、8种肠杆菌属、1种克雷伯氏菌属、3种芽孢杆菌属和1种葡萄球菌属的28种细菌菌株具有活性。LysKB317的活性在pH 6左右最佳，但在pH 4.5 - 7.5范围内具有广泛的活性和稳定性，至少持续48小时。在50°C下观察到最大活性，至少持续72小时。此外，LysKB317在30%乙醇中至少72小时内保持稳定。在实验感染的玉米醪发酵中，1 μM内溶素使细菌载量降低了3个对数级变化，而0.01 μM使细菌减少了2个对数级变化。用≥0.01 μM LysKB317处理的感染培养物的发酵产物（乙醇、残留葡萄糖、乳酸和乙酸）浓度与未受污染的对照相似。
外源添加的LysKB317内溶素在燃料乙醇发酵罐中常见的条件下具有功能，可开发作为燃料乙醇发酵过程中控制污染的抗生素替代品。
