Characterization of Lactobacilli Phage Endolysins and Their Functional Domains-Potential Live Biotherapeutic Testing Reagents

Phage endolysin-specific binding characteristics and killing activity support their potential use in biotechnological applications, including potency and purity testing of live biotherapeutic products (LBPs). LBPs contain live organisms, such as lactic acid bacteria (LAB), and are intended for use as drugs. Our approach uses the endolysin cell wall binding domains (CBD) for LBP potency assays and the endolysin killing activity for purity assays. CBDs of the following five lactobacilli phage lysins were characterized: CL1, Jlb1, Lj965, LL-H, and Phi JB. They exhibited different bindings to 27 LAB strains and were found to bind peptidoglycan or surface polymers. Flow cytometry based on CBD binding was used to enumerate viable counts of two strains in the mixture. CL1-lys, jlb1-lys, and Phi JB-lys and their enzymatic domains (EADs) exhibited cell wall digestive activity and lytic activity against LAB. Jlb1-EAD and Phi JB-EAD were more sensitive than their respective hololysins to buffer pH and NaCl changes. The Phi JB-EAD exhibited stronger lytic activity than Phi JB-lys, possibly due to Phi JB-CBD-mediated sequestration of Phi JB-lys by cell debris. CBD multiplex assays indicate that these proteins may be useful LBP potency reagents, and the lytic activity suggests that CL1-lys, jlb1-lys, and Phi JB-lys and their EADs are good candidates for LBP purity reagent development.