Abstract
The binding affinity of the N-terminal peptidoglycan binding domain of endolysin KZ144 (PBD KZ), originating from Pseudomonas aeruginosa bacteriophage ϕKZ, has been examined using a fusion protein of PBD KZ and green fluorescent protein (PBD KZ-GFP). A fluorescence recovery after photobleaching analysis of bound PBD KZ-GFP molecules showed less than 10% fluorescence recovery in the bleached area within 15 min. Surface plasmon resonance analysis confirmed this apparent high binding affinity revealing an equilibrium affinity constant of 2.95 × 10 7 M −1 for the PBD KZ-peptidoglycan interaction. This unique domain, which binds to the peptidoglycan of all tested Gram-negative species, was harnessed to improve the specific activity of the peptidoglycan hydrolase domain KMV36C. The chimeric peptidoglycan hydrolase (PBD KZ-KMV36C) exhibits a threefold higher specific activity than the native catalytic domain (KMV36C). These results demonstrate that the modular assembly of functional domains is a rational approach to improve the specific activity of endolysins from phages infecting Gram-negatives.
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