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Novel lytic enzyme of prophage origin from Clostridium botulinum E3 strain Alaska E43 with bactericidal activity against Clostridial cells

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cris.lastimport.scopus2024-02-12T19:51:41Z
dc.abstract.enClostridium botulinum is a Gram-positive, anaerobic, spore-forming bacterium capable of producing botulinum toxin and responsible for botulism of humans and animals. Phage-encoded enzymes called endolysins, which can lyse bacteria when exposed externally, have potential as agents to combat bacteria of the genus Clostridium. Bioinformatics analysis revealed in the genomes of several Clostridium species genes encoding putative N-acetylmuramoyl-l-alanine amidases with anti-clostridial potential. One such enzyme, designated as LysB (224-aa), from the prophage of C. botulinum E3 strain Alaska E43 was chosen for further analysis. The recombinant 27,726 Da protein was expressed and purified from E. coli Tuner(DE3) with a yield of 37.5 mg per 1 L of cell culture. Size-exclusion chromatography and analytical ultracentrifugation experiments showed that the protein is dimeric in solution. Bioinformatics analysis and results of site-directed mutagenesis studies imply that five residues, namely H25, Y54, H126, S132, and C134, form the catalytic center of the enzyme. Twelve other residues, namely M13, H43, N47, G48, W49, A50, L73, A75, H76, Q78, N81, and Y182, were predicted to be involved in anchoring the protein to the lipoteichoic acid, a significant component of the Gram-positive bacterial cell wall. The LysB enzyme demonstrated lytic activity against bacteria belonging to the genera Clostridium, Bacillus, Staphylococcus, and Deinococcus, but did not lyse Gram-negative bacteria. Optimal lytic activity of LysB occurred between pH 4.0 and 7.5 in the absence of NaCl. This work presents the first characterization of an endolysin derived from a C. botulinum Group II prophage, which can potentially be used to control this important pathogen.
dc.affiliationUniwersytet Warszawski
dc.contributor.authorKaczorowski, Tadeusz
dc.contributor.authorWyrzykowski, Dariusz
dc.contributor.authorMorzywolek, Agnieszka
dc.contributor.authorPlotka, Magdalena
dc.contributor.authorSzadkowska, Monika
dc.contributor.authorMakowska, Joanna
dc.contributor.authorWaters, Jerel J.
dc.contributor.authorSwift, Steven M.
dc.contributor.authorDonovan, David M.
dc.contributor.authorKozłowski, Łukasz
dc.contributor.authorKaczorowska, Anna-Karina
dc.date.accessioned2024-01-25T13:52:23Z
dc.date.available2024-01-25T13:52:23Z
dc.date.copyright2021-09-02
dc.date.issued2021
dc.description.accesstimeAT_PUBLICATION
dc.description.financePublikacja bezkosztowa
dc.description.number17
dc.description.versionFINAL_PUBLISHED
dc.description.volume22
dc.identifier.doi10.3390/IJMS22179536
dc.identifier.issn1422-0067
dc.identifier.urihttps://repozytorium.uw.edu.pl//handle/item/113956
dc.languageeng
dc.pbn.affiliationcomputer and information sciences
dc.relation.ispartofInternational Journal of Molecular Sciences
dc.relation.pages1-21
dc.rightsCC-BY
dc.sciencecloudnosend
dc.subject.enN-acetylmuramoyl-L-alanine amidase
dc.subject.enClostridium botulinum
dc.subject.enendolysin
dc.subject.enprophage
dc.subject.enlipoteichoic acid
dc.titleNovel lytic enzyme of prophage origin from Clostridium botulinum E3 strain Alaska E43 with bactericidal activity against Clostridial cells
dc.typeJournalArticle
dspace.entity.typePublication