PMID:22524239

Citation	Park, J, Yun, J, Lim, JA, Kang, DH and Ryu, S (2012) Characterization of an endolysin, LysBPS13, from a Bacillus cereus bacteriophage. FEMS Microbiol. Lett. 332:76-83
Abstract	Use of bacteriophages as biocontrol agents is a promising tool for controlling pathogenic bacteria including antibiotic-resistant bacteria. Not only bacteriophages but also endolysins, the peptidoglycan hydrolyzing enzymes encoded by bacteriophages, have high potential for applications as biocontrol agents against food-borne pathogens. In this study, a putative endolysin gene was identified in the genome of the bacteriophage BPS13, which infects Bacillus cereus. In silico analysis of this endolysin, designated LysBPS13, showed that it consists of an N-terminal catalytic domain (PGRP domain) and a C-terminal cell wall binding domain (SH3_5 domain). Further characterization of the purified LysBPS13 revealed that this endolysin is an N-acetylmuramyl-l-alanine amidase, the activity of which was not influenced by addition of EDTA. In addition, LysBPS13 demonstrated remarkable thermostability in the presence of glycerol, and it retained its lytic activity even after incubation at 100 °C for 30 min. Taken together, these results indicate that LysBPS13 can be considered a favorable candidate for a new antimicrobial agent to control B. cereus.
Links	PubMed Online version:10.1111/j.1574-6968.2012.02578.x
Keywords	Anti-Bacterial Agents/chemistry; Anti-Bacterial Agents/metabolism; Bacillus cereus/chemistry; Bacillus cereus/virology; Bacteriophages/genetics; Binding Sites; Detergents; Edetic Acid; Endopeptidases/chemistry; Endopeptidases/genetics; Endopeptidases/metabolism; Enzyme Stability; Genes, Viral; Glycerol; Hydrogen-Ion Concentration; Muramic Acids/metabolism; N-Acetylmuramoyl-L-alanine Amidase/chemistry; N-Acetylmuramoyl-L-alanine Amidase/genetics; N-Acetylmuramoyl-L-alanine Amidase/metabolism; Osmolar Concentration; Peptidoglycan/metabolism; Protein Structure, Tertiary; Recombinant Proteins/chemistry; Recombinant Proteins/genetics; Recombinant Proteins/metabolism; Temperature; Viral Proteins/chemistry; Viral Proteins/genetics; Viral Proteins/metabolism
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:J9PU17		GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000314:		F		Peptidoglycan of B. cereus was incubated with 5 micrograms LysBPS13 for 30 min to determine the amidase activity of LysBPS13 by the amount of free N-acteylmuramic acid liberated. The N-acteylmuramic acid product was first degraded to lactic acid, and then acetaldehyde to be identified colorimetrically with p-hydroxydiphenyl (PHD). The results showed an increase in free N-acteylmuramic acid from cleavage of the bond between N-acetylmuramic acid and L-alanine (Figure 2), demonstrating LysBPS13 has N-acetylmuramyl-L-alanine amidase activity. A glycosidase assay ruled-out LysBPS13 as a glucosaminidase or a muramidase by showing free reducing sugars were not generated by the peptidoglycan after LysBPS13 treatment. Additionally, when 5 micrograms/milliliter of purified, recombinant LysBPS13 was added to B. cereus cells, almost all the B. cereus cells were effectively lysed within 10 min (Figure 1.c), further supporting N-acetylmuramoyl-L-alanine amidase activity of LysBPS13.	complete CACAO 10884
9CAUD:J9PU17	enables	GO:0008745: N-acetylmuramoyl-L-alanine amidase activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

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