67 × 10−15) (Fig. 1a). This C-terminal domain was also found in the protein of B. cereus AH676 (ZP 0419059), the Bacillus phages TP21-L (Ply21, CAA72267) and bg1 (LysBG1, ABX56141), and the Lactobacillus phage LL-Ku (AAV30211). However, this domain was not fully characterized. Recombinant LysBPS13 was cloned and expressed in E. coli and purified. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of the purified endolysin (Fig. 1b). As expected, the purified, recombinant LysBPS13 showed lytic activity against B. cereus cells. As little as 5 μg mL−1
5-Fluoracil of LysBPS13 effectively lysed B. cereus cells within 10 min (Fig. 1c). Viable cell counting showed that there was approximately 3-log reduction U0126 by 5 μg of LysBPS13 under this reaction condition after 5 min (data not shown). Because blastp analyses showed that LysBPS13 had high similarity to a number of N-acetylmuramyl-l-alanine amidases, the amidase activity of LysBPS13 was evaluated by measuring free N-acetylmuramic acid liberated from peptidoglycan (Hadzija, 1974). When peptidoglycan of B. cereus was treated with LysBPS13 for 30 min, a significant increase in free muramic acid was detected resulting from cleavage of the bond between N-acetylmuramic acid and l-alanine (Fig. 2).
This demonstrates that LysBPS13 has N-acetylmuramyl-l-alanine amidase activity. Because the glycosidase assay revealed that free reducing sugars were not generated from peptidoglycan after LysBPS13 treatment, this enzyme is not a glucosaminidase or a muramidase. Four genera of Gram-positive bacteria, including Bacillus sp., and five genera of Gram-negative bacteria were examined for their susceptibility to LysBPS13 (Fig. 3). LysBPS13 exhibited the strongest
activity against B. cereus ATCC 10876, and it could lyse all of the tested Bacillus species, including pathogenic B. cereus and Bacillus thuringiensis. However, other tested Gram-positive bacteria, such as Listeria monocytogenes, Enterococcus faecalis, Cediranib (AZD2171) Staphylococcus aureus, and Staphylococcus epidermidis, were not lysed by LysBPS13. Among the tested Gram-negative bacteria, LysBPS13 was active against Salmonella, E. coli, Cronobacter sakazakii, and Shigella strains, when these bacteria were treated with EDTA (data not shown). The relative lytic activity against these bacteria was as strong as it was against Gram-positive bacteria (74 ~84%). However, the endolysin did not show lytic activity against these Gram-negative bacteria in the absence of EDTA treatment. To determine the optimal conditions for LysBPS13 function, the exogenous lytic activity of LysBPS13 was examined under different conditions. Lytic activity was highest at pH 9.5 and significantly decreased at pH > 10.5 and < 7.5 (Fig. 4a). The optimal temperature for lytic activity was 42–45 °C (Fig. 4b). The effect of ionic strength on the lytic activity of LysBPS13 was assessed with different concentrations of NaCl (Fig. 4c).
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