, 1995; Loeffler et al, 2003; Schmelcher et al, 2012) This may

, 1995; Loeffler et al., 2003; Schmelcher et al., 2012). This may be an advantage of this endolysin, as these ionic conditions correspond to the salt concentration of many food products. LysBPS13 seems to need no metal ions for its lytic activity, because the addition of EDTA (300 mM) did not affect its lytic activity (Fig. 4d), nor did the presence of metal ions (1 mM MgCl2, CaCl2, ZnCl2, or MnCl2) (data not shown). This result was unexpected because the three Zn2+-binding residues in the PGRP domain were completely conserved

in LysBPS13. While T7 lysozyme that belongs to the PGRP family has Zn2+-dependent amidase activity (Gelius et al., 2003; Kim et al., 2003), another report found a Zn2+-independent amidase (ORF9) in GSK126 the E. faecalis BKM120 bacteriophage EF24C (Uchiyama et al., 2011). Like LysBPS13, E. faecalis ORF9 has a PGRP domain at its N-terminus, and blastp analysis

indicated Zn2+-binding sites, but Zn2+ did not seem to be essential for its activity. Yet, we cannot rule out the possibility that Zn2+ or other metal cofactors are bound to LysBPS13 too tightly to be removed by EDTA. Therefore, further study is necessary to elucidate the structure of the PGRP domain in endolysins, particularly the Zn2+-binding site. When LysBPS13 was tested in combination with various detergents (Fig. 4d), LysBPS13 showed full or higher activity in the presence of zwitterionic (CHAPS) RVX-208 and nonionic detergents (Triton X-100, Tween-20). However, both anionic (SDS) and cationic (CTAB) detergents inactivated LysBPS13. Thermostability of phage endolysins would be advantageous for applications as biocontrol agents

that undergo heat treatment. B. cereus food poisoning is often associated with cooked rice products, because B. cereus spores are able to endure high temperatures and germinate when cooling down (Stenfors Arnesen et al., 2008). Most endolysins are labile to heat (Lavigne et al., 2004). However, to date, only a few lysins have been reported to be thermostable, including Gp36 from the Pseudomonas aeruginosa bacteriophage φKMV (Lavigne et al., 2004); the lysins HPL118, HPL511, and HPLP35 from Listeria bacteriophages (Schmelcher et al., 2012); and the GVE2 lysin (EF079891) from Geobacilllus phage GVE2 (Ye & Zhang, 2008). Gp36 has extremely high thermostability, retaining 21% of its activity after autoclaving at 121 °C for 20 min; other lysins have milder thermostability (Lavigne et al., 2004; Ye & Zhang, 2008). LysBPS13 appeared to be highly stable, as the protein retained full lytic activity after a week-long incubation in storage buffer at room temperature. The thermostability of LysBPS13 was further assessed after pre-incubation of the enzyme at temperatures between 4 and 100 °C (Fig. 5). LysBPS13 demonstrated lytic activity after incubation for 30 min at all tested temperatures.

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