Orito Y, Morita M, Hori K, Unno H, Tanji Y
Department of Bioengineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, 226-8501Yokohama, Japan.
Appl Microbiol Biotechnol. 2004 Jul;65(1):105-9. doi: 10.1007/s00253-003-1522-1. Epub 2004 Jan 9.
To determine the function of the C-terminal region of Bacillus amyloliquefaciens phage endolysin on Pseudomonas aeruginosa lysis, the permeabilization of the outer membrane of P. aeruginosa was analyzed. Glu-15 to His (E15H) and Thr-32 to Glu (T32E) substitutions were introduced into the Bacillus phage endolysin. Neither E15H nor T32E substitution induced enzymatic and antibacterial activities. These two, Glu-15 and Thr-32, were considered to be the active center of the enzyme. The addition of purified E15H and T32E proteins to P. aeruginosa cells induced the release of periplasmic beta-lactamase from the cells, indicating that both proteins enhance permeabilization of the outer membrane. However, the addition of E15H and T32E proteins to P. aeruginosa cells did not induce the release of cytoplasmic ATP from the cells. These results indicate that the antibacterial activity of the endolysin requires both the C-terminal enhancement of the permeabilization of the P. aeruginosa outer membrane and N-terminal enzymatic activity.
为确定解淀粉芽孢杆菌噬菌体溶菌酶C端区域对铜绿假单胞菌的裂解功能，分析了铜绿假单胞菌外膜的通透性。将芽孢杆菌噬菌体溶菌酶中的Glu-15替换为His（E15H）以及Thr-32替换为Glu（T32E）。E15H和T32E替换均未诱导酶活性和抗菌活性。Glu-15和Thr-32这两个位点被认为是该酶的活性中心。向铜绿假单胞菌细胞中添加纯化的E15H和T32E蛋白可诱导细胞周质β-内酰胺酶释放，这表明这两种蛋白均增强了外膜的通透性。然而，向铜绿假单胞菌细胞中添加E15H和T32E蛋白并未诱导细胞释放细胞质ATP。这些结果表明，溶菌酶的抗菌活性既需要增强铜绿假单胞菌外膜的通透性，也需要N端的酶活性。
