Protein size marker is indicated on the left. (B) The purified VirB1-89KCHAP protein after Ni+ affinity chromatography and gel filtration. Lytic activity and biochemical characterization of VirB1-89KCHAP To determine the muramidase activity of the purified VirB1-89KCHAP protein, peptidoglycan hydrolase activity was analyzed by using zymography with S. suis peptidoglycan as substrate. After SDS-PAGE,

the positive control hen egg white lysozyme, p38 MAPK signaling pathway the negative control BSA protein, and the VirB1-89KCHAP protein could be seen after staining with Coomassie blue (Figure 3A). The gel was then stained with methylene blue to detect peptidoglycan hydrolase activity as a clear zone against a dark blue background. We noticed that VirB1-89KCHAP exhibited apparent enzyme activity

as the positive control did, while the negative control BSA did not (Figure 3B). These zymography data suggested that the VirB1-89KCHAP protein could solubilize the cell wall of S. suis 2. Figure 3 Lytic activity detection of VirB1-89KCHAP. Zymography analysis of peptidoglycan hydrolase activity of VirB1-89KCHAP. The gel was stained with Coomassie blue (A) and then overstained with Methylene blue (B). (C) Bacteriostatic activity of VirB1-89KCHAP. Proteins used: 1, hen egg white lysozyme; 2, BSA; 3, VirB1-89KCHAP. In another set of experiments, the bacteriostatic activity of VirB1-89KCHAP Seliciclib solubility dmso was determined with slip diffusion method to confirm its peptidoglycan hydrolase activity. We found that both the VirB1-89KCHAP protein and the hen egg white lysozyme could suppress the growth of S. suis 2, while the BSA control could not

(Figure 3C). To reveal the basic biological characteristics of VirB1-89KCHAP, we examined the optimum reaction condition of VirB1-89KCHAP by using Micrococcus lysodeikticus cells as substrate. Results showed that on increasing the pH, peptidoglycan hydrolase activity of VirB1-89KCHAP increases and reaches maximum at pH 8.0 (Figure 4A). When the pH exceeds 9.0, the relative activity decreased sharply. VirB1-89KCHAP functions best at an optimal temperature of 40°C. The enzyme Tangeritin activity rapidly declined at temperatures above 50°C and only 25% of the maximal activity was measured at 60°C (Figure 4B). From the thermal stability data, the relative activity is higher at 30°C than at 40°C, suggesting that pre-incubation of VirB1-89KCHAP at 30°C causes lower decay in relative activity compared to the enzyme pre-incubated at 40°C (Figure 4C). With increasing temperature, pre-incubation of VirB1-89KCHAP caused increasing decay in the relative activity of the enzyme. Figure 4 Dynamic changes in lytic activity of VirB1-89KCHAP at different pH values or temperatures. (A) The effect of pH on enzyme activity of VirB1-89KCHAP. (B) The effect of temperature on enzyme activity of VirB1-89KCHAP. (C) Thermostability of the VirB1-89KCHAP protein.