As shown in Table 3, the MICs of both aculeacin A and Aac-treated aculeacin A for check details Candida tropicalis F-129 were 0.05 μg·ml-1. palmatic acid (M+H m/z = 257), were not found in the ESI-MS analysis (data not shown).
These results revealed that Aac is not an aculeacin A acylase. Table 3 The minimal inhibitory concentrations of aculeacin A for Candida tropicalis F-129 OD600 a at serial diluted aculeacin A (μg·ml-1) Aculeacin A 0 0.001 0.005 0.01 0.05 0.1 0.5 1 Untreated 0.592 ± 0.036* 0.615 ± 0.088* 0.255 ± 0.096* 0.126 ± 0.029* 0.045 ± 0.006 0.047 ± 0.008 0.043 ± 0.002 0.041 ± 0.004 Aac treated 0.629 ± 0.032* 0.634 ± 0.047* 0.297 ± 0.030* 0.093 ± 0.017* 0.070 ± 0.035 0.054 ± 0.007 0.044 ± 0.002 0.042 ± 0.005 a Values represent the averages ± standard errors from triplicate independent assays. By one-tailed student’s t-test (P < 0.05), the value with an asterisk (*) designates a significant difference from the 1 μg·ml-1 of aculeacin A test shown in the same row. There
is no significant difference between Aac-untreated aculeacin A and Aac-treated aculeacin A in the same column Aac is active against BTK inhibition AHLs with acyl side chains greater than 6 carbons To determine the substrate specificity and enzymatic activity of the AHL-acylase Aac in E. coli DH10B (pS3aac), a range of AHLs were mixed with cells of E. coli DH10B (pS3aac) to perform the HSL-OPA assay. As shown in Table
4, E. coli DH10B (pS3aac) could not degrade the short-chain AHLs, C4-, C6-, or 3OC6-HSLs; Tau-protein kinase however, E. coli DH10B (pS3aac) exhibited activities against long-chain AHLs, C7-, C8-, 3OC8-, MRT67307 nmr C10-HSLs. The AHLs of more than ten carbon-acyl chains, i.e. C12-HSL and C14-HSL, could not be determined due to the poor solubility of their substrate. These results indicate that the substrate specificity of the AHL-acylase Aac is within the limit of more than six carbon-acyl chain AHLs. Table 4 Substrate specificities of the AHL-acylase Aac against AHLs AHL-acylase activities (nmol·min-1·ml-1)a AHLs E. coli DH10B (pBBR1MCS-3) E. coli DH10B (pS3aac) C4-HSL 0.26 ± 0.15 (7.2%) 0.37 ± 0.13 (10.3%) C6-HSL 0.31 ± 0.15 (8.7%) 0.38 ± 0.10 (10.5%) 3OC6-HSL 0.37 ± 0.09 (10.5%) 0.35 ± 0.09 (10.5%) C7-HSL 0.23 ± 0.15 (6.4%) 3.60 ± 0.31 (100.0%)* C8-HSL 0.21 ± 0.16 (5.7%) 1.63 ± 0.21 (45.4%)* 3OC8-HSL 0.22 ± 0.17 (6.1%) 2.56 ± 0.04 (71.1%)* C10-HSL 0.25 ± 0.15 (7.1%) 3.10 ± 0.25 (86.1%)* a Values represent the averages ± standard errors from triplicate independent HSL-OPA assays. The relative activity in parentheses is based on defining C7-HSL-degrading activity exhibited by E. coli DH10B (pS3aac) as 100%. The value with an asterisk (*) is the significant difference from the E.