1 on RP-HPLC.
Active peak is boxed. Table 1 Purification of mutacins F-59.1 and D-123.1 by hydrophobic chromatography. Step Volume (mL) Activity (AU/mL) Total Protein (mg) Total activity (AU.103) Specific activity (AU/mg) Yield (%) Purification (fold) mutacin F-59.1 Culture supernatant 1000 400 10000 400 40 100 1 Sep-Pak C18 95 3200 3000 304 101 76 2.5 C18 RP-HPLC 2 16000 0.1 32 3.2 × 105 8 8 × 103 mutacin D-123.1 Culture supernatant 675 200 4320 135 31 100 1 Sep-Pak C18 50 1600 8 80 LY3009104 1 × 104 59 320 C18 RP-HPLC 1 800 0.005 0.8 1.6 × 105 0.2 5120 A total of 25 amino acids were sequenced for mutacin F-59.1 and its identity with pediocin-like bacteriocins was confirmed by multiple alignment (Figure 3). The sequence revealed high levels of similarity to class IIa bacteriocins with the presence of the five residues of the common consensus sequence -YGNGV- and the two conserved RG7112 chemical structure cysteine residues at positions 9 and 14. The substitution of unidentified amino acids (annotated X) in the mutacin
F-59.1 sequence with consensus amino acids found in our alignment (Figure 3) and those of others [2, 13], revealed that the following N-terminal sequence KYYGNGVTCGKHSCSVDWSKATTNI matches the molecular mass determined by MALDI-TOF MS analysis (2720 Da +/- 2 Da, due to the formation of selleck inhibitor the current disulfide bridge found between C9 and C14 in pediocin-like bacteriocins , (Figure 4)). The isoelectric point of mutacin F-59.1 (pI = 8.71) and secondary structure prediction with this sequence correlate well with other class IIa bacteriocins (Figure 3) [2, 4]. Figure 3 Multiple sequence alignment of mutacin F-59.1 with homologous class IIa bacteriocins. Consensus sequence appears in bold. Some of the leader sequences are shown with the double glycine
motif. Underneath appears in italic the predicted secondary structure Sitaxentan for mutacin F-59.1 and pediocin PA-1. Output classification is as follows: H, alpha-helix; E, extended strand; T, turn; C, the rest . Accession numbers refer to bacteriocins in the protein database from the NCBI (AAC60413, ; AAB23877, ; AAG28763, ; AAL09346, ; P35618, ; P80953, ; ACD01989, ; BAB88211, ; AAQ95741, ). Figure 4 MALDI-TOF-MS spectra obtained for pure mutacin F-59.1. The molecular mass for mutacin D-123.1 was computed to be 2364 Da (Figure 5). However, sequencing of the mutacin D-123 proved to be problematic. Edman degradation of native mutacin D-123.1 was blocked after the first residue (F). The sequence of only the first 9 amino acids was clearly obtained after the derivatisation procedure, but with at least two peaks at each cycle. Figure 5 MALDI-TOF-MS spectra obtained for pure mutacin D-123.1. The growth of M. luteus ATCC 272 was inhibited immediately following the addition of a purified preparation of mutacin F-59.1 at 160 AU/mL as the viable count decreased rapidly and dropped to zero compared to the control.