albicans strains was present mainly in the fraction precipitated with 85% ammonium sulfate (Figure 1b). Fractions precipitated with 30% and 50% ammonium sulfate exhibited weak inhibition. The supernatant obtained after 85% ammonium sulfate precipitation clearly did not exhibit any antifungal activity. The GW-572016 ic50 antifungal substance present in the 85% cut-off also inhibited germ tube formation in C albicans NCIM 3471 (data not shown). As is clear from Table 3,

ammonium sulfate precipitation resulted in an approximate 2-fold increase in specific activity. After ion- exchange chromatography using DEAE Sepharose, the adjacent fractions 31–35 in the chromatogram, showed biological activity (Figure 3), and the specific activity increased 17-fold. After gel filtration, the recovery was

approximately 22-fold. Based on the purification steps summarised in Table 3, it was concluded that the total active antimycotic protein recovered was 0.45% only. Table 3 Summarised Purification steps of ACP Purification stage Volume (mL) Activity (AU mL-1) Protein (mg mL-1) Specific activity (AUmg-1protein) Purification factor Recovery (%) Culture Supernatant 400 1600 0.4025 39751 1 100 Ammonium sulfate selleck screening library and dialysis 10 3200 0.0444 72072 1.8 11 Ion Exchange Chromatography 6 1600 0.0023 695652 17.5 0.57 Gel Filtration 2 1600 0.0018 888888 22.4 0.45 Figure 3 Chromatogram of antimycotic protein ACP produced by E. faecalis on DEAE Sepharose, absorbance of fractions taken at 280 nm. Fractions (31–35) showing biological activity. Direct detection of activity on PAGE After gel filtration, partially purified active pooled fractions (30 μL), were loaded onto Tricine gel containing 10% resolving and 5.0% stacking gel. A clear zone of inhibition on the C. albicans MTCC 3958 overlaid gel was shown in a Petri dish (Figure 4), wherein a simultaneously silver stained gel showed a corresponding band that 3-oxoacyl-(acyl-carrier-protein) reductase was responsible for the biological activity. Based on the polypeptide molecular weight marker, the molecular mass of the active peptide was estimated to be approximately 43 kDa (Figure 4). We did not observe any biological activity of the bands using glycine Native PAGE. Figure 4 Tricine-PAGE

of ACP purification fractions and gel overlay with C. albicans (MTCC 183). Lane 1, molecular weight marker. Lane 2, dialyzed concentrate after 85% ammonium sulfate fractionation. Lane 3, pooled active fractions collected through DEAE Sepharose matrix. Lane 4, silver stained fractions after gel filtration using Sephadex-G 75. Lane 5, Inhibition zone by antimycotic protein (ACP) on the overlay gel. Amino acid sequencing The first 12 amino acid residues of the N-terminal were determined by Edman degradation. The minor sequence obtained from the twice repeated N-terminal sequencing was GPGGPG, and the same partial sequence was matched for homology. Complete homology was not found in the NCBI BLAST result. However, the GPGG sequence matched a known ABC transporter, i.e.