Then it was centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was collected and stored at −80°C until use. The Antimicrobial activity of the supernatant was tested against C. albicans MTCC 3958, P. aeruginosa MTCC 741, S. aureus MTCC 737. Physicochemical properties of the anti-Candida compound Sensitivity to heat, pH,
and hydrolyzing enzymes Temperature stability was evaluated by incubating the CFS at various temperatures: 60°C for 90 see more min, 90°C for 20 min, 100°C for 20 and 30 min or autoclaved. Residual anti-Candida activity was determined by a well-diffusion assay against C. albicans. The effect of pH was determined using a pH range from 2 to 10 adjusted with diluted HCl or NaOH. After incubation at 37°C for 1 h, the resulting CFS was subjected to an agar-well diffusion assay to record the loss or retention of biological activity. Resistance to several proteolytic enzymes was tested by incubating the dialysed concentrate with pepsin, α-amylase, pronase E, trypsin, lipase and proteinase K at a final concentration of 1.0 mg mL-1. Buffers were used as controls. Samples were incubated at 37°C for
90 min. The residual activity was determined by cut-well agar assay. Effect of organic solvents, surfactants, and storage The sensitivity of dialyzed concentrate of ACP was tested in the presence of several organic solvents (methanol, ethanol, isopropanol, hexane, formaldehyde, chloroform, acetone and acetonitrile) at a final concentration of 25% (v/v). After incubation for 2 h at 37°C, the OSI-906 purchase Protein tyrosine phosphatase organic solvent was evaporated using a speed vac system (Martin Christ), and the residual antimicrobial
activity was determined. An untreated dialysed concentrate sample was taken as control. The effect of various surfactants, including Triton X-100, Tween-20, SDS, urea, EDTA, PMSF, and DTT (1.0% each) on the dialyzed concentrate was also tested. To assess whether the antifungal activity was due to the oxidation state of cysteine residues, β-mercaptoethanol (1 and 2 mmol) was used. The heat-treatment at 80°C was given for 10 min. In order to determine the stability, the CFS, dialyzed concentrate and partially purified ACP samples were stored for 1 year at low temperatures (4, −20 and −80°C) and the antimicrobial activity was compared to the freshly purified preparation. Partial purification of the anti-Candida compounds E. faecalis was cultured in mTSB medium at 14°C for 48 h. Cells were harvested by centrifugation at 12,000 rpm for 30 min at 4°C, and the CFS was filtered through 0.45 μm membranes. The culture supernatant was subjected to sequential ammonium sulphate precipitation to achieve 30%, 50% and 85% saturation at 4°C with constant and gentle stirring for 1 h. The precipitated proteins were pelleted by centrifugation at 12,000 rpm for 30 min. The protein pellet was dissolved in sterile 20 mmol sodium phosphate buffer pH 8.