Purification and characterization of the anti-Candida toxin of Pichia anomala WC 65

Pichia anomala WC 65 secretes a toxin that is inhibitory to a variety of yeasts, including strains of the animal pathogen Candida albicans. The toxin was purified to homogeneity by ultrafiltration, ethanol precipitation, ion-exchange chromatography with a Mono Q column, and gel permeation chromatography with a Superose 12 column. The toxin had a molecular weight of 83,300 as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gradient gels and a molecular weight of 85,290 as determined by gel permeation chromatography. The isoelectric point of the toxin was pH 5.0. The toxin was stable between pH 2.0 and 5.0. Chemical analysis of the purified toxin indicated that the toxin was a glycoprotein composed of about 86% protein and 14% carbohydrate. At high concentrations, the toxin showed a tendency to aggregate, with loss of biological activity against C. albicans, Pichia bimundalis, and Saccharomycodes ludwigii. Purified toxin expressed killing activity against C. albicans in contrast to the static activity of the crude toxin.