A Novel Killer Toxin Produced By The Marine-derived Yeast Wickerhamomyces Anomalus YF07b

Killer toxin is the antimicrobial exotoxin that is produced by many killer yeastsand is usually the protein or glycoprotein with small molecular mass. Killer toxinusually combines with the first receptors on the sensitive yeast cell walls and kills thesensitive yeast indirectly. The killer strains are insensitive to their own toxins, butremain susceptible to the toxins secreted by other killer yeasts.The marine yeast Wickerhamomyces anomalus YF07b was isolated from the gutof sea squirts (Ascidia sp.) with wide spectra of the killing activity and is able toproduce killer toxin against the pathogenic yeast Metschnikowia bicuspidata WCY.In this research study, the best cultivation conditions for the marine-derived yeastW. anomalus YF07b to produce killer toxin were pH3.5, NaCl concentration2.0%(w/v), temperature20℃, cultivation time for3days. The best action conditions forthe crude killer toxin were pH3.5, NaCl concentration4.0%(w/v), temperature16℃.In our previous study, it was found that the killer toxin produced by themarine-derived yeast W. anomalus YF07b has both killing activity andβ-1,3-glucanase activity and the molecular mass of it is47.0kDa. In this study,another killer toxin was purified from the prepared cell culture supernatant of themarine yeast W. anomalus YF07b by dialysis, DEAE Sepharose Fast Flowanion-exchange chromatography, ultrafiltration with a5-kDa cutoff membrane witha Labscale TFF System (Millipore), Sephadex G-75chromatography, concentratedfiltration through an AmiconYM3(MW cutoff10kDa) membrane and SDS-PAGE.The purified killer toxin only had killing activity against some yeast strains, but hadno β-1,3-glucanase activity and the molecular mass of it was67.0kDa. This meantthat the yeast W. anomalus can produce different kinds of killer toxin. So the purifiedkiller toxin in this study is a novel killer toxin. The optimal pH, temperature and NaCl concentration for action of the newly purified killer toxin were3.5,16℃and4.0%(w/v). The newly purified killer toxin had killing activity against Yarrowia lipolytica,Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis,Candida albicans and Kluyveromyces aestuartii.Only the whole cells of the pathogenic yeast M. bicuspidata WCY was killed,suggesting that the cell wall of the pathogenic yeast is required for the activity of thenewly purified killer toxin. The newly purified killer toxin could be bound only by thewhole cells of the sensitive yeast. This may suggest that the primary receptor for thekiller toxin was located in the cell wall of the sensitive yeast. Scanning electronmicroscopy and Fluorescence microscopy showed that the newly purified killer toxincan destroy cell wall and change the permeability of plasma membrane of thesensitive yeast. The killing mechanisms of the newly purified killer toxin may be thatthe novel killer toxin interacts with cell wall receptor of the sensitive yeast, causingdefective integrity of the cell wall, damage of the plasma membrane function and celldeath.