Kluyveromyces phaffii killer toxin active against wine spoilage yeasts: purification and characterization

Abstract

The killer toxin secreted by Kluyveromyces phaffii (KpKt) is active against spoilage yeast under winemaking conditions and thus has potential applications in the biocontrol of undesired micro-organisms in the wine industry. Biochemical characterization and N-terminal sequencing of the purified toxin show that KpKt is a glycosylated protein with a molecular mass of 33 kDa. Moreover, it shows 93 % and 80 % identity to a b-1,3-glucanase of Saccharomyces cerevisiae and a b-1,3-glucan transferase of Candida albicans, respectively, and it is active on laminarin and glucan, thus showing a b-glucanase activity. Competitive inhibition of killer activity by cell-wall polysaccharides suggests that glucan (b-1,3 and b-1,6 branched glucans) represents the first receptor site of the toxin on the envelope of the sensitive target. Flow cytometry analysis of the sensitive target after treatment with KpKt and K1 toxin of S. cerevisiae, known to cause loss of cell viability via formation of pores in the cell membrane, suggests a different mode of action for KpKt. INTRODUCTIONKiller toxins were first discovered in Saccharomyces cerevisiae by Makower & Bevan (1963) and they have since been found in numerous other yeast genera (Philliskirk & Young, 1975; Stumm et al., 1977;Rosini, 1983Rosini, , 1985Starmer et al., 1987). Through the following years, killer yeasts and their toxins found applications in several fields. For example, killer yeasts have provided an interesting model for studying the mechanisms involved in the processing and secretion of extracellular proteins, and the identification of killer toxin receptors on the envelope of sensitive targets has helped in the elucidation of the structure and function of the yeast cell wall. Killer yeasts have been used to combat contaminating wild yeasts in the food and fermentation industries, and to control wooddecay basidiomycetes and plant-pathogenic fungi (Young, 1987; Boone et al., 1990;Palpacelli et al., 1991, van Vuuren & Jacobs, 1992Walker et al., 1995). In the medical field, these yeasts have been used in the biotyping of pathogenic yeasts and yeast-like fungi (Morace et al., 1989;Boeckhout & Scorzetti, 1997;Buzzini & Martini, 2001), and in the development of novel antimycotics for the treatment of human and animal fungal infections Seguy et al., 1998; Conti et al., 2002;Schmitt & Breinig, 2002).Recently, it has been shown that Kluyveromyces phaffii (since reclassified as Tetrapisispora phaffii) (Ueda-Nishimura & Mikata, 1999) produces a killer toxin (KpKt) that is active on wine spoilage yeasts (Ciani & Fatichenti, 2001). KpKt has an extensive anti-Hansenispora/Kloeckera activity under winemaking conditions and, therefore, is of particular interest for its potential application as an antimicrobial agent in the wine industry (Ciani & Fatichenti, 2001). At present, the inhibition of wild spoilage yeast at the prefermentative stage is achieved by the addition of SO 2 to freshly pressed must. This antiseptic agent, which has been shown to have a toxic action on humans, is also r...

β-1,3-Glucanase from Delftia tsuruhatensis Strain MV01 and Its Potential Application in Vinification

“…Such a high addition of protein could require a subsequent fining of the wine. Furthermore, the application of killer toxins produced by various yeasts (56) may be possible due to their high specific action against yeasts, though killer toxins inhibit only a minor group of sensitive yeast strains and are not effective against a broad range of different species (11,12,13). The ␤-1,3-glucanase from Delftia tsuruhatensis strain MV01 showed activity against all tested yeast species.…”

Enzymic Activity of the K5-Type Yeast Killer Toxin and Its Characterization

“…10) In the killer assay medium supplemented with 1 M sorbitol, the cytocidal effect of the toxin was abolished, indicating that hyper-osmotic medium protected the cells from lysing which leads to cell death. This finding further confirmed that -1,3-glucanase activity kills the cells.…”

“…8) In some cases the toxin interferes with cell wall synthesis by inhibiting -1,3 glucan synthase 9) or by hydrolyzing the major cell wall component -1,3 glucans. 10,11) The killer character confers a considerable advantage on yeast cells in competitive situations against other sensitive microbial cells in their ecological niches. 1) This advantage has scientific and commercial significance, since killer yeasts and their toxins have found several applications.…”

Non-Saccharomyces Killer Toxins: Possible Biocontrol Agents Against Brettanomyces in Wine?

“…These killer toxins exhibit broader anti-yeast spectra compared to those of Saccharomyces (Palpacelli et al, 1991;. For instance, the species Tetrapisispora phaffii and Kluyveromyces wickerhamii display killer activity against the apiculate yeast Hanseniaspora uvarum and species of the Brettanomyces/Dekkera genus respectively (Ciani & Fatichenti, 2001;Comitini et al, 2004a). Since the first record of a killer toxin inhibiting an apiculate yeast (Ciani & Fatichenti, 2001) (Comitini et al, 2004b;Santos et al, 2009;.…”

“…the polysaccharide chitosan) have been tested for controlling B. bruxellensis growth and were proven to have limited efficiency (Suárez et al, 2007). In addition, hypersensitivity to SO 2 in some wine consumers has spurred the demand for the use of nonchemical preservatives (Comitini et al, 2004a;Lustrato et al, 2006). Alternative methods therefore currently are being sought to control the growth of B. bruxellensis.…”