New killer toxins of halophilic Hansenula anomala.

Abstract

Twostrains of killer yeasts, both identified as Hansenula anomala, were isolated from shoyu moromi. Both killer yeasts showed killer activity toward Zygosaccharomyces rouxii EAunder high salt concentration conditions. The killer toxins produced by these strains were purified by ultra filtration and ion-exchange chromatography followed by gel filtration.The molecular weights of the toxins were about 300kd and both toxins were glycoproteins. The isoelectric point of the toxin, Kh-I, produced by one strain was pH 2.9 and that of the toxin, Kh-II, produced by the other strain was pH 3.6. The amino acid composition of Kh-I was different from that of Kh-II. Kh-II was more thermolabile than Kh-I. The killer activities of both toxins were not observed in the absence of NaCl in the medium but increased with increasing NaCl concentration. The killer spectra of Kh-I and Kh-II were different from those of the killer toxins known as K^-K^. The killer activities of these strains were not abolished by cycloheximide treatment and by cultivation at 37°C. No plasmid was detected in either killer yeast. Hansenula mrakii10) and Hansenula saturnusl l) have been reported. But the existence of a killer character in halophilic yeasts has not previously reported/There are many kinds of killer strains in a wide variety of yeasts. This suggests that there should be killer strains amonghalophilic yeasts. If halophilic killer yeasts are isolated, they will be useful for microbial control in shoyu making. We tried to isolate killer yeasts from shoyu moromi and successfully isolated two different killer yeast strains, and found that they produced killer toxins with unique characteristics, whose activities appeared in mediumcontaining a high concentration of NaCl, but not in its absence.This paper describes the purification and various physicochemical properties of the killer toxins produced by these yeasts. MATERIALS AND METHODSYeast strains. Zygosaccharomyces rouxii EAfrom the yeast collection of Higeta Shoyu Co., Ltd. was used as the test strain for the detection of killer activity. S. cerevisiae NCYC235, S. cerevisiae NCYC738, S. capensis NCYC 761, C. glabrata NCYC388, H. anomala NCYC434, K. fragilis NCYC587, C. valida NCYC327, H. anomala NCYC435, H. mrakii NCYC500, K. drosophilarum NCYC575 and C. glabrata ATCC15126 were used as standard killer strains with different killer sensitivities.2'12)Media. YEPD-1 medium (glucose 2%, polypeptone 2%, yeast extract 1%, 0.1 m citrate phosphate buffer, pH 4.8) and YEPD-2 medium (YEPD-1 medium containing NaCl 8%) were used for cultivation of the killer yeasts. YEPDMB1agar (YEPD-1 medium containing methylene blue 0.03% and agar 2%) and YEPD-MB2 agar (YEPD-MB1 agar supplemented with 8% NaCl) were used for the isolation of killer yeasts. For the curing test on the killer activity, YMagar (glucose 1%, polypepton 0.5%, yeast extract 0.3%, malt extract 0.3%, agar 2%) was used.Detection of killer yeasts. The detection of killer yeasts was performed by the cross-streak test,13) as fol-

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

“…The P. anomala WC 65 toxin was also larger than other polypeptide killer toxins reported for Hansenula mrakii (Mr 8,900) (1), H. saturnus (Mr $,500) (11), and S. cerevisiae K1 toxin (M, 20 (21), and the halophilic toxin of H. anomala (Mr ca. 300,000) (8) are larger. This last glycoprotein toxin was purified from two halophilic isolates of H. anomala.…”

“…This last glycoprotein toxin was purified from two halophilic isolates of H. anomala. This toxin was active (only in the presence of sodium chloride) against Zygosaccharomyces rouxii (8). Whether these large, halophilic toxins are active against C. albicans is not known.…”

“…Although killer phenotypes have been reported for strains of the genera Saccharomyces, Candida, Cryptococcus, Debaryomyces, Hansenula, Kluyveromyces, Pichia, and Torulopsis (15,22,25), killer toxins of only three isolates of Saccharomyces cerevisiae (12)(13)(14)28), one isolate each of Kluyveromyces lactis (21,23), Hansenula mrakii (1), Hansenula saturnus (11), and Pichia kluyveri (10), and two halophilic isolates of Hansenula anomala (8) have been purified. Extensive studies in purification and characterization of killer toxins have been accomplished only with toxins of Saccharomyces cerevisiae (26,28).…”

Past, present and future research directions with Pichia anomala

“…There is an amazing diversity of characterised killer proteins, with molecular masses ranging from 8 kDa to more than 300 kDa (De Ingeniis et al 2009;Kagiyama et al 1988); however, this diversity in protein size may arise mainly from different levels of glycosylation. An extremely heat-stable killer protein has been observed in one strain (Vustin et al 1989).…”

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

“…SW-55 (Yokomori et al, 1988) and Hansenula anomala (Kagiyama et al, 1988), KpKt is a glycosylated protein. The estimated mass of the carbohydrate part of KpKt is similar to that exhibited by K28 killer toxin of S. cerevisiae (Pfeiffer & Radler, 1982).…”