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-
