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Some organisms capable of lysing viable yeast cells were isolated from brewery sewage by the enrichment culture method by intermittent feeding with brewer's yeast. The strain (B 111-1) showing the highest lytic activity was identified as Arthrobacter luteus.The activity of Arthrobacter luteus, B 111-1, to lyse viable yeast cells appeared in the culture medium in the early stationary phase of the bacterial growth.The pattern of change in lytic activity during cultivation was different from those of IS-1, 3-glucanase and protease activities suggesting that some additional factors are essential for the lysis of viable yeast cells.The pH value of culture medium during cultivation influenced the development of the activity considerably.The pH value first decreased and then increased with the appearance of lytic activity.The activity to lyse viable yeast cells developed adaptively when the organism was cultivated in a medium containing yeast cells or 3-1, 3-glucan, suggesting that the latter causes induction.Cell-free culture fluid of the organism showed a lytic activity-on cells of brewer's yeast at all stages of growth and on viable cells of yeast strains belonging to a wide range of genera. However, it did not lyse viable cells of the yeasts Rhodoturula, Pichia, and Hansenula, or viable and heat-treated cells of bacteria and molds.The lytic activity of the cell-free culture fluid was not lost by dialysis. It was salted out at 0.395 saturation of ammonium sulfate and was lost on incubation at 60° for 5 min. It was also inactivated by such protein denaturing agents as mercuric chloride, sodium laurylbenzene sulfonate, and silver nitrate, indicating that the lytic activity was due to enzymes.The optimum pH for lysis of viable yeast cells was 7.0-8.0. The lytic activity was relatively stable between pH 5 and 10.Since GIAJA's work (1) on snail gut juice, there have been 57 many re-

The susceptibilities of various strains of yeast to a yeast cell wall lytic enzyme produced by

An enzyme which lysed viable yeast cells was purified about 75-fold from the culture fluid of Arthrobacter luteus by procedures including salting out with ammonium sulfate and Biogel CM-100 column chromatography. The purified enzyme appeared homogeneous on electrophoresis and ultracentrifugation, and had a molecular weight of about 21,000. This enzyme lysed viable yeast cells in the absence of any other enzymes or additives and was tentatively named zymolyase. The optimum pH for lysis of viable yeast cells was 7.0-7.5, the optimum temperature was 35°, and the enzyme was relatively stable at pH 5-11. About 70 % of its activity was lost on incubation at 50° for 5 min, and all its activity was lost on incubation at 60° for 5 min. Studies on the hydrolyses of j3-1,3-glucans, i.e., yeast glucan, pachyman, curdlan, laminarin, and laminaran, showed that zymolyase is an endo-jS-1,3-glucanase, which specifically releases laminaripentaose as the minimum product unit. 1-lowever, it behaves like an exo-enzyme in hydrolysis of high molecular jS-1,3-glucan, which shows strong molecular aggregation like yeast glucan, releasing laminaripentaose units. Zymolyase shows much more affinity for insoluble glucan than for soluble glucan. Previously we reported that the culture broth of a strain of Arthrobacter luteus accumulated lytic activity for viable yeast cells only when the culture medium contained j3-1,3-glucan, and that the pattern of accumulation of this activity was quite different from that of-1,3-glucanase (EC 3.2.1.6) (1). These differences 323 324 KTTAMURA, KANEKO, and YAMAMOTO VOL. 20 suggested that some essential factors, other than fi-1,3-glucanase, might be involved in the lyses of viable yeast cells. This paper describes the purification, properties, and mode of action of an enzyme of Arthrobacter luteus, which lyses viable yeast cells but differs from fl-1, 3-glucanase (EC 3.2.1.6). MATERIALS AND METHODS Strain, medium, cultivation and crude preparation of the yeast-lytic enzyme. Arthrobacter luteus B 111-1 was cultivated in dried yeast medium (dried yeast 2 %, yeast extract 0.2 %, NH4NO3 0.2 %, K2HPO4 0.1 %, MgSO4.7H2O 0.1 %, and Fe2(S04)3 • xH2O 0.01 %, pH 10) at 30° for 24 hr with aeration. The culture broth was centrifuged at 8,000 rpm for 10 min. The crude enzyme preparation was obtained from the supernatant by the same method as described in a previous report (1). fl-1,3-Glucanase. Endo-fl-1 ,3-glucanase was prepared by gel filtration on Sephadex G-50 from Glucanase G-4, kindly provided by Dr. Y. Takagi, Amano Seiyaku Co., Ltd. Carbohydrates. Yeast glucan was prepared from brewery yeast by the method of PEAT et al. (2). Pachyman was prepared from Poria cocos as follows : The powdered fungus was added to 0.1 N NaOH solution to 0.5 % concentration, the mixture was stirred for 30 min at room temperature, and then filtered. Pachyman was precipitated from the filtrate by neutralization with 0.2 N HCI. After being washed twice with water, the precipitate was dissolved in 0.1 N NaOH solution and centrifuged. Pachyman...