RELEASE OF NITROGENOUS SUBSTANCES BY BREWER'S YEAST

Transcription

1 JOURNAL OF BACTERIOLOGY Vol. 87, No. 6, pp June, 1964 Copyright 1964 by the American Society for Microbiology Printed in U.S.A. RELEASE OF NITROGENOUS SUBSTANCES BY BREWER'S YEAST III. SHOCK EXCRETION OF AMINO ACIDS M. J. LEWIS AND H. J. PHAFF Department of Food Science and Technology, University of California, Davis, California ABSTRACT LEWIS, M. J. (University of California, Davis), AND H. J. PHAFF. Release of nitrogenous substances by brewers' yeast. III. Shock excretion of amino acids. J. Bacteriol. 87: When Saccharomyces carlsbergensis (two strains) and S. cerevisiae (one strain) were grown in static culture and the harvested, washed cells were suspended in a solution of glucose, amino acids were suddenly released and then rapidly reabsorbed in a space of about 2 hr. The phenomenon of amino acid release, which was termed shock excretion, varied in intensity with the strain of yeast and was shown to be dependent on the sie of the pool of free amino acids within the cells. Shock excretion was independent of osmotic pressure of the suspending medium, but required the presence of a fermentable sugar. D-Galactose and maltose caused shock excretion only when yeast was previously adapted to these sugars. Limiting glucose concentrations prevented reabsorption of amino acids, and a further decrease in glucose concentration also limited excretion. Shock excretion was strikingly reduced when the temperature of the suspending medium was lowered. Received for publication 20 January 1964 During the course of some preliminary investigations in this laboratory (Delisle and Phaff, 1961) into the excretion of nitrogen-containing compounds by pregrown cells of a strain of Saccharomyces carlsbergensis, it was observed that a sample of the medium, taken very early after yeast was suspended in a solution of glucose, often contained an unexpectedly high level of a-amino nitrogen. The yeast used in these experiments was well washed with water after growth, so that there was no contamination of the suspending medium by carry-over from the growth medium. The present work has established that the rapid release of amino acids proceeded for about 1 hr after yeast was suspended in glucose solution, and was followed by a period of rapid reabsorption of most of the a-amino nitrogen. Since this cycle was complete in about 2 to 3 hr, the phenomenon is not observed if samples are taken, for example, 6 hr apart. Under the above conditions of fermentation, we previously showed (Lewis and Phaff, 1963) that compounds absorbing strongly at 260 m,u are also released at an accelerated rate as compared with storage in water or in a nonmetaboliable sugar. These compounds, however, were never observed to be reabsorbed, and they remain in the medium at the end of an experiment. The source of the excreted amino acids and nucleotides was shown to be in the free pools of these two classes of compounds within the cell (Lewis and Phaff, 1963). This paper is concerned with an examination of some of the factors which control the phenomenon of sudden release of amino acids, which we have called shock excretion, and their subsequent reabsorption MATERIALS AND METHODS Composition of the growth medium and yeasts used. All yeast strains were grown on the following semidefined medium (per cent, w/v): glucose, 5; peptone (Difco), 0.5; yeast autolysate (Albimi Laboratories, Inc., Flushing, N. Y.), 0.5; KH2PO4, 0.05; MgSO4 (anhydrous), The ph was adusted to 5.0 with phosphoric acid. Three yeast strains were used: (i) S. carlsbergensis 57-42, a nonflocculent Danish bottom yeast, previously used by Delisle and Phaff (1961); (ii) S. carlsbergensis L, a flocculent, American east coast, commercial bottom yeast; and (iii) S. cerevisiae 62-5, a commercial baking yeast. Grouing and harvesting of the cells. Unless specified otherwise, a 100-ml portion of the above medium in a 250-ml Erlenmeyer flask was inoculated directly from the stock slope of the desired yeast. After 2 days at 25 C, without agitation, this starter culture was transferred

2 1390 LEWIS AND PHAFF J. BACTERIOL. into 1.5 liters of the same medium in a 3-liter Fernbach flask. Incubation was continued for an additional 3 days; the yeast was roused daily. At the end of this period, the cells were harvested and washed four times with sterile water by centrifugation. Thus, young but mature and fully grown cells were obtained for use in the excretion experiments. The dry matter yield of cells was about 4.5 g per flask. Dry weight of cells. The washed yeast cells were diluted with water to a concentrated suspension of known volume. The optical density of a suitable dilution of the suspension was determined in a Klett-Summerson colorimeter with a red (' 66) filter, and the corresponding yeast concentration (dry weight per ml) was read from a previously constructed calibration curve. Suitable volumes of the concentrated yeast suspension were then measured into the test solutions, to yield a yeast concentration (dry weight basis) of 10 mg/ml. Most test solutions were of 60 ml final volume and were contained in 125-ml Erlenmeyer flasks. Sampling and extraction of pool contents. The suspensions were stored at room temperature (24 to 26 C) without agitation. Samples of 5 or 10 ml of the test solutions were taken when required, after dispersing the yeast evenly in suspension by swirling the flasks. The samples were then centrifuged, and the clear supernatant liquid was decanted and centrifuged once more. After decanting again, the supernatant sample was froen until ready for analysis. FIG. 1. Shock excretion shown for three yeasts. Yeast concentration, 10 mg/ml (dry weight basis). Suspending medium, 10% glucose. Room temperature (24 to 26 C). After washing the initial sediment with distilled water, the free amino acids and nucleotides in the intracellular pools were extracted from the pellet of cells by bringing the small amount of liquid associated with the cells rapidly to a boil over an open flame. About 5 ml of water were then added, and the extraction was completed by heating for 10 min in a boiling-water bath (Spiegelman, Halvorson, and Ben-Ishai, 1955). The extract was cooled and made to known volume, and the debris was removed by centrifugation. This sample was also froen until it was analyed. Ultraviolet absorption. The ultraviolet absorption of solutions was determined in a Beckman model DU spectrophotometer at 260 m,u with distilled water as blank. a-amino nitrogen. The ninhydrin method of Moore and Stein (1954) was used in the determination of a-amino nitrogen. The blue color obtained in the reaction was read in a Klett- Summerson colorimeter with a red (# 66) filter. L-Leucine was used to construct the calibration curve so that the values reported are leucinenitrogen equivalents. Sugar. Reducing sugar was determined by the ferricyanide method of Hassid (1937). D-Galactose and maltose used for suspending yeasts consisted of special lots "substantially glucose-free" and were purchased from Sigma Chemical Co., St. Louis, MIo. RESULTS Effect of strain. First, for comparison, the phenomenon of shock excretion was measured with several yeasts. The variations observed are illustrated (Fig. 1) for three Saccharomyces yeasts which were grown and tested under identical conditions. Although there was considerable variation in the height of the shock excretion curves, the phenomenon is not the property of a particular strain. S. cerevisiae 62-5 showed the least shock excretion and S. carlsbergensis L, the most. Differences in the height of the curves were not entirely attributable to a variable content of free amino acids in the cells, since differences in the level of these substances were small (Fig. 2). The graphs in Fig. 2, which are based on the same experiment shown in Fig. 1, show a rapid drop in the internal concentration of free amino acids immediately after the yeasts were placed in glucose solution. During this

3 VOL. 87, 1964 SHOCK EXCRETION OF AMINO ACIDS BY BREWER'S YEAST 1391 :1= 20 YEASTS, 10mg/ml YEAST L -J oo YEAST57-42 X~~~-- YEAST FIG. 2. Changes in internal a-amino nitrogen during shock excretion. Data based on the experiment shown in Fig. 1. phase, lasting about 1 hr, internal amino acids were released to the medium and at the same time partially synthesied into protein. Synthesis is evident because the decrease in internal level of amino acids was greater than the increase in the medium. In the second hour of shock excretion, there was a slowing down of the depletion of the free cell contents, the degree of decrease depending on the yeast strain; this slowing is attributed to reabsorption of amino acids from the medium, while synthesis continues. This break in the curve was most marked in yeast L, which had the greatest amount of material to reabsorb. As reabsorption continued, the cell contents of free amino acids fell again to the end of the experiment (synthesis). As the shape of the shock excretion curve itself was determined by the two opposing forces of amino acid release and reabsorption, so these two factors, together with protein synthesis, determined the rate of depletion of the cellular content of free amino acids. E 50H_ 40_ Thus, the curves for shock excretion in Fig. 1 are interestingly reflected in the curves of Fig. 2, which depict the changes in the free amino acids in the cellular pool. Since S. carlsbergensis L exhibited shock excretion most strikingly, further experiments were done only with this yeast. Effect of oxygen supply during growth of the yeast. Within any one experiment using the same batch of cells, the shock excretion curves were reproducible, but with different batches of cells grown under essentially similar conditions there was some variation in the height and, to some extent, the position of the peak of the curve. The influence of the previous history of the cells on subsequent shock excretion was therefore investigated briefly. The effect of oxygen supply was tested first (Fig. 3). It was found that cells grown in a nonagitated medium (see Materials and Methods) readily exhibited shock excretion, whereas those cells grown in the same medium in shake culture, with an adequate supply of oxygen, did not release amino acids appreciably. The free amino acid pool of cells grown aerobically was quite small compared with that of cells grown anaerovv O 0 YEAST L GROWN AEROBICALLY INTERNALO(-NH2 10 I YEAST L GROWN _ ANAEROBICALLY INTERNAL * a-nh2 - EXTERNAL - a-nh2 2 3 FIG. 3. Effect of aeration during growth of yeast L on shock excretion and internal a-amino nitrogen pool. Yeast concentration, 10 mg/ml (dry weight basis). Suspending medium, 10% glucose. Room temperature (24 to 26 C). I

4 1.9,92 LEWIS AND PHAFF J. BACTERIOL, bically. This may be owing to the high yield of yeast obtained under aerobic conditions, which would probably result in exhaustion of the supply of nitrogen before that of sugar. The rather low level of internal amino acids in the aerobic yeast is probably the reason for the absence of E , ~2O CD~~~ ug/ml 68Mg/mi 27Mg/mI FIG. 4. Effect of the sie of the internal amino acid pool on shock excretion. Pool sie at the start of the experiment, indicated by the figures above the curves, is expressed as,ug/ml of internal a-amino nitrogen in the test suspension. Yeast L, 10 mg/ml. Room temperature (24 to 26 C). Yeast was grown on a basal medium (see text) supplemented as follows: for curves A, B, C, and D, 0.0, 0.1, 0.6, and 2.0% peptone; for curve E, 2.0% peptone and 1.0% yeast autolysate. a shock excretion curve. It was not possible, therefore, to take advantage of the increased yield of cells achieved in shake culture. Nitrogen content of the medium of growth. The effect of nitrogen supply during growth was tested, since, from the previous experiment, there appeared to be a relationship between the cell content of free amino acids and shock excretion. Yeast L was grown without aeration in a basal medium consisting of 5% glucose, 0.1% yeast autolysate, 0.05% KH2PO4, and 0.02% MgSO4. This medium was fortified with peptone and extra yeast autolysate as indicated in Fig. 4. The results obtained, when these cells were subsequently suspended in 10% solutions of glucose, show a striking relationship between the internal amino acid pool level and the height of the shock excretion cruves. With an adequate pool level (curves C, D, and E) approximately one-third of the original pool contents was released in shock excretion. Effect of osmotic pressure of the suspending medium. In the previous experiments, washedyeast suspensions were transferred into test media which consisted of 10% solutions of glucose. The osmotic environment of the cells was, therefore, changed suddenly, and since the release of amino acids in shock excretion occurred immediately after transfer to glucose solution it seemed possible that the phenomenon was a response to the external change in osmotic pressure. To test this possibility, yeast L was grown on glucose and exposed (i) to glucose; (ii) to a potentially metaboliable sugar (maltose), but one to which the yeast was not adapted; and (iii) to a nonmetaboliable sugar (lactose). Shock excretion was observed in glucose as expected, but amino acid release in 10% maltose or lactose was not greater than the release observed in water (Fig. 5). Similar results were obtained in 10% solutions of purified D-galactose. Although this experiment demonstrated satisfactorily that osmotic pressure alone was incapable of inducing shock excretion, the participation of osmotic pressure in the presence of a metaboliable sugar could not be excluded. Yeast L was therefore suspended in solutions of galactose (10%) or lactose (10%) to which was added a small amount of glucose (0.2%) to stimulate metabolism. Glucose (10%) and water served as controls. The media which contained 0.2%

5 VOL. 87, 1964 SHOCK EXCRETION OF AMINO ACIDS BY BREWER'S YEAST 1393 FIG. 5. Effect of maltose and of lactose on shock excretion by yeast L grown on glucose. Suspension in 10% glucose served as control. GLUCOSE 105 GLUCOSE 0.2% GLUCOSE 0.2% GLUCOSE 0.25 YF^ATl lnn/.l 1^f'.TAqC I Lr.AI ArTnCC Inq 0 2 FIG. 6. Effect of addition of small amounts of glucose to lactose and galactose on shock excretion by unadapted yeast L placed in solutions of lactose and galactose. The lower curves are controls without glucose. glucose supported greater release of amino acids than did water, but the further addition of high levels of galactose or lactose did not have an additive effect on the release of amino acids (Fig. 6). Amino acid release, therefore, was a function of the presence of a metaboliable sugar, and the phenomenon was not influenced by a high osmotic pressure of the cell environment. Response of adapted cells. The fact that S. carlsbergensis can readily be adapted to utilie galactose or maltose permitted a confirmatory experiment to show that shock excretion was dependent upon the ability of a sugar to be metabolied by the cells. Yeast L was grown on glucose, galactose, and maltose. Shock excretion was measured under the conditions indicated in Fig. 7. It is evident that only cells adapted to galactose or maltose demonstrated shock excretion when placed in 10% solutions of the corre- sponding sugars. The height of these curves was lower than that of the glucose curve at the left of Fig. 7, since, as analyses showed, the cells grown on galactose and maltose contained a smaller pool of free amino acids than did the cells grown on glucose. Effect of sugar concentration. As shown in the last experiment, availability of metaboliable sugar appears to be a principal requirement for shock excretion to occur. The next experiment was designed to determine whether there was a relationship between the amount of sugar present in the medium (energy supply) and the quantity of amino acids released from the cell. Figure 8 shows the results when yeast L was suspended in glucose solutions ranging in concentration from 0.2 to 3.5%. Release of amino acids increased with initial glucose concentration until r= 11' GLUCOSE 10% GALACTOSE 10 X MALTOSE lo YEAST L lomg/ml YEAST L 10mg/ml YEAST L 10mg/ml GROWN ON GLUCOSE -.--AMINO-N lo ' P- ADAPTED ADAPTED 0.1 /,b c L...L.. t4on-d~pted NOAU-AEDPJOD 0-O P FIG. 7. Shock excretion of yeast L when grown on galactose or maltose. Nonadapted cells were used as control. Room temperature (24 to 26 C). 0 A G 3.5 0I 2;S GLUCOSE GLU S GLUCOSE 3% GLUOSUCOSLUOS 1.1% ; AER SD '.o -> FIG. 8. Effect of glucose concentration on shock excretion by yeast L. Yeast concentration, 10 mg/mi (dry weigbht basis). Room temperature (24 to 26 C). -J 0

6 1394 LEWIS AND PHAFF the latter reached approximately 0.9 %; interestingly, this increase was roughly linear at the lowest levels of glucose tested (up to 0.4%). When the glucose concentration was increased above 0.9%, reabsorption of amino acids became progressively more pronounced. The graphs clearly show (especially when the glucose concentration was limiting) that the extent of reabsorption was dependent upon the amount of glucose which remained in the supernatant liquid after excretion had been completed. If there was no residual glucose in the external medium after the excretion curve of amino acids had reached a maximum, there was no reabsorption (up to 0.7% glucose). At higher concentrations (0.9 to 3.5% glucose), there was a progressive increase in the amount of a-amino nitrogen reabsorbed during the 3-hr test periods. Although of no direct concern to the subject matter in this paper, it may be mentioned that the release of 15 - a. 10 2F CD 0 i 5 30 c ~~~20 c FIG. 9. Effect of temperature on shock excretion with yeast L (10 mg/ml) when placed in 10% glucose solutions. J. BACTERIOL. nucleotides (dotted lines) was also increased with the availability of metaboliable sugar. Effect of temperature. Experiments already described in this paper showed that shock excretion and reabsorbtion are processes dependent upon the availability of a metaboliable sugar, and can be strongly influenced by limiting concentrations of sugar (Fig. 8). The amount of sugar available to the cell in the presence of excess sugar is governed by the rate of fermentation, which in turn is strongly influenced by temperature. It was of interest, therefore, to study the effect of temperature on shock excretion in 10% glucose solutions. The extent of shock excretion was determined at 10-degree intervals from 0 to 30 C, and at room temperature for comparison, since all previous tests were conducted at that temperature. Shock excretion and reabsorption of amino acids were very sensitive to the temperature of the suspending medium (Fig. 9). Amino acid release and reabsorption were greatest at 30 C, and were strongly depressed below 20 C. The lower fermentation rate at reduced temperatures could well explain this temperature sensitivity, and suggests that shock excretion is dependent upon energy supply. Effect of storage. During the course of this work, it was often important to know whether or not the same same batch of cells would yield reproducible results on succeeding days. It was of practical interest, therefore, to examine the effect of storage of the yeast at 4 C on subsequent shock excretion. A batch of yeast L was grown in the usual way, harvested, washed, and diluted to a known concentration (60 mg/ml). After reaching the storage temperature of 4 C, a sample (10 ml) of this suspension was tested immediately in 10% glucose solution (50 ml) and thereafter at 4 hr, 2 days, and 5 days. The shapes and heights of the shock excretion curves were essentially identical after each period of storage, so that any changes in cell structure or viability which may have occurred during storage were of no importance in those mechanisms which control shock excretion. For comparison, excretion of ultraviolet-absorbing materials was also measured. The similarity in the shape of the latter curves after the various storage periods confirms previous findings by Delisle and Phaff (1961) that storage of another strain of S. carlsbergensis for 15 days

7 VOL. 87, 1964 SHOCK EXCRETION OF AMINO ACIDS BY BREWER'S YEAST at 2 C resulted in very little release of materials absorbing at 260 m,u. DISCUSSION The quantitative reproducibility of the phenomenon of shock excretion of amino acids by yeast requires rigid control of experimental conditions. The three principal factors which have been shown to affect shock excretion in the present study were the sie of the internal amino acid pool (which in turn was governed by growing conditions), the temperature of the suspending medium, and a sufficient supply of a metaboliable sugar. A fourth variable, the osmotic pressure of the medium used for suspending the yeast, was shown to have no effect on shock excretion. This variable was tested at length, since shock excretion occurred immediately upon changing the environment of the yeast from distilled water to sugar solution. It is possible that the amino acids subject to shock excretion form part of a special pool (Halvorson and Cohen, 1958), which may be similar to the "expandable pool" described by Cowie and McClure (1959). This pool is more readily exchanged with the external medium than is the true internal or conversion pool. The large variation in sie of the extractable pocl as a function of the organic nitrogen content of the medium of growth (Fig. 4) would be explainable in terms of an expandable pool. Shock excretion also varied with yeast strain, even if the extractable amino acid pools were of comparable sie and other experimental conditions were the same, so that some additional, strain-dependent factor strongly influences shock excretion. Since excretion and reabsorption of amino acids is complete within 2 to 3 hr after yeast cells are suspended in solutions of fermentable sugar, the phenomenon is one that may be easily overlooked. The a-amino nitrogen released represents a relatively small proportion of the total nitrogen content of the yeast, although it amounts to roughly one-third of the free amino acid pool (Fig. 4). Since, in the presence of glucose without an external source of nitrogen, yeast can grow to a significant extent at the expense of that nitrogen free within the cell, it is somewhat surprising that a large part of this nitrogen should first be excreted before it is subsequently reabsorbed and utilied for protein synthesis. The possibility that the phenomenon under study is due to the presence of dead cells or is the result of autolysis of part of the cells must be considered. Shock excretion was observed only in the presence of a fermentable sugar, which seems a less likely situation for autolysis to occur than when yeast is suspended in water or in a nonutiliable sugar, since glucose stimulates the synthetic activities of the cell. The cells used in the experiments were freshly harvested, and at no time were they allowed to stand at elevated temperatures which would encourage autolysis. In addition, prolonged storage had no effect on shock excretion. The released amino nitrogen was associated with small molecules which are dialyable (Delisle and Phaff, 1961) and which are not precipitable by 12.5% trichloroacetic acid (unpublished data). Autolysis causes the appearance in the medium of proteinaceous material, which is partially trichloroacetic acidprecipitable and is not readily assimilated by living cells. In view of these facts, we consider shock excretion to be a property of living yeast cells, and to be independent of autclysis. The experiments described in this paper indicate that shock excretion is a dynamic process, or is the result of dynamic processes. In particular, the phenomenon only occurs in the presence of fermentable sugar and is sensitive to conditions which limit energy supply, such as low temperature or a limiting concentration of sugar. The energy requirement of shock excretion is currently under investigation. ACKNOWLEDGMENT Grateful acknowledgment is made to the Brewing Industry Research Institute for financial support of this project. LITERATURE CITED 1395 COWIE, D. B., AND F. T. MCCLURE Metabolic pools and the synthesis of macromolecules. Biochim. Biophys. Acta 13: DELISLE, A. L., AND H. J. PHAFF The release of nitrogenous substances by brewers' yeast. Am. Soc. Brewing Chemists Proc., p HALVORSON, H. O., AND G. N. COHEN Incorporation des amino-acides endogenes et exogenes dans les prot6ines de la levure. Ann. Inst. Pasteur 95: HASSID, W. Z Determination of sugars in

8 1396 LEWIS AND PHAFF J. BACTERIOL plants by oxidation with ferricyanide and ceric sulfate titration. Ind. Eng. Chem. Anal. Ed. 9: LEWIS, M. J., AND H. J. PHAFF Release of nitrogenous substances by brewers' yeast. 2. Effect of environmental conditions. Am. Soc. Brewing Chemists Proc., p MOORE, S., AND W. H. STEIN A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211: SPIEGELMAN, S., H. 0. HALVORSON, AND R. BEN- ISHAI Free amino acids and the enymeforming mechanism, p In W. D. McElroy and B. Glass [ed.], Amino acid metabolism. Johns Hopkins Press, Baltimore.