STUDIES ON THE CELL WALL LYTIC ENZYMES PRODUCED BY STREPTOMYCES SPECIES PART 1. THE STRAINS AND THEIR LYTIC ACTIVITY TOWARD SACCHAROMYCES

Transcription

1 J. Gen. Appl. Microbiol. Vol. 6, No. 1, 1960 STUDIES ON THE CELL WALL LYTIC ENZYMES PRODUCED BY STREPTOMYCES SPECIES PART 1. THE STRAINS AND THEIR LYTIC ACTIVITY TOWARD SACCHAROMYCES AKIRA FURUYA and YONOSUKE IKEDA Institute of Applied Microbiology, University of Tokyo Received for publication, November 30, 1959 INTRODUCTION Many reports concerning bacteriolytic substances have been published (1), especially lysozyme obtained from egg white has been studied by many investigators (2). But enzymes which attack cell walls of yeasts and fungi are not known as well. Only " Snail enzyme " isolated from the gut of snails has been found to cause lysis of cell walls of yeasts and fungi. Recently H0RIK0SHI and SAKAGUCxi (3) discovered that the lytic enzyme produced by Bacillus circulans is capable of lysing the cell walls of several species of fungi such as Aspergillus and Penicillium, and is also relatively active toward Saccharomyces cerevisiae. These investigations were intended to obtain lytic enzymes from microorganisms which are capable of lysing yeast cell walls. In this paper, the method of isolation of the microorganisms and the properties of the lytic factor produced by a strain of Streptomyces isolated from soil is described. EXPERIMENTAL I) Isolation of the cell wall decomposing microorganisms from soil Medium: Y-medium consisted of compressed baker's yeast, 100 g, K2HPO4, 2 g, MgSO4.7H2O, 1 g, distilled water 11, adjusted to ph 6.8. Y-G medium consisted of compressed baker's yeast, 50 g, KH2PO4, 2 g, MgSO4.7H2O, 1 g, glucose, 5 g, distilled water 11, adjusted to ph 6.5. Sterilization was carried out 120 for 5 minutes in autoclave. a) Initial screening test The soil sample (approx. 5 g) was suspended in 10 ml of sterilized water and the upper layer was further diluted to to 0.2 ml of the diluent was spread on Y-agar plate and incubated for one week at 30 C. After two or three days incubation, a number of colonies appeared on the surface of the agar. The few colonies surrounded by a clear zone were selected and streaked on another agar plate. The growths were purified by repeating the single colony isolation on Y-agar plates. In this way about 150 strains of bacteria and Streptomyces were isolated from soil. It appeared that the microorganisms which formed clear zones on the Y-agar 40

2 1960 Cell Wall Lytic Enzymes Produced by Streptomyces sp. 41 plates produced some substances that caused lysis of the heat-treated yeast cells contained in the agar. 5 strains of bacteria and 5 strains of Streptomyces with large and sharp zones were selected from the isolated 150 strains and were cultured at 30 in shake-flasks containing 100 ml of Y-G medium. The lytic activities of their culture liquids toward heat-treated yeast cells were measured at 24 hr. intervals by the turbidimetric method. None of them showed activity. It therefore became necessary to select from the isolated 150 strains those which produced the lytic substances in submerged culture. b) Second screening test The 150 isolates were inoculated into a large test tube containing 20 ml of Y-G medium and were shaken at 30. The lytic activity toward heattreated yeast cell was measured by the following method. 15 ml of Y-medium was poured into a Petri dish and incubated for 48 hr. at 37 to dry the agar. Six to seven steel cylinders of the type used in the assay of antibiotic activity were placed on the treated Y-agar plate. At 24 hr. intervals, the culture broths were withdrawn, poured into the cups and incubated for 48 hr. at 30. Around the cups of the lytic active broth, lysed zones were formed as shown in Photo. 1. By this method it was possible to measure Lysed Photo. 1. Cup-assay of lytic zones formed by lytic active activities broth after of culture broths. 48 hr. incubation at 30. semi-quantitatively the lytic activities of the culture broths toward heattreated yeast cells. As a result, eight strains of Streptomyces and nine strains of bacteria were found to show rather strong lytic activities in their culture broths after two or three days of culture. In Table 1, the diameters and degree of clarity of the lysed zones are listed. Generally, the lysed zones of Streptomyces were larger and clearer than those of bacteria. Moreover since most of culture liquids of bacteria were very viscous, it was inconvenient to measure

3 42 A. FURUYA and Y. IKEDA VOL. 6 Table 1. Lytic activities of Streptomyces and bacteria isolated Lytic activities were measured by the cup-assay method. The shaken at 30 for two or three days in Y-G medium. from strains soil. were the lytic activities by the turbidimetric method and to isolate the lytic factor from them. The following experiments were performed with the eight selected strains of Streptomyces. c) The lytic activities of the Streptomyces culture broths The spores of each strain on Y-agar slants were transferred into test tubes containing 10 ml of Y-G medium and shaken at 30 for 24 hr ml of these culture broths was transferred into a fresh flask containing 100 ml of Y-G medium and incubated on a shaker at 120 r.p.m. and at 30. At daily intervals, the lytic activity was measured by the cup-assay method previously described. The results are shown in Table 2. The lytic activities of the culture broths reached a maximum on the second to third Table 2. Changes in activity of culture broths of Streptomyces. Lytic activity was measured by cup-assay method.

4 1960 Cell Wall Lytic Enzymes Produced by Streptomyces sp. 43 day. Strain strains. no. 62 especially showed activity for a longer period than other On the second day, the culture liquids were taken, centrifuged, and filtered throug h a Seitz filter. The lytic activites of the filtrates were measured by the turbidimetric method using a electrophotometer. Significant decrease in turbidity of every sample was observed within one to two hours, clearly indicating that these eight strains of Streptomyces produced some lytic factor active against heat-treated yeast cells. (see Fig. 1) The lytic activity of the broths toward living yeast cells was also measured by the same turbidimetric method. No decrease in turbidity was observed during three hours incubation. By conducting the initial and second screening tests, it became possible to isolate those microorganisms which produced lytic factors against heat-treated yeast cells in submerged cultures. The following experiments were performed with strain no. 62, bacause 1) its lytic activity was greater than the other strains, 2) it produced lytic factor in culture broth for an extended time, 3) it did not produced dark brown pig ments as did strain nos. 22, 55, 61 and 71, which disturbed the accurate tur bidimetric assay of the lytic activity. d) Identification of the strains w hick produced lytic factors Identification of the typical th ree strains-nos. 55, 59, and 62-among the eight strains of Streptomyces was made by Dr. SHIGEO ABE and Mr. FUKUSAarch Laboratory of Kyowa Fermentation BURO OKAMOTO of the Tokyo Rese Industry Co. Ltd. As a result of their experiments, it was found that strain no. 55 belongs to Streptomyces oliv aceus, strain no. 59 resembles St. griseoflavus and strain no. 62 resembles St. albus. According to the classification of WAKSMAN and LECHVALIER (4), the aerial mycelium of St. albus is a spiral form and its conidiospore is sphe rical but the aerial mycelium of strain no. 62 is a straight form and the con idiospore is elliptical. However the other properties and physiological morphological of strain no. 62 is found to be identical with those of St. albus. II) Lytic factor produced by Streptomyces no. 62 strain Culture of the microorganism : the strain was grown in the same medium and method described in part 1-c. Shake flasks containing 100 ml of Y-G

5 44 A. FURUYA and Y. IKEDA VOL. 6 medium were inoculated with 0.5 ml of the seed culture and were shaken at 30. Preparation of the yeast cell suspension : a loop of vegetative cells of Saccharomyces cerevisiae no. 6 strain was transferred to a shake flask containing 100 ml of LINDEGREN's complete medium (5) and shaken for 24 hr. at 30. The cells were collected by centrifugation, washed twice with distilled water and suspended in physiological saline. The suspension was heat-treated at 60 in a water bath for 30 minutes. The cell number of the suspension was adjusted to 3.3 x 108 per ml. a) Assay of lytic activity The culture liquid was centrifuged to separate the mycelia and filtered through a Seitz filter. Assay of the lytic activities was performed by following the decrease in turbidity of yeast cell suspension. One ml of yeast cell suspension was added to 10 ml of the filtrate and the mixture was incubated at 37 in a water bath with mild shaking. Turbidities were measured by a Kotaki electrophotometer 5-D Type at 10 minute intervals for a period of two hours. Fig. 2 illustrate a typical experiment with an active filtrate and shows the relation of the fall in turbidity to the decrease of cell number measured by the THOMAS hemocytometer. For estimation of lytic activities of various preparations, the turbidity reduction rate were calculated according to the following equation. Turbidity reduction rate= dt- Do x 100 o- Do dt = turbidity of reacti n mixture at time t o Do= turbidity of filtrat e before addition of yeast cell suspension do= turbidity of reacti on mixture at time zero The progressive curves for turbidity reduction rate of yeast cell suspenn of the lytic factor are shown in Fig. 3. The sion in various concentratio lytic factor was diluted wit h M/20 phosphate buffer, ph 7.6. The control experiment was performed u sing the same buffer instead of the lytic factor. The slope of the linear porti on of the curves are related to the concentration of the lytic factor so that a reasonable accurate estimation of various prepa- ration can be made,

6 1960 Cell Wall Lytic Enzymes Produced by Strcptomyces sp. 45 tivities in the culture liquids using Y-G culture liquid showed the strongest activity. Under the conditions described above, one unit of the lytic activity is defined as the amount of the lytic factor which causes one percent fall in the turbidity reduction rate of yeast cell suspension per ten minutes. According to this definition, the lytic activity of the culture liquid used in the experiment of Fig. 3 is 1.5, 1.5, 1.3 or 0.9 unit per ml which were calculated from the curves D-1, D-2, D-3 and D-4 respectively. b) Lytic activity and age of culture The changes of lytic acmedium were measured at daily It appeared The results are intervals. shown that Fig. the 4. third day's c) Lysis of various heat-treated yeast cells To clarify the effect of heat treatment on lysis of yeast cells by the lytic factor, yeast cell suspension was heat-treated under various conditions: 120 (autoclaved), 100 (heated to 100, immediately cooled by running water), 100 for 10 minutes (heated to 100, kept at the same temperature for 10 Fig. culture 4. Lytic activity changes in the liquid of strain no. 62. yeast minutes), 60 for 60 minutes (heated at 60 in a water bath for 60 minutes) and 60 for 30 minutes. The cell number of each heat-treated suspension was adjusted to 3.3 x 10g per ml. The results are shown in Fig. 5. It Fig. 5. Lysis cells. c v vv -rv vv ~~~~~ ~, of various heat-treated

7 46 A. FURUYA and Y. IKEDA VOL. 6 appeared that severe heat treatment made yeast cells more susceptible to the lytic factor. In the screening experiments, it was found that the eight strains of Streptomyces showed lytic activity toward heat-treated yeast cells but was not active toward living cells. In the experiment of Fig. 5, the cells heattreated at 100 for 10 minutes are lysed about twice as fast as those heattreated at 60 for 30 minutes. Moreover, as shown in Fig. 3, the slope of D-3 curve in that reaction mixture, the amount of the lytic factor was about half of that in D-1, is much less when compared to the slope of the latter. Therefore, it seems probable that the reason living cells were not lysed by the factor was not because the lytic factor was inactive against living cells but because the living cells were more resistant to the lytic factor and also the amount of the lytic factor in culture broth was not enough to cause lysis of living cells in a short period. In order to elucidate these points, separation and concentration of the lytic factor was carried out in the following experiments. d). Separation of the lytic factor Separation of the lytic factor from culture broth was done according to the method described in Fig. 6. The fraction precipitated by 70 per cent saturation with ammonium sulfate was collected and dissolved in phosphate Fig. 6, Separation process of lytic factor from the culture broth

8 1960 Cell Wall Lytic Enzymes Produced by Streptomyces sp. 47 buffer. sphate. The The colored material was removed by adsorption on calcium pho- lytic activities of B, E-1, and E-2 preparation were 1.4, 11, 80 unit per ml, giving a concentration of the lytic factor about sixty times that of the original broth. Fig. 7. Lysis of living yeast cells by the lytic factor. Lytic factor E-1 fraction 4 ml M/20 Phosphate buffer ph Suspension of living yeast cells 1 (cell number 3.3 x 108/ml) Lysis was measured at 37. e) Lysis of living yeast cell The lysis of living yeast cells by E-1 preparation was tested. Saccharomyces cerevisiae no. 6 strain was cultured by the same method described in the beginning of Part II. The harvested cells were washed and suspended in M/20 phosphate buffer ph 7.6. Cell number was adjusted to 3.3 x 108/ml. Lysis of living cells was measured by following the decrease in turbidity of the suspension and counting the cell number at ten minute intervals. The results are shown in Fig. 7. It was found that living yeast cells were also lysed by the lytic factor in a short time. SUMMARY In order to obtain microorganisms which produce lytic enzyme active against cell walls of Saccharomyces cerevisiae, samples collected from soil were spread on an agar medium containing heat-treated baker's yeast. In the initial screening, about 150 colonies which showed clear zones on the medium around them were selected. In the second test the lytic activity of the culture liquid of these isolates were measured by means of the cupassay method. Among them eight strains of Streptomyces were found to be active and nine strains of bacteria active to a lesser extent. By means of the turbidimetric assay, the culture liquids of all eight strains of Streptomyces also exhibited lytic activity toward heat-treated cells of Saccharomyces cerevisiae in two hours incubation. Among the eight strains of Streptomyces, strain no. 62 was selected and studied further, because it showed the strongest lytic activity and produced the lytic factor for an extended time. The lytic factor produced by the strain was concentrated by ammonium sulfate precipitation. Living cells of Saccharomyces cerevisiae proved to be lysed by the concentrated lytic factor in short period.

9 48 A. FURUYA and Y. IKEDA VOL. 6 ACKNOWLEDGEMENTS The authors wish to thank Dr. SHIGEO ABE and Mr. FUKUSABURO OKAMOTO of the Tokyo Research Laboratory, Kyowa Fermentation Industry Co. Ltd. for their identification experiments of the isolated strains of Streptomyces. The authors are also indebted to the Waksman Foundation of Japan for a grant which enabled the research to be carried out. REFERENCES (1) M. WELSCH: J. gen. Microbiol., 18, 491 (1958). (2) M. R. J. SALTON: Bact. Rev., 21, 82 (1957). (3) K. HORIKOSHI and K. SAKAGUCHI: J. Gen. Appl. Microbiol., 4, 1 (1958). (4) S. A. WAKSMAN and H. A. LECHEVALIER: Actinomycetes and their Antibiotics (1953). Williams and Wilkins. (5) C. C. LINDEGREN: The Yeast Cell, Its' Genetics and Cytology (1949). Educational Publisher's Inc., Saint Louis, P