APPLIED MICROBIOLOGY, Sept.

, 1965 Copyright @ 1965 American Society for Microbiology

Vol. 13, No. 5 Printed in U.S.A.

Fermentation Studies with Streptomyces griseus

I.

Carbohydrate Sources for the Production of ProteIase and Streptomycin

SHIROH SHIRATO AND CHIZUKO NAGATSU 2nd Laboratory, Kaken Chemical Co., Ltd., Komagome Kamefujimae-cho, Bunkyo-ku, Tokyo, Japan Received for publication 8 March 1965

AB3STRACT SHIRATO, SHIROH (Kaken Chemical Co., Ltd., Bunkyo-ku, Tokyo, Japan), AND CHIZUKo NAGATSU. Fermentation studies with Streptomyces griseus. I. Carbohydrate sources for the production of protease and streptomycin. Appl. Microbiol. 13:669-672. 1965.-The effects of four carbon sources and inorganic phosphate on the production of streptomycin and protease by a strain of Streptomyces griseus were studied. Protease production was increased in fermentations with comparatively rapid consumption of carbohydrate, and streptomycin was produced under conditions of moderately slow consumption. Starch was consumed more rapidly than glucose, and, in fermentations with starch as a carbon source, good yields of protease were associated with poor yields of streptomycin. The effect of the concentration of inorganic phosphate varied with the sugar source; the rate of consumption of glucose or fructose increased with the addition of inorganic phosphate, and the utilization of starch or maltose was not affected.

The utilization of various carbohydrates by 1952a; Nakayama et al., 1952b) was grown on Streptomyces griseus has been the subject of many Krainsky yeast agar. After an incubation of 2 at 26 C, spores were transferred to a investigations. Although there are several reports weeks medium and grown for 1 week. After wheatdrying grain (Hubbard and Thornberry, 1946; Saunders and vacuo, cultures were stored in a refrigerator. Sylvester, 1947; Dulaney, 1948; Karrow et al., in The inoculum was cultured for 30 hr at 26 C 1952; Perlnan and Wagman, 1952; Hockenhull with shaking in medium of the following composiet al., 1954; Hunter and Hockenhull, 1955; Perl- tion, per liter of tap water: glucose, 20 g; meat man and O'Brien, 1956; Silverman and Rieder, extract, 10 g; sodium chloride, 5 g. Transfers 1960; Hockenhull, 1960) pertaining to the rela- were then used to inoculate 25 ml of the following tionship between carbohydrate utilization and medium in 500-ml flasks. The medium contained streptomycin production by S. griseus, it appears (per liter): carbohydrate, 35 g; defatted soybean ammonium beer yeast, powder, that no study has considered the relationship be- sulfate, 225 g; dried carbonate, 2 3g;g;sodium chlog; tween elaboration of streptomycin and protease ride, 2 g; soy calcium 2.4 g; KH2PO4 was added at bean oil, by this organism and the type of carbohydrate concentrations of 0.02 and 0.08 g per 100 ml of used as the carbon source. broth. Glucose, fructose, maltose, and soluble Nomoto and Narahashi (1956, 1959a, b, c) re- starch were used as carbohydrate sources. Sugar ported that the broth of a streptomycin fermenta- solutions were sterilized separately, but soluble tion contained a new type of protease, and sug- starch was added before sterilization. Analytical methods. After 24, 48, 72, and 96 hr gested that this enzyme may be utilized for many purposes. This strongly active and specific pro- of fermentation, flasks were sampled, and pH, tease has had wide practical as well as laboratory mycelial growth, residual sugar concentration, streptomycin potency, and protease activity were application. measured by the following procedures. The pH The purpose of this series of investigations was measured with a Beckman-type pH meter. was to study factors affecting protease production Mycelial growth was recorded as per cent (by by S. griseus, in an effort to obtain higher yields volume) of precipitate after centrifugation at of the enzyme. The relationship between produc- 3,000 rev/min for 15 min. Residual sugar concention of streptomycin and protease by this or- tration was determined by Bertrand's method. ganism and the type of carbohydrate used is Starch and maltose were hydrolyzed with acid prior to analysis. Streptomycin content was asdiscussed. sayed by the cup method (microbiological assay) with Bacillus subtilis PCI 219 as the test organism. MATERIALS AND METHODS
Protease activity was determined by the method Organism and culture conditions. S. griseus of Nomoto and Narahashi (1956), and was exstrain K-1 (Nakayama, Ikeda, and Kawasaki, pressed as PU (pronase units). 669

670
RESULTS

SHIRATO AND NAGATSU

APPL. MICROIBJOL.

Glucose medium. Changes during the course of the fermentation are shown in Table 1 and Fig. 1. These data are taken from duplicate flasks and represent the average of three fermentations. Glucose was consumed rather slowly when inorganic phosphate was not present in the medium. Residual sugar at 96 hr in this case was approximately half of the starting concentration. When 0.02 or 0.08% KH2P04 was added, the consumpTABLE 1. Effect of KH2PO4 on growth and pH with Streptomyces griseus in glucose medium
No KH2PO4
Tim e-_

tion of glucose was accelerated. The formation of both cells and protease was also increased, but streptomycin production was highest with moderate glucose consumption in the medium
with 0.02% KH2P04.

Starch medium. Starch was consumed very rapidly, as shown in Fig. 2. Changes during the course of this fermentation are shown in Table 2 and Fig. 2. Streptomycin was produced only in small amounts, whereas the yields of protease were good and occurred only at an early stage in
30

0.02% KH2PO4
pH

0.08% KH2PO4
20
pH

Timeycelial pH Mgowth
hr

Mycelial

growth
%
-

Mycelial

growth
%

10 _

0 24 48 72 96

7.8 8.4 8.6 8.6

8.6

20 26 23 25

7.8 7.6 8.3 8.2 8.0

7.8
7.2

27 31 29 31

7.4 7.2
8.2

38 30 27 27

100

3.

80

60-

40

1,2

20

24

48

72

96

Time (hr)
FIG. 2. Effect of KH2PO4 on the production of streptomycin and protease, and the consumption of carbohydrate, with Streptomyces griseus in starch medium. Symbols: 0 protease, PU/ml; * = streptomycin, mg/30 ml; o = residual sugar, mg/2.5 ml. KH2PO4 added: 1, none; 2, 0.02%; 3, 0.08%.
TABLE 2. Effect of KH2PO4 on growth and pH with Streptomyces griseus in starch medium
No KH2PO4 Time
0
24

0.02% KH2PO4
pH

0.08% KH2PO4
pH

48

72

96

pH
hr

Mycelial growth
%
-

Mycelial growth
%
-

Mycelial
growth

Time (hr)
FIG. 1. Effect of KH2PO4 on the production of streptomycin and protease, and the consumption of carbohydrate, with Streptomyces griseus in glucose medium. Symbols: 0 = protease, PU/ml; 0 = streptomycin, mg/30 ml; = residual sugar, mg/2.5 ml. KH2PO4 added: 1, none; 2, 0.02%; 3, 0.08%.

0 24 48 72 96

7.8 6.3 8.0 8.2 8.4

32 38 23 17

7.8 5.9 8.0 8.3 8.5

27 26 26 16

7.8 5.6 8.0 8.4 8.5

27 26 21 15

VOL. 13, 1965 PRODUCTION OF PROTEASE AND STREPTOMYCIN BY S. GRISEUS

671

the fermentation cycle. The addition of inorganic phosphate had no influence upon the production of antibiotic or protease or upon the consumption of starch. Fructose medium. Since S. griseus has the enzyme system of the Embden-Meyerhof-Parnas pathway (Wang, Bialy, and Gilmour, 1956), it was considered of interest to compare fermentations having glucose and fructose as carbon sources. Typical data are presented in Table 3 and Fig. 3.
TABLE 3. Effect of KH2PO4 on growth and pH with Streptomyces griseus in fructose medium
No KH2PO4
Time
H

In general, fermentations with fructose appeared similar to those with glucose. However, fructose disappeared more rapidly at the same level of inorganic phosphate. The highest streptomycin yields with fructose were produced at phosphate levels lower than in the case of glucose. Protease production was highest in both media with 0.08 % phosphate. Maltose medium. The data shown in Table 4 and Fig. 4 are similar to those obtained in the starch medium. Carbohydrate disappeared rapTABLE 4. Effect of KH2PO4 on growth and pH with Streptomyces griseus in maltose medium
No KH2PO4

0.02% KH2PO4
H

0.08% KHP204
P
H

0.02% KH2PO4
pH

0.08% KH2PO4
pH

Mycelial growth

Mycelial

growth

Mycelial
growth

TimeI
pH
hr

hr

0 24 48 72 96

7.4 7.2 7.5 7.2 7.8

23 27 27 27

7.4 7.8 6.9 7.6 8.0

28 30 23 29

7.4 7.4 6.6 7.5 7.5

Mycelial growth %

Mycelial
growth

Mycegial
growth

28 24 22 18

7.4

24 48 72 96

7.8 6.9 7.2 8.0

26 34 32

25

7.4 7.3 7.0 7.5 8.0

37 31 31 28

7.4 7.1 7.1 7.4 8.0

45 55 41 30

301
20-

~~~~~~~2

'3

10-

'2
1

100

80

60

'2
*3

40

3
20

24

48

72

96

24

Time (hr)
FIG. 3. Effect of KH2PO4 on the production of streptomycin and protease, and the consumption of carbohydrate, with Streptomyces griseus in fructose medium. Symbols: 0 = protease, PU/ml; 0 = streptomycin, mg/30 ml; o = residual sugar, mg/2.5 ml. KH2PO4 added: 1, none; 2, 0.02%; X, 0.08%.

Time (hr)

48

72

96

FIG. 4. Effect of KH2PO4 on the production of streptomycin and protease, and the consumption of carbohydrate, with Streptomyces griseus in maltose medium. Symbols: 0 = protease, PU/mI; * = streptomycin, mg/30 ml; O = residual sugar, mg/2.5 ml. KH2PO4 added: 1, none; 2, 0.028%6; 8, 0.08%.

672

SHIRATO AND NAGATSU

APPL. MICROBIOL.

idly, and the concentration of phosphate in the medium was without effect. Higher streptomycin yields were obtained in the absence of inorganic phosphate, whereas higher protease concentrations were present in medium with phosphate.
DISCUSSION

Perlman and Wagman (1952) showed that the velocity of glucose consumption was increased and was accompanied by decreased streptomycin production when phosphate was added to the streptomycin fermentation. This was confirmed in our experiments, which also included the fermentation of fructose, maltose, and starch at various phosphate concentrations. In the case of glucose, the addition of phosphate increased carbohydrate consumption. In the case of starch, the concentration of inorganic phosphate had almost no effect on the velocity of carbohydrate metabolism. An interesting relationship was observed between streptomycin production and protease elaboration. Good yields of streptomycin were obtained at the expense of protease under conditions in which carbohydrate consumption was cormparatively slow. More protease, on the other hanid, was produced when carbohydrate was consumed more rapidly. In the starch medium, the plf level increased considerably throughout the course of fermentation. Since this enzyme is inactivated in aqueous solution at high alkaline levels (Nomoto and Narahashi, 1959d), this may be one of the reasons for the reduced yield of protease in spite of a good production curve at the earlier stage of fermentation. Perlman and O'Brien (1959) also reported on the relationship of carbohydrate utilization by four strains of S. griseus which were derived from the same original strain. Two strains utilized glucose, maltose, and starch, and produced streptomycin in good yield; the other two strains utilized glucose rapidly but not starch or maltose, and they produced little or no streptomycin in medium containing starch or maltose. In the present study, S. griseus K-1, which was isolated from soil collected in Japan, utilized starch well but produced only a very small amount of streptomycin.
ACKNOWLEDGMENTS We express our sincere thanks to T. Yabuta and Y. Ikeda of Tokyo University and H. Kubo and H. Nakayama of Kaken Chemical Co., Ltd., for their guidance. We also thank S. Ida of Kaken Chemical Co., Ltd., for his encouragement throughout this study.

LITERATURE CITED DULANEY, E. L. 1948. Observations on Streptomyces griseus. III. Nitrogen carbon sources for growth and streptomycin production. J. Bacteriol. 56:305-313. HOCKENHULL, D. J. D. 1960. The biochemistry of streptomycin production. Progr. Ind. Microbiol. 2:131-165. HOCKENHULL, D. J. D., K. H. FANTES, M. HERBERT, AND B. WHITEHEAD. 1954. Glucose utilization by Streptomyces griseus. J. Gen. Microbiol. 10:353-370. HUBBARD, C. V., AND H. H. THORNBERRY. 1946. Utilization of various carbohydrates by Streptomyces griseus for production of streptomycin and growth. Trans. Illinois State Acad. Sci. 39:57-64. HUNTER, G. D., AND D. J. D. HOCKENHULL. 1955. Actinomycete metabolism-incorporation of C14-labeled compounds into streptomycin. Biochem. J. 59:268-272. KARROW, E. O., R. L. PECK, C. ROSENBLUM, AND D. T. WOODBURY. 1952. Microbiological synthesis of C14-labeled streptomycin. J. Am. Chem. Soc. 74:3056-3059. NAKAYAMA, H., Y. IKEDA, AND M. KAWASAKI. 1952a. On the streptomycin producing Streptomycete. Rept. Sci. Res. Inst. (Tokyo). 28(6): 387-394. NAKAYAMA, H., Y. IKEDA, K. KAJIKAWA, M. KAWASAKI, AND Y. SERIZAWA. 1952b. Rept. Sci. Res. Inst. (Tokyo). 28(6) :395-404. NOMOTO, M., AND Y. NARAHASHI. 1956. A proteolytic enzyme of Streptomyces griseus. J. Biochem. (Tokyo) 46:653-667. NOMOTO, M., AND Y. NARAHASHI. 1959a. An improved method for purification of a protease of Streptomyces griseus. J. Biochem. (Tokyo) 46839-847. NOMOTO, M., AND Y. NARAHASHI. 1959b. A proteolytic enzyme of Streptomyces griseus. III. Homogeneity of the purified enzyme preparation. J. Biochem. (Tokyo) 46:1481-1487. NOMOTO, M., AND Y. NARAHASHI. 1959c. A proteolytic enzyme of Streptomyces griseus. IV. General properties of Streptomyces griseus protease. NOMOTO, M., AND Y. NARAHASHI. 1959d. Rept. Sci. Res. Inst. (Tokyo) 35(1):84-89. PERLMAN, D., AND E. O'BRIEN. 1956. Utilization of carbohydrates by strains of Streptomyces griesus. J. Bacteriol. 72:214-218. PERLMAN, D., AND G. H. WAGMAN. 1952. Studies on the utilization of lipids by Streptomyces griseus. J. Bacteriol. 63:253-262. SAUNDERS, A. P., AND J. C. SYLVESTER. 1947. Synthetic media for the production of streptomycin. Abstr. 112th Meeting Am. Chem. Soc., p. 9A-10A. SILVERMAN, M., AND S. RIEDER. 1960. The formation of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus. J. Biol. Chem. 235:
1251-1254.

WANG, C. H., J. J. BIALY, AND C. M. GILMOUR. 1956. Federation Proc. 15:378-380.