Effect of Cobalt-Porphyrin on the Growth of Vitamin B12-Producing Microbes1

EFFECT OF COBALT-PORPHYRIN ON THE GROWTH OF VITAMIN B12-PRODUCING MICROBES1

YOSHIKAZU SAHASHI, TOSHIO MUTO, NOBUO SENO, TAKAO SUZUKI, AND TADASHI HIRABAYASHI

Laboratory of Biochemistry, Tokyo Agricultural University, Setagaya-ku, Tokyo

(Received January 16, 1957)

In previous papers (1) the biosynthesis of vitamin B12 by B12-nonproducing microbes in B12-free basal medium containing cobalt-porphyrin and synthetic 5, 6-dimethylbenzimidazole was reported and the biochemical significance of cobalt-porphyrin was discussed from various points of vitamin B12 biochemistry. Further experiments have thereafter been attempted on the effect of cobalt porphyrin on the growth of vitamin B12-producing microorganisms. In the present paper the outline of the authors' researches performed in 1955-1956 will be briefly described.

EXPERIMENTAL

1. Effect of Co-porphyrin on the Growth of Streptomyces olivaceus PD 61 Experiments with Streptomyces olivaceus PD 61 (Sakai) were carried out by an ordinary shaking culture in a medium containing cobalt-porphyrin (2). Composition of Vitamin B12-free Basal Media

(pH 7.4-7.5)

Cultivation was continued for 144 hours, and the vitamin B12 content of the final fermentation was estimated by an ordinary Euglena method. A marked growth of Streptomyces olivaceus was always seen in the case of the synthetic medium supplemented with Co-porphyrin, but no additive eff ets were

1 Biochemical Studies on Vitamin B12 . Ⅹ Ⅲ.

56 Vol. 3 EFFECT OF COBALT-PORPHYRIN 57 observed by the addition of 5, 6-dimethylbenzimidazole (DBI). The growth of the microbes was estimated photometrically.

TABLE Ⅰ The Net Increase in Vitamin B12 in the Brotha at the End of Fermentation

a The content of vitamin B12 was measured after removing the Co-porphyrin

initially added. b The content of vitamin B12 was tested using Lactobacillus lactis Dorner .

FIG. 1 The Growth of Streptomyces oli FIG. 2 The Growth of Streptomyces oli vaceus in Dulaney Medium Containing Co vaceus in the Waksman Medium Containing Porphyrin Co-Porphyrin Ⅰ, Negative control. Ⅱ, 140mg/l Co- Ⅰ, Negative control. Ⅱ, 140mg/l Co porphyrin was added. Ⅲ, Both 140mg/l porphyrin was added. Ⅲ, Both 140mg/l co-porphyrin and 20mg/l DBI were added. Coporphyrin and 20mg/l DBI were added. Ⅳ, 20mg/l DBI was added. Ⅳ, 20mg/l DPI was added.

2. Effect of Co-Porphyrin on the Growth of the Infusoria from Goats. The second experiment was performed with the inf usoria from the gastric juice of goats (3). At first, infusoria were isolated from the gastric juice of a previously fistulated goat. The microflora thus obtained were washed with sterilized water, and the gastric juice was removed by centrifugation from the sediment, yielding 5ml of the microflora . 5ml of the infusoria thus obtained were suspended in 100ml of a vitamin 58 SAHASHI, MUTO, SENO, et al. 1957

B12-free basal culture medium. Composition of Basal Culture Medium (in g): D-Glucose, 1.5; NaCl, 6.4; MgCl2, 0.08; KCl, 0.16, CaCl2, 0.2; NaH2PO4 , 0.01; sodium L-glutamate, 2.0; NaHCO3, 0.2; H2O, 1000ml. (pH 7.0-7.2) To the suspension of infusoria were added vari ous amounts of the synthetic medium supplemented with Co-porphyrin and DBI, and afteri ncubating the mix ture at 400 living cells were accurately determined. Composition of Co-Por phyrin-Containing Medium: Co-porphyrin 10.5mg DBI 1.5mg Basal culture medium 5.0ml FIG. 3 Effect of Chlorophyll Ration on the Fecal Microbes of Cattle The concentration of ○ Main experiment, * Chlorophyll ration Co-porphyrin was previou , △ Control. sly calculated from the pre liminary comparative expe riments with standard vitamin B12 solution (10mƒÁ/ml).

TABLE Ⅱ Living Test of Isolated Infusoria in the Synthetic Medium Supplemented with Co-porphyrin and DBI

3. Experiments on the Growth of Intestinal Microbes of Cattle Fed the Diet Supplemented with Chlorophyll-rich Ration. Experiments were first carried out on the cases of "Kiriyoi disease" in Shimane Prefecture (4). The animals were previously fed a chlorophyll deficient diet in winter season. When Actinomycetes disappeared from the Vol. 3 EFFECT OF COBALT-POBPHYRIN 59

TABLE Ⅲ Living Test of Isolated Infusoria in the Synthetic Medium Supplemented with Vitamin B12

feces, chlorophyll-rich supplement was administered, and the growth of vitamin B12-producing microbes was tested by an ordinary plate culture method. Chlorophyll supplementation always indicated a marked growth of Acti nomycetes (Fig. 1-3). The results may also be due to the physiological significance of Co-porphyrin for the vitamin B12-producing microbes.

TABLE Ⅳ Effect of Chlorophyll Ration (Aucuba japonica) on the Fecal Actinomyces of Cows

Recently, similar experiments were carried out by Suzuki and Hirabayashi in Mobara Province, Chiba Prefecture. Since many years ago, cows have been administered with green leaves of evergreen, Aucuba japonica Thumb., as an efficient nutrient for increasing lactation in both Shizuoka and Chiba Prefectures. This effect was assumed by Suzuki (5) to be due to the benefi cial effect of vitamin B12 on the growth of the gastric microbes, and from this point of view the excretion of Actinomycetes in feces was tested. From his careful screening test of microorganisms it was found that the administra tion of the leaves of evergreen to cows always resulted in the growth of B12

producing microbes (Table ‡W). This seems to add an another evidence in 60 SAIIASHI, MUT(), SRNO, et・a1. 1957

establishing a new special significance of Co-porphyrin for the biosynthesis of vitamin B12.

SUMMARY

1. The possibility of the importance of Cobalt-porphyrin on the growth of vitamin B12-producing strains was suggested by the observations of a marked and constant increase in Streptomyces olivaceus PD 61, goat-gastric Infusoria and Actinomycetes in cows on chlorophyll-containing ration. 2. The biosynthesis of vitamin B12 by vitamin B12-forming organisms seems to be concerned with cobalt-porphyrin rather than 5, 6-dimethylbenzimidazole.

REFERENCES

1. Sahashi, Y., Muto, T., and Asai, S., Bull. Agr. Chem. Soc. Japan 19, 129, 282 (1955); Muto, T., and Sahashi, Y., J. Vitaminol. 3, 50 (1957). 2. Sahashi, Y., Muto, T., and Asai, S., Vitamins 9, 443 (1955); 10, 140 (1956). 3. Sahashi, Y., Muto, T., Seno, N., and Asai, S., J. Agr. Chem. Soc. Japan 30, (1956).4 . Sahashi, Y., and Seno, N., Vitamins 9, 370 (1955). 5. Sahashi, Y., Suzuki, T., and Hirabayashi, T., ibid. 12, 117 (1957)