Streptomycin

Summary Streptomyces griseus DTH-2 was grown in 5 1 fermentors on complex media containing calcium carbonate as a buffering agent. It was shown that automatic pH control (4N KOH) could substitute the calcium carbonate giving higher yields of streptomycin. The yield was further increased by omitting inorganic phosphate from the medium and by differential addition of glucose/ammonium sulphate during the fermentation. The maximal yield obtained was 8.5 g of streptomycin per liter.     

Streptomycin and lysozyme

Résumé Les microorganismes sensibles au lysozyme ne subissent pas l'agglutination par la streptomycine. Inversement, la streptomycine empêche la clarification des suspensions microbiennes par le lysozyme. Cet effet est attribuable à la prévention de la dissolution des noyaux. Les mutants résistants à la streptomycine restent sensibles au lysozyme et se comportent, en présence de streptomycine plus lysozyme, comme les souches-mères.

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Lysozyme-sensitive microorganisms are not subject to streptomycin-agglutination. Conversely, streptomycin inhibits the clarification of bacterial suspensions by lysozyme. This effect is mainly due to a prevention of nuclear dissolution. Streptomycin-resistant mutants remain lysozyme-sensitive and behave towards streptomycin plus lysozyme as do parent-cells.

     

Biosynthesis of Streptomycin

A natural precursor (L) of streptomycin which had no antibiotic potency was obtained from mycelium suspension of Streptomyces griseus in glucose solution and was transformed to streptomycin by H enzyme obtained from mycelium of the organism. This transforming reaction was carried out most effectively at slightly alkaline pH and inhibited by inorganic phosphate and ethylenediaminetetraacetate. L component was considered to be a phosphorylated compound and liberation of the phosphoric acid was essential for L component to be transformed to streptomycin. This transformation was performed not only by H enzyme but also by intestinal alkaline phosphatase, although some difference in the reaction mechanism was supposed to be between those two enzymes.     

Biosynthesis of Streptomycin

A method for the accumulation of the streptomycin precursor (L) in the culture broth of Streptomyces griseus was developed and the precursor was successfully isolated from the broth.

When the microorganism was cultured under shaking in the glucose-meat extract-peptone medium (0.5% glucose, 0.2% yeast extract, 0.2% meat extract, 0.4% peptone, 0.5% sodium chloride, 0.025% magnesium sulfate, pH 7.0), the accumulation of the precursor in the broth was induced by the addition of supplementary glucose (e.g., 2 g glucose per 100 ml broth) 24 hr after inoculation followed by further cultivation for 48 hr. Increased accumulation of L component was obtained merely by increasing glucose content in the culture medium (e.g., 5% glucose-containing medium in the above-indicated one) instead of glucose supplement on the way of fermentation. For the accumulation of a large amount of L component in a culture broth, it looked to be necessary for pH value of the broth to be maintained between 6 and 7 during fermentation.

L component was isolated from the culture broth by adsorption on Amberlite IRC-50 and elution with 2% NaCl solution. The L component was separated on this column from contaminated streptomycin which requires 5% NaCl solution to be eluted. The L component in the 2% NaCl eluate was adsorbed on active carbon at neutral or slightly alkaline pH and eluted with 95% methanol at acidic pH, Partially purified L component precipitated as hydrochloride by addition of acetone to the methanol extract which had been concentrated in vacuo.