Biosynthesis of Streptomycin

Part V Purification and Properties of a Streptomycin Precursor

Abstract

The streptomycin precursor (L) was purified and its properties were studied.
The precursor L in the crude aqueous solution was first precipitated as its reineckate, and then recrystallized from hot water. The L reineckate was decomposed to L sulfate and the protonated reineckate by acidification with sulfuric acid. After removing the protonated reineckate with Amberlite XAD-2, the L sulfate solution was subjected to column chromatography on Sephadex C-25. Gradient elution was performed with aqueous ammonium acetate solution. The L-containing fractions were dried thoroughly in vacuo till no free ammonium acetate remained. The acetate form of L thus obtained was changed to sulfate form with sulfuric acid and acetone, obtaining white powder or L sulfate.
The L gave the positive maltol reaction, the intensity of which was equal to that given by the same amount of streptomycin on the basis of bioassay. It was bioautographically shown that dephosphorylation of L by alkaline phosphatase or lanthanum hydroxide gave rise to formation of streptomycin. The molar concentration of the phosphorus in L was same as that of streptomycin derived from the L. These results suggested that the L was phosphorylated streptomycin; one mole phosphoric acid being attached to one mole streptomycin with ester bond.