Bleomycin
Hydrochloride,Nippon Kayaku, Co.,Japan
To a medium having a composition of 6.4 % of millet jelly, 0.5 % of glucose,
3.5 % of soybean powder, 0.75 % of corn steep liquor, 0.3 % of sodium
chloride, 0.1 % of potassium secondary phosphate, 0.05 % of zinc sulfate,
0.01 % of copper sulfate, 0.2 % of sodium nitrate and 0.01 % of Toho No. 1
(trade name for a surface active agent composed of polyoxyethylene
manufactured by Toho Chemical Industry Co. Ltd., Japan) was added 3-aminopropyl-
dimethylsulfonium bromide hydrobromate in a proportion of 0.4 mg/ml
to adjust the pH of the medium to 6.5.
Each 100 ml of the thus treated medium was separately charged into a
Sakaguchi flask and was then sterilized. Subsequently, Streptomyces
verticillus (ATCC No. 15003) was inoculated in the medium and was cultured
at 27°C for 8 days with stirring at 130 r.p.m. Thereafter, the culture liquors
(4.5 L) were collected and filtered to obtain 3.0 L of a filtrate (potency 38.8
mg/ml, total potency 416.4 mg). This culture filtrate was passed through and
adsorbed on a column packed with 200 ml of Amberlite IRC-50 and was
washed with water and was eluted with 0.5 N hydrochloric acid. 1.0 L of the
eluate was neutralized, was passed through and adsorbed on a column packed
with 100 ml of active carbon, was washed and was then eluted by use of a
1:1 (by volume) mixture of acetone - 0.02 N aqueous hydrochloric acid
solution, and fractions active to Mycobacterium 607 were collected and
concentrated to dryness. The resulting residue was dissolved in 5 ml of an 80
% aqueous methanol solution and was charged into a column packed with 30
ml of neutral alumina, followed by elution with an 80 % aqueous methanol
solution. Subsequently, bleomycin-containing fractions were collected and
concentrated to dryness to obtain 195 mg of bleomycin hydrochloride
(potency 650.7 mcg/mg, total potency 172 mg). The yield from the culture
filtrate was 30.5 %.
