Prumycin produced by Bacillus cereus.

The isolation of prumycin from cultured broths of Streptomyces kagawaensis has been reported by HATA et al.') It inhibits the growth of some bacteria and fungi,') and shows antitumor action.') The structure determined by OMURA et al.3,4) to be 4-D-alanylamino-2-amino-2,4-dideoxy-Larabinose. We isolated this antibiotic from cultured broths of a strain BMG366-UF5 which was isolated from a soil sample collected at Suginami, Tokyo, and classified as Bacillus cereus.®) Bacillus cereus BMG366-UF5 was deposited with the Fermentation Research Institute, Agency of Industrial Science and Technology, Japan, as FERM P-6395. The spores on an agar slant were suspended with sterilized water (120 ml). The suspension (1.2 ml) was inoculated into a medium (110 ml) containing 3.0 % glycerol, 2.0 % fish meal and 0.2 % CaCO3 (adjusted to pH 7.4 with 4 M NaOH) in a 500-m1 baffled Erlenmeyer flask, and the culture was grown at 27°C for 3 days on a rotatory shaker (180 r.p.m.). The cultured broth (20 flasks) was filtered and the antibiotic in the filtrate (2,000 ml, pH 6.8, 404 teg/m1 determined by the cylinder-plate method against Escherichia coli K-12) was adsorbed on a column of Amberlite IRC-50 (40 Na+, 230 ml). After washing the column with water (1,000 ml), the antibiotic was eluted with 0.4 M HCI and the active eluate (390 ml) was concentrated to dryness (13 g, 40 peg/mg). The residue was extracted with methanol (20 ml) to remove the salt, and the extract was concentrated to dryness (7.25 g, 81 pg/mg). The residue was dissolved in 0.2 M NaCl (6 ml) and the antibiotic in the solution was adsorbed on a column of CMSephadex C-25 (200 ml, equilibrated with 0.2 M NaCI). After washing the column with 0.2 M NaCI (600 ml), the antibiotic was eluted with 0.4 M NaCl and the active eluate (390 ml) was concentrated to dryness. The residue was extracted with methanol (20 ml) and the extract wass concentrated to give a crude powder (1.24 g, 343 rig/mg). The powder was further purified by readsorption on a column of CM-Sephadex C-25 (90 ml, equilibrated with 0.4 M NaCI) followed by elution with 0.4 M NaCI. The active eluate (200 ml) was concentrated, the residue was extracted with methanol (15 ml) and the extract was concentrated to yield a powder (871 mg, 505 fig/mg). The methanol solution (10 ml) of the powder was passed through a column of Sephadex LH-20 (90 ml, developed with methanol) and the active eluate (25 ml) was concentrated to give a powder (629 mg, 706 pg/mg, 55 % yield from the broth filtrate) of prumycin hydrochloride. Crystallization from a methanol solution (I ml) of the powder (310 mg) gave the (3-anomer of prumycin dihydrochloride (40 mg, 1,000 pg/mg), mp 189192°C (decomp.), [a] /° +111° (c 1, methanol) [lit.') mp 198-.200°C (decomp.), [a]0 +115° (c 0.5, methanol)]. All spectral data of the antibiotic were identical with those of prumycin.