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Production of bacillin by Bacillus sp. strain no. KM-208 and its identity with tetaine (bacilysin).

In the course of screening for new antibiotics, we isolated antibiotic KM-208 possessing an inhibitory activity against certain grampositive and gram-negative bacteria from fermentation broth of Bacillus sp., strain No. KM-208. Production of KM-208 was carried out using a 100-liter tank fermentor for 40 hours at 27'C, in a medium containing glucose 2.0 °, , soybean meal 2.0 %, dry yeast 0.3 %, KCl 0.05%, MgSO4.7H2O 0.05%, K2HPO4 0.1%, NaCl 0.3% (w/v, pH 7.0). The activity was determined by a paper disc-agar method using Staphylococcus aureus FDA 209P as a test organism. The isolation and purification of KM-208 was performed by the following method: Whole broth was adjusted to pH 2.5 with 6 N HCl and centrifuged, and the supernatant was decolorized with activated charcoal. The solution was adsorbed on Amberlite IR-120(H+), followed by elution with 0.5N NH,OH. The active eluate was concentrated in vacuo and then lyophilized to give a brown crude powder (6.0g). Further, the crude powder was chromatographed over carbon (eluted with 50% MeOH), silica gel (EtOH-H2O, 10:1) and then CM-Sephadex (H2O) to yield a white powder (75 mg). The IR spectrum of KM-208 showed the presence of peptide linkage at 3500 3000 cm-I and 17001500 cm-1 (Fig. 1). The result from the amino acid analysis of the hydrolyzate (6N HCl, 105°C, 18 hours) of KM-208 revealed the presence of alanine and tyrosine, in a molar ratio of 1 : 0.65. These data suggested a similarity between KM-208 and the antibiotics tetaine1) (bacilysin) and bacillin2,3) which was reported by BOROWSKI et al. (1952) and WOODRUFF et al. (1946) respectively. The structure of bacilysin4,5,6) has been determined by WALKER et al. as shown in Chart 1, but that of bacillin has not been reported. In order to establish the identity of KM-208, bacilysin and bacillin, bacillin was isolated in a similar method to KM-208 from the cultured broth of a bacillin-producing strain (No. MB-155) supplied from the Merck Sharp & Dohme Research Laboratories. On paper and thin-layer (silica gel and avicel) chromatography, KM-208, tetaine and bacillin gave the same Rf values in various