Isolation and characterisation of an antimicrobial substance produced by Streptomyces violatus

Abstract

Streptomyces violatus produced an antimicrobial substance (268 µg/ml) after 7 days incubation at 30°C in static cultures. Fifteen litres of the culture filtrate were extracted with ethyl acetate and concentrated till dryness. The oily extract was subjected to silica gel column chromatography. Gradient elution was carried out using n-hexane-ethyl acetate (4:1→1:14) and ethyl acetate-ethanol (7:3→1:10). The fractions showing the highest antimicrobial activity were pooled and subjected to a second silica gel column chromatography. The active fractions obtained from the second column were pooled and further purified using preparative high performance liquid chromatography (HPLC). The major peak was collected, concentrated and subjected to spectroscopic analysis (300 MHz 1 H-NMR, 2D COSY, 75 MHz 13 C-NMR, DEPT, UV, IR, Mass spectrometry, elemental analysis) in order to characterize the antimicrobial compound. 1 H-NMR spectra indicated the presence of a di-substituted benzene ring with two aliphatic side chains. The molecular formula of C15H23NO3 with a molecular weight of 276 was estimated from the elemental analysis and mass spectra. Spectroscopic analysis of the purified fraction indicated that the antibiotic produced by S. violatus belongs to an aromatic family of antibiotics, but this compound was not identical to similar antibiotics described in the literature and was designated MSW2000. Gram-positive bacteria were more sensitive than Gram-negative to MSW2000. The antibiotic weakly affected the growth of Saccharomyces cerevisiae and Mucor meihei. X-ray microanalysis of S. aureus treated with the antibiotic MSW2000 showed a reduction in element levels in the cells, especially K, P and S, which may indicate that the antibiotic affects the cell membrane.