No abstract
Carbazomycins G (I) and H (II), new congeners of the carbazomycin complex, have been isolated from the culture broth of Streptoverticillium ehimense. They have proved to contain a unique quinol moiety in the molecule. Their structures have been elucidated by mass and NMR spectrometries and X-ray crystallographic analysis. Carbazomycin G showed moderate antifungal activity against Trichophyton species.In a previous paper1}, we have reported the isolation of carbazomycins C, D, E and F as the minor components of carbazomycin complex. The structures of carbazomycins C (IV) and D (V) were determined by spectroscopic and chemical means, and in the present paper are confirmed by an X-ray crystallographic analysis of IV.In our successive search for other active substances produced by the same microorganism, we have found two new components named carbazomycins G (I) and H (II) which are shown to possess a unique quinol moiety in the molecule. In this paper, we report the isolation, physico-chemical and antimicrobial properties, and structure elucidation of I and II by mass,^-H and 13C NMRspectrometries and X-ray crystallographic analysis. The confirmation of the structures of IV and V by X-ray crystallography of IV is also reported.Results and Discussion Isolation of Carbazomycins G and H Streptoverticillium ehimense was cultured in the same wayas reported previously0. The broth filtrate was extracted with ethyl acetate and the extract was fractionated by silica gel columnchromatography using the solvent composed of «-hexane and ethyl acetate (7 : 1~1 : 1). The last fraction eluted with «-hexane and ethyl acetate (1 : 1) was concentrated and further purified by repeated preparative TLCon precoated Silica gel plates (Merck Art. No. 5715) developed with «-hexane and ethyl acetate (2 : 3). Under a UV-light (365 nm), I gave strong blue fluorescence and n gave dark yellow one, and both components gave the same brownish purple color on silica gel TLCby heating after spraying with 10 % sulfuric acid.
The 3beta-hydroxysteroid oxidase produced by Streptomyces violascens was purified from the culture broth by procedures including batch-wise treatment on DEAE-cellulose, ammonium sulfate fraction, gel filtration on Sephadex G-75, and column chromatography on DEAE-cellulose. The highly purified enzyme preparation exhibited no significant absorption maxima in the visible region other than a maximum at 280 nm. Optimum pH and temperature for the enzyme activity were approximately pH 7.5 and 50 degree, respectively. The Michaelis constant (Km) for cholesterol determined under two different experimental conditions were 4.5 and 6.7 X 10(-4) M. The enzymatic activity was remarkably inhibited by various metal salts such as FeC1(3), FeSO4, AgNO3, etc. On the other hand, neither EDTA nor Fe-chelating agents had any inhibitory effect on the enzymatic activity, while other metal-binding agents, KCN and NaN3, caused significant inhibition. The enzyme activity was inhibited almost completely by N-bromosuccinimide and iodine but not p-chloromercuribenzoate. The highly purified enzyme did not require any external electron acceptors other than oxygen. In addition, the activity was not influenced by the addition of external electron donors. The enzyme showed a high substrate specificity for 3beta-hydroxysteroids and the relative oxidation rates were 100 for cholesterol, 91 for 5alpha-cholestan-3beta-ol, 83 for pregn-5-en3beta-ol-one, 80 for androst-5-en-3beta-ol-17-one, 64 for 5alpha-androstan-3beta-ol-17-one, ect. The oxidation of cholesterol by the enzyme was remarkably inhibited by the addition of 5alpha-cholestan-3beta-ol, 5alpha-cholestan-3-one, 5beta-cholestan-3beta-ol, 5alpha-cholestane3beta, 5alpha-doil or 5alpha-lanosta-8, 24-den 3beta-ol. These findings indicate the present enzyme belongs to the class of 3beta-hydroxysteroid oxidase but some of its physical and enzymatic properties obviously differ from those of 3beta-hyroxysteroid oxidase of Brevibacterium.
An unidentified Streptomyces, tentatively designated as Strain H 1051-MY 10, was proved to produce viomycin and two new antibiotics. The new antibiotics were extracted from the cultured mycelia with acetone and transferred to ethyl acetate after acetone was removed in vacuo. The extracted antibiotics were separated into two components by alumina column chromatography and named carbazomycins A and B, because both antibiotics were proved to contain a carbazole nucleus. The molecular formulae of carbazomycins A and B were determined to be C16H17NO2 and C15H15NO2, respectively. Further, carbazomycin B was methylated with diazomethane to give carbazomycin A. Carbazomycins inhibited the growth of phytophathogenic fungi and further showed weak antibacterial and antiyeast activities. THE JOURNAL OF ANTIBIOTICS JULY
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