A species of
Micromonospora, Micromonospora floridensis
NRRL 8020, has been found to produce an actinomycin complex consisting of at least 25 active components. After solvent extraction of the complex, separation of the individual components was carried out by preparative thin-layer chromatography. Hydrolysis and subsequent electrophoretic and chromatographic identification of the amino acid content of each of the isolated components have shown differences from known actinomycins, and the probability exists that these contain a number of amino or imino acids not previously found in other members of this group of antibiotics.
Micromonosporasp. 69-683 produces neomycinB and except for an early report of production of actinomycin from a culture not well defined, is presently the only other knownexampleof a major Streptomyces-iproducQdantibiotic also produced by a memberof the genus Micromonospora.A variety of carbon and nitrogen sources were found to be suitable substrates for Micromonospora sp. 69-683. The antibiotic is removedfrom the fermentation broth with a cationic-exchange resin and is purified by passing the mixture through a Dowex1x2 resin column. Chemical, physical and biological data confirm that it is identical to neomycin B.The isolation of neomycin in 1948 resulted from a comprehensive screening of streptomyces which have yielded many important chemotherapeutic agents. In 1963 Weinstein et al4) reported on gentamicin, the first important antibiotic produced by a member of the genus Micromonospora. This report concerns the production of neomycin by Micromonospora sp. 69-683 and is presently the only known example of an important streptomyces-produced antibiotic which is also produced by a memberof the genus Micromonospora.
Materials and Methods
Preliminary TaxonomyColonies of Micromonospora sp. 69-683 on an organic agar medium are plicate (folded) and initially are a reddish orange turning a deep dark brown as abundant sporulation develops in the colony. A light yellowish diffusible pigment is typically produced. Anelectron-micrograph of a thin section of the spore of Micromonospora sp. 69-683 attached to its sporophore is shown in Figure 1. The sporophore has a diameter of0.5 micron. The spore surface appears minutely warty. Rupture of the thin cell wall will facilitate release of the mature spore.Detailed taxonomic studies will be reported elsewhere. Antibiotic Production The mediumused for the inoculum preparation is detailed in Table 1. The culture was grown for 72hours at 28°C on a rotary shaker. A 5 % inoculum was then used for all fermentation studies. The initial soil isolate produced only 20 mcg/ml but by a series of mutation steps utilizing exposure to UVlight and natural selection the potency was increased to approximately 400mcg/ml.The mediumused for fermentation of the culture is detailed in Table 2. Flask fermentations were carried out in 500-ml Erlenmeyer flasks containing 100 ml of medium. Maximumantibiotic yields of 300^400mcg/mlwere reached after 96-hour fermentation at 28°C on a rotary shaker run at 280rpm. Other carbohydrate and nitrogen sources were studied and several can be used which
Gentamicin, an aminoglycoside antibiotic, occurs both free in the fermentation filtrate and bound to the mycelium of the producing organism, Micromonospora purpurea. The bound gentamicin, which represents the major portion of the total quantity present in the fermentation broth, was released after exposure to acid, alkali, or sonic disruption. Washing the mycelium with distilled water, heat treatment, and the addition of sodium chloride to the fermentation medium were not effective methods for releasing bound gentamicin. Gentamicin and other aminoglycoside antibiotics were effectively adsorbed from neutral aqueous solutions by the acid-extracted mycelium of M. purpurea, In addition, the acid-extracted mycelium of actinomycetes other than M. purpurea were shown to be able to adsorb gentamicin from solution.
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