ABSTRACTObjectives: Pseudonocardia alni exhibits antimicrobial activity against tested pathogenic Staphylococcus aureus, Microsporum canis, and Trichophytonmentagrophyte. The present paper aimed to optimize various cultural conditions for antimicrobial metabolite production, purification, andcharacterization of the active substance.Methods: The effects of various parameters such as culture media, carbon and nitrogen sources, phosphate concentration, pH, temperature, incubationperiod, and

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... on rate on bioactive metabolite production were studied using a flask scale with varying single parameter. The active substanceswere purified by adsorption chromatography using Silica gel column and Sephadex LH 20 column, and the physical, chemical, and biological propertieswere characterized.Results: The metabolite production by P. alni was greatly influenced by various cultural conditions. It produced high levels of the antimicrobialsubstance in International Streptomyces project-2 broth compared with that in potato dextrose broth. The optimum parameters for antimicrobialproduction from the actinobacterium occurred in the production medium consisting of glucose (1%) and tryptone (1%), 0.001 M of K2HPO4 and0.05M glycine at initial pH 8.5 and incubated at 30°C for 4 d in stand incubator. The higher concentration of phosphate buffer salts (˃0.01M) repressedthe bioactive production. The purified active substance had relative factor Rf=0.53 in the mobile phase of a thin layer chromatography system andthe maximum absorbance (λ max) at 216 nm. The results of infrared spectra Fourier transform-infrared spectroscopy analysis indicate that it may beregarded to glycopeptide antibiotic. The purified substance had antibacterial and antifungal activities as well as cytotoxic activity in breast cancer cellline Michigan Cancer Foundation-7 and normal hepatic cell line (WRL-68) at a percentage up to 23.7% and 7.64%, respectively.Conclusions: The actinobacterium P. alni was a novel strain having the ability to produce antimicrobial and anticancer substances.Keywords: Pseudonocardia alni, Antifungal–antibacterial, Anticancer optimization, Production, Purification, Characterization.