Production, Purification, and Characterization of Bioactive Metabolites Produced from Rare Actinobacteria Pseudonocardia Alni
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Online - 2455-3891 Vol 9, Suppl. 3, 2016 Print - 0974-2441 Research Article PRODUCTION, PURIFICATION, AND CHARACTERIZATION OF BIOACTIVE METABOLITES PRODUCED FROM RARE ACTINOBACTERIA PSEUDONOCARDIA ALNI RABAB OMRAN1*, MOHAMMED FADHIL KADHEM2 1Department of Biology, College of Science, University of Babylon, Babel, Al-Hillah, Iraq. 2Department of Pharmacology, Ibn Hayyan College, Karbala, Iraq. Email: [email protected], [email protected] Received: 30 August 2016, Revised and Accepted: 12 September 2016 ABSTRACT Objectives: Pseudonocardia alni exhibits antimicrobial activity against tested pathogenic Staphylococcus aureus, Microsporum canis, and Trichophyton mentagrophyte. The present paper aimed to optimize various cultural conditions for antimicrobial metabolite production, purification, and characterization of the active substance. Methods: The effects of various parameters such as culture media, carbon and nitrogen sources, phosphate concentration, pH, temperature, incubation period, and agitation rate on bioactive metabolite production were studied using a flask scale with varying single parameter. The active substances were purified by adsorption chromatography using Silica gel column and Sephadex LH 20 column, and the physical, chemical, and biological properties were characterized. Results: The metabolite production by P. alni was greatly influenced by various cultural conditions. It produced high levels of the antimicrobial substance in International Streptomyces project-2 broth compared with that in potato dextrose broth. The optimum parameters for antimicrobial production from the actinobacterium occurred in the production medium consisting of glucose (1%) and tryptone (1%), 0.001 M of K2HPO4 and the bioactive production. The purified active substance had relative factor Rf=0.53 in the mobile phase of a thin layer chromatography system and 0.05M 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) repressed regarded to glycopeptide antibiotic. The purified substance had antibacterial and antifungal activities as well as cytotoxic activity in breast cancer cell the maximum absorbance (λ max) at 216 nm. The results of infrared spectra Fourier transform-infrared spectroscopy analysis indicate that it may be Conclusions:line Michigan CancerThe actinobacterium Foundation-7 P.and alni normal was a hepaticnovel strain cell line having (WRL-68) the ability at a topercentage produce antimicrobial up to 23.7% and and 7.64%, anticancer respectively. substances. Keywords: Pseudonocardia alni, Antifungal–antibacterial, Anticancer optimization, Production, Purification, Characterization. © 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons. org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2016.v9s3.14961 INTRODUCTION optimizing the fermentation conditions to achieve the maximum yield of antibiotic production from the rare actinobacterium P. alni, as well as Most of the antimicrobial drugs in use today are derivatives of purification and characterization of an antimicrobial substance. natural products of actinomycetes and fungi. Antibiotics produced by microorganisms have been evolving for one billion years [1]. The activity METHODS of antibiotics is estimated against pathogenic microbes based on their ability to inhibit target macromolecules and biosynthesis pathways. On Bacterial strains the other hand, these microorganisms develop resistance systems to P. alni was previously isolated from soil in Biotechnology Laboratory survive, therefore the screenings for the new antimicrobial producer had the ability to produce are endless [2,3]. Although the successive discoveries of antibiotics antimicrobial substances against pathogenic bacteria and fungi, resulted in increasing the number of known antibiotics reaching to includingat Babylon StaphylococcusUniversity, Iraq aureus[7], and, Escherichiait coli, Morganella 22500, only about 150 antibiotics are in direct use in the human and morganii, Microsporum canis, and Trichophyton mentagrophyte veterinary treatment and agricultural applications. About 45% of these The pure cultures were maintained in AMES medium at 4oC for further metabolites are produced from actinomycetes such as Streptomyces sp., studies. [7]. and rare actinobacteria [4]. Antibiotic production by microbial strains Pathogenic isolates S. aureus and T. mentagrophyte were obtained cancomprising be significantly 74% and influenced the rest containing by different other nutrition actinomycetes and cultivation species from Biotechnology Laboratory to use as a test organism in all conditions. Therefore, the medium constitutions together with the experiments which were performed in the same laboratory at metabolic capacity of the producing microorganism greatly have an Babylon University. impact on bioactive metabolite synthesis. Several environmental factors, such as temperature, pH, aeration, agitation, and incubation Optimization of production parameters The effect of various parameters, including culture media, carbon In a previous study, Pseudonocardia alni as a rare actinobacterium was and nitrogen sources, phosphate concentration, pH, temperature, isolatedperiod, play from a amajor soil sample, role in thewhich production was identified of antimicrobial depending agents on cultural, [5,6]. incubation times, and an agitation rate of production medium, on physiological, and biochemical characteristics. This isolate had the bioactive metabolite production, was studied by varying a single parameter at a time. All experiments were performed in 250 ml objectives of this study were formulating a production medium and conical flasks, each one containing 50 ml of variant tested medium in ability to produce antifungal and antibacterial metabolites [7]. The Omran and Kadhem Asian J Pharm Clin Res, Vol 9, Suppl. 3, 2016, 264-272 triplicate. All media were sterilized by autoclave at 121°C for 20 min. Initial pH of production medium The flasks were inoculated with 1 ml of P. alni inoculum. The inoculum The optimal initial pH of the medium for maximum antibiotic was prepared as a seed culture that was grown under optimal growth production was determined by preparing fermentation media, in conditions (International Streptomyces project [ISP-2] broth at pH8 and incubated at 30°C for 3 d). triplicate, with 0.05 M available buffer, including citrate buffer at pH 6 Production media formulation aand seed 6.5, culture Tris buffer of P. alniat pH 7, 7.5, and 8, and glycine buffer at pH 8.5, P. alni was grown separately in two sterilized media, the first, potato antimicrobial9, 9.5, and 10 activity[3,4]. Fermentation of fermentation media filtrates were wasinoculated tested withby agar 1 ml well of aged 3 d and incubated at 30°C for 7 d. The dextrose broth (PDB) which was prepared from 2% glucose and 20% Optimumdiffusion method incubation [9]. temperature thepotato supernatant extract (20 was g supplemented of potato slice with boiled 2 g glucose, in 90 ml and distilled finally thewater pH To determine the optimum temperature for antibiotic production, the (DW) for 10 min, after cooling to room temperature, it was filtrated, 2HPO4 and KH2PO4 optimal fermentation media in triplicate were inoculated with 1 ml of a to reach final concentration 0.01 M, and the volume was completed to seed culture of P. alni aged 3 d and incubated at different temperatures was adjusted to 7.5 using phosphate buffer salts (K which was prepared according to the ISP-2, ISP-2 broth comprised activity of fermentation filtrates was determined by agar well diffusion 0.4%100 ml glucose, with DW). 1% Theyeast second extract, medium, and 0.4% yeast malt extract-malt extract dissolved extract in broth, 0.01 from 20°C to 50°C with an interval of 5° for 7 d [5,6]. The antimicrobial Optimummethod [9]. incubation period that,M of phosphatethe cell-free buffer filtered at pH supernatant 7.5 [6,8]. Both was mediaseparated were from inoculated the culture with Optimal period of antibiotic production was determined by inoculating broth1 ml of by a seedcooling culture centrifugation aged 3 d and at 8000×then incubated g for 10 min,at 30°C and for then 7 d. it After was optimal fermentation media, in triplicate, with 1 ml of a seed culture of filtered by Millipore filter (0.22 µm). Antagonistic activity of P. alni P. alni cultural filtrate was estimated against the pathogenic test organism by activity of fermentation filtrates was tested every day by agar well aged 3 d and incubated at 30°C for 10 d [5,6]. The antimicrobial Agaragar well well diffusion diffusion method method [9]. Agitationdiffusion method [9]. Agar well diffusion method was performed according to Perez et al The effect of shaking rate of fermentation media was studied after using Mueller-Hinton agar (MHA) medium for S. aureus determined the incubation period. in triplicate, at shaker incubator T. mentagrophyte. The inoculum of the test organisms was prepared. [9] in different shaking rotations ranging from 40 to 200 rpm, also it separately by mixing three microorganism colonies from the and exponential PDA for is incubated at the stand incubator under optimum fermentation phase with 10 ml of sterile nutrient broth, and the turbidity was compared parameters. The antimicrobial activity of fermentation filtrates was with that of the standard 0.5 McFarland solution which is equivalent to Thermalassayed by stability