Drug Discovery in Soil Bacteria from Unique Ecosystems of India and Indonesia Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/8k71nk003

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  • Natural products isolated from bacteria can be developed into new therapeutic agents. The soil bacteria actinomycetes, specifically the Streptomyces species, are prolific producers of antibiotics. This thesis is composed of two projects which focus on novel drug discovery from soil bacteria collected from the Indonesian Black Water Ecosystem (the ICBB strains) and soil bacteria from the abandoned Hundung Cement Factory in India (the MBRL 201 and 251 strains). The ICBB strains were cultured in Yeast Peptone Glucose Malt Extract and Modified Bennet media and the MBRL strains were cultured in Yeast Malt Extract Glucose medium. Following a week of cultivation, the culture broths were centrifuged. The supernatant was extracted sequentially with EtOAc and BuOH. The remaining aqueous solution after the extraction process was lyophilized and prepared for biological activity testing. The mycelia of the MBRL strains were also subjected to further extraction using a mixture of MeOH and acetone. Biological assays were carried out against several microbial and fungal organisms. Active extracts from the ICBB strains were noted, but due to time constraints no follow-up experiments were carried out. On the other hand, the active extracts from the MBRL strains were extensively investigated using bioassay-guided isolation and purification. On the basis of the sequence of their 16S rRNA genes, MBRL 201 and 251 strains were identified to be Streptomyces sp. and Pseudomonas sp., respectively. Active extracts from these strains were subjected to bioassay-guided fractionations. The MBRL 251 BuOH extract and the MBRL 201 EtOAc extract were investigated most extensively because the TLC and bioassay results indicated the presence of bioactive natural products in these extracts. The MBRL 251 BuOH extract was fractionated and purified with a series of open column chromatography. The active compound was analyzed using mass spectrometry, ¹H and ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy. In addition, ANTIBASE®, a searchable database that contains descriptive, physico-chemical and spectroscopic data, was used to quickly identify known compounds. The results revealed that the bioactive compound in MBRL 251 is phenazine-1- carboxylic acid. The MBRL 201 extract was fractionated with preparative thin layer chromatography and purified with size exclusion chromatography. However, at this time, further experiments are necessary to determine the chemical structure of the active MBRL 201 compound.
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