Isolation And Identification Of Actinomyces From Soil And Analysis Of Their Metabolites

Because of their production of antibiotics, enzymes and their inhibitors and other useful biologically active substances, actinomycetes has great economic value, which has found 20,000 kinds of bioactive substances, about 70% is produced by the actinomycetes. The soil is the most abundant actinomycetes place, so development of actinomycetes in soil is of great significance. Screening of multi-antibiotic resistances actinomycetes with selective medium is an efficient method with strong purpose 3 kinds of antibiotics were useed for screen the actinomycetes isolated from soil samples in this work, in order to provide a convenient and efficient way for strain screening works, at the same time develop more microbial pesticides. The detailed studies as follows:(1) Isolation of actinomyces. Selected five soil samples, which came from Dazhou city in Sichuan Province, Wuhan City in Hubei Province, Linyi City in Shandong Province, Haicheng city in Liaoning Province, Harbin City in Heilongjiang Province, the five areas of natural drying of soil samples after pretreatment with selective isolation medium humic medium wih 20μg/mL nalidixic acid for the selective isolation of actinomycetes. A total of 54 strains were isolated from soils.(2) Screening of high activity strains. Streptomycin, gentamicin and rifampin triple-antibiotic resistance selection method were obtained for further screening. Eventually the activity of four actinomycetes is very high, which were strain-10, strain-49, strain-50 and strain-52, and further study was carried out.(3) Fermentation of crude extracts of bacteria and fungi inhibition. The four actinomycete fermentation extract were carried out on two kinds of pathogenic bacteria and 9 fungis for screening antimicrobial activity, the results showed that the inhibition zone of bacteria reached more than 20mm, and the inhibitory activity of 9 fungus also showed varying degrees, and some even reached to 95%.(4) The herbicidal activity of metabolites. Also were two kinds of herbicide target enzyme and two weeds were used for screening the activity of actinomyces. The results showed that the fermentation extract of strain-10 can inhibit asparagine synthetase, and had a very strong inhibition of Echinochloa crusgall and Abutilon theophrasti medic, the fermentation extracts of strain-50 can inhibit acetyl-carboxylic acid synthase, and had a very strong inhibition of Abutilon theophrasti medic. (5) The thermal stability of metabolites. The thermal stability of fermentation extracts by these four actinomycetes were carried out , the results was that the temperature stability of strain-10, strain-50 and strain-52 were better, when the temperature to 90℃, they also has strong antibacterial activity; the temperature stability of strain-49 was weak, when the temperature reached to 30℃, its antibacterial activity was completely lost.(6) The taxonomy of actinomyces. Combined with classical taxonomy and molecular biology 16SrDNA classification, these four categories of actinomycetes with its morphology, cultural characteristics, physiological and biochemical characteristics were consistent with Streptomyces; strain-10 with the genus of Streptomyces lavendulae 99% similarity; strain-49 with 99% similarity of Streptomyces violaceolatus; strain-50 with 99% similarity of Streptomyces olivaceus; strain-52 with 100% similarity of Streptomyces pactum strain 173848. Further confirm that four actinomycetes belong to the genus of Streptomyces.(7) Isolation of metabolites. Fermentation extracts of these four actinomycetes were isolated, purified and identified. The results showed that six kinds of compounds were isolated from strain-10; the compound fogacin was isolated from strain-49; the anti-cancer, antibacterial, herbicidal activity and other uses of the active compound borrelidin was isolated from strain-50; the separation process of the fermented extracts of strain-52 was under way.(8) The proof of compounds. A compound borrelidin herbicidal and antibacterial activity test, results showed that the compound borrelidin has a strong herbicidal activity and antibacterial activity.