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Studies On The Bioactive Compounds Of Cyclopeptides From Microbes Of East China Sea

Posted on:2008-04-25Degree:DoctorType:Dissertation
Country:ChinaCandidate:H P ZhuFull Text:PDF
GTID:1100360218458835Subject:Biochemistry and Molecular Biology
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The world's oceans are endowed with tremendous bio-resources and marineenvironment provide the primary and important site for bioprospecting. First, morethan 70%of the planet is covered by seas and oceans, most of which are deeper than2,000 m, and the marine environment is far from being uniform but compriseshydrothermal vents, cold and hydrocarbon seeps, saturated brines, carbonate mounds,mineral nodule fields and, most commonly, sediments of various geological originand depths. Second the accepted view' that life first evolved in the oceans withinwhich is found the greatest degree of biodiversity. Most importantly the enormousbiodiversity in the oceans appears to reflect a similarly extensive chemical diversity.Recent drugs screening projects based on marine actinomycetes, for example, haveuncovered numerous novel chemical structures with a discovery rate that is greaterthan that from their terrestrial relatives. Therefore, the marine microorganisms haveattracted considerable attention because of chemical unique, rich physiologicalproperties and thus potential as important drugs of their metabolites.Among our four sea area, the Bohai Sea, the Yellow Sea and the South China Seahave been researched much of their microbial resources, but there is little report aboutlarge scale and large extent resource investigation, active strains screening of theChina East Sea area. In the past five years our team collected 28 sea water and seasediment samples 35 nautical miles far from the seacoast of the China East Sea, and1043 microorganism cultures were isolated from them with dilution and plate streaktechnique. With Pyricularia oryzae as primary activity screening model, 216 cultureswith potent inhibition on Pyricularia oryzae were obtained. In this dissertation, astrain with better activity, F9947, was genomic identified based on its 16S rDNA.Various physiological and biochemical aspects were characteried. Its optimumfermentation conditions were studied. After large scale fermentation, its secondarymetabolites were isolated and purified, their structures were elucidated byspectrometry (NMR, MS etc)and their bioactivities were also tested in differentmodels. The result of the presentation shows that the strain F9947 was a lower halophilicmicrobe and its most optimum salt concentration is 2.5%. Further study showed thatthe activity reachs up to the peak on the condition of 25℃, with the shake frequencyof 120 r/min and the original fermentation pH value of 6.0, the inoculation dose of 5%(V/V) and harvest at 72 to 96 hours after fermentation. The fermentation mediumingredients include 0.4%of peptone, 0.4%of yeast extraction, 1%of beef cream and1%of glucose.The 16S rDNA amplification and sequence alignment and evolutionary treeestablishment showed that the strain F9947 was most intimate with Bacillus subtilisBFAS on the tree and had a identity of 99.67%with it, so F9947 was identified asBacillus subtilis. The study of physiological and biochemical characteristics alsodemonstrated that this strain had some same essential sepects.with the terrene Bacillussubtilis.According to the optimum fermentation conditions, 30 liter culture broth wereharvested. After centrifugation, the thallis were removed, and the broth showed goodinhibition ability aganist Pyricularia oryzae, the maximum active dilution was 1:256.The 30 liter broth was extracted by the same volume of Ethyl Acetate and n-Butanolin turn respectively three times, then boiled to dry and 10.3 gram of Ethyl Acetateextract and 48.2 gram n-Butanol extract were obtained. Being checked by Pyriculariaoryzae, their minimum active concentration were 7.8μg/mL and 0.98μg/mL. TheEthyl Acetate extract were fractionated by silicon column chromatograghy repeatedly,and eluted by the gradient of chloroform and methanol and then be purified by highperformance liquid chromatagroghy (HPLC), 28 compounds were obtained in all.Using nuclear magnetic resonance (NMR) and mass spectrometry (MS) technology,and complemented with routine physical and chemical methods, 13 compounds wereelucidated. They were cyclo(D-Pro-D-Ile), cyclo(L-Pro-L-Val), cyclo(L-Pro-D-Leu),cyclo(L-Val-L-Leu), cyclo(D-Pro-D-Phe), cyclo(L-Ala-L-Ile), cyclo(L-Ala-L-Leu),cyclo(Gly-Tyr), Uridine,cyclo(L-Pro-L-Ala), cyclo(L-Ala-L-Phe), cyclo(Gly-Ala) andcyclo(Ala-Gly-Ser-Val-Gln-Tyr-Pro-Asn-Gly-Asn-Gly).Cyclo(Ala-Gly-Ser-Val-Gln-Tyr-Pro-Asn-Gly-Asn-Gly) is a novel firstly isolated compound, with inhibiting activity against pancreatic cancer cell 1990 and livercancer cell hul-7, their ED50 were 2.12 and 3.22μg/mL, respectively. All of thecyclopeptides showed strong anti- Pyricularia oryzae ability, the MIC<1μg/mL.Being reported, cyclo(D-Pro-D-Ile), cyclo(L-Pro-D-Leu) and cyclo (D-Pro-D-Phe)had strong anti-Vibrio anguillarm abiiity, their MICs were 0.05, 0.12 and 0.03μg/mL,respectively; cyclo(L-Pro-L-Val)had antibiotic and cytotoxic activity, and cyclo-(L-Ala-L-Leu)had virus-static activity.
Keywords/Search Tags:Marine microorganism, Pyricularia oryzae, Fermentation, 16S rDNA, HPLC, Cyclopeptide, Structure elucidation, Cytotoxicity
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