Çeşitli habitatlardan izole edilen endosporlu Basillerin antimikrobiyal aktivite açısından taranarak metabolitlerin saflaştırılması
Screening and purification for biological active metabolites of endospor forming bacilli isolated from different habitats
Yazar: FERDAĞ ÇOLAK
Danışman: DOÇ. DR. SEMRA İLHAN
Eskişehir Osmangazi Üniversitesi / Fen Bilimleri Enstitüsü / Biyoloji Ana Bilim Dalı
Derece: Doktora, 2006
Konu: Biyoloji = Biology

Özet:
ÇE TL HAB TATLARDAN ZOLE ED LEN ENDOSPORLU BAS LLER NANT M KROB YAL AKT V TE AÇISINDAN TARANARAK METABOL TLER NSAFLA TIRILMASIFERDA ÇOLAKÖZETAntibiyotikler mikroorganizmalar taraf ndan üretilen, yüksek seyreltmelerde di ermikroorganizmalar n büyümesine antagonistik etki yapan maddeler olarak tan mlanmaktad r.Çoklu ilaç direncine sahip bakteriler birden fazla antibiyoti e dirençli olanlard r vemikroorganizmalar n dirençli hale geldikleri antibiyotiklerden art k etkilenmezler.Çal mam zda çe itli habitatlardan 414 endospor olu turan basil izole edilmi tir. Buizolatlar n aktif metabolit aç s ndan tarama i lemi iki a amada gerçekle tirilmi tir. Birincia amada piece agar yöntemi kullan lm ve ikinci a amada ise klinik izolatlara kargösterdi i aktivite piece agar ve disk difüzyon yöntemine göre yap lm t r. %kinci taramadaizolatlar n % 23'ü en az ndan bir klinik izolata kar aktivite göstermi tir.Seçilen izolatlar VITEK sistem ile identifiye edildi inde 4'ü Bacillus subtilis, 4'ü B.cereus, 2'si B. amyloliquefaciens, Ya asidi profillerine (FAME) göre identifiye edildi inde7'si B. cereus, 1'i B. amyloliquefaciens, 2'si Paenibacillus macerans olarak tan mlanm t r.S cakl k, çalkalama h z , pH, karbon kayna , azot kayna ve mineral madde tipininseçilen izolat n geli imi ve antibakteriyal ve antifungal metabolit üretimi üzerine etkisiresponce surface modeline göre incelenmi tir. 200 rpm çalklama h z nda, 35-42 ºC'de ve pH7 de en fazla hücre geli imi ve metabolit üretimi görülmü tür. Modele göre karbonkayna n n en önemli faktör oldu u bulunmu tur.Endasimbiyotik stain (P. macerans) taraf ndan üretilen metabolitler etil asetat ileekstrakte edilerek ince tabaka kromotografisi ve kolon kromoto rafisi ile safla t r lm t r.Aktif maddenin etkisi minimum inhibisyon konsantrasyonu ve minimum sidalkonsantrasyonu ile de erlendirilmi tir.Anahtar Kelimeler: Bacillus, antimikrobiyal aktivite, FAME, surface responce, VITEK,%TK, KK, M%K

Abstract:
SCREENING AND PURIFICATION FOR BIOLOGICAL ACTIVEMETABOLITES OF ENDOSPOR FORMING BACILLI ISOLATED FROMDIFFERENT HABITATSFERDA ÇOLAKSUMMARYAntibiotics have been defined as substances produced by microorganisms that,in high dilution, are antagonistic to the growth or life of other microorganisms. Multidrug resistant bacteria are those that are resistant to more than one antibiotic, meaningthat the microorganisms are no longer affected by the antibiotics to which they havebecome resistant.In this study a total of 414 endospor forming bacilli were isolated from differenthabitats. Screening for biological active metabolite was carried out two stage. In firstscreening, 221 active isoltes were obtained using piece agar method, in terms of theirgeneral inhibition effects to some test bacteria, yeast and molds. Second screening ofthe isolates was carried out disc diffusion and piece agar method in terms of theirgeneral inhibition aganist to some multidrug resistant microorganisms. 23% of theisolates shown inhibition effects aganist at least one multi drug resistantmicroorganisms.Ten endospor forming bacillus strains selected were identified as 4 Bacillussubtilis, 4 B. cereus and 2 B. amyloliquefaciens by VITEC system and as 7 B. cereus, 1B. amyloliquefaciens and 2 Paenibacillus macerans according to faty acids profiles(FAME).A surface responce model was used to study the effect of pH, temperature,agitation, carbon source, nitrojen source and mineral matter source on growth of P.macerans and its antifungal and antibacterial metabolite production. 200 rpm agitation,temperature 35-42ºC?de and pH 7 favoured cell growth and prouction metabolite.According to the model it was found that carbon source was the most important factor.The metabolites produced by the endosymbiotic strain (P. macerans) wereextracted with ethyl acetate. Active metabolite was isolated and purified by thin layerchromotogrphy and column chromotography. The effect of active substans on testmicroorganisms was evaluated by minimum inhibition consentration ve minimum cidalconcentration.Key words: Bacillus, antimicrobial activity, FAME, surface responce, VITEK,TLC, CC, MIC