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Çukurova Üniversitesi Fen Bilimleri Enstitüsü ÇUKUROVA ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ DOKTORA TEZİ Ashabil AYGAN HALOALKALOFİL BACILLUS SP. İZOLASYONU, AMİLAZ, SELÜLAZ VE KSİLANAZ ENZİMLERİNİN ÜRETİMİ, KARAKTERİZASYONU VE BİYOTEKNOLOJİK UYGULAMALARDA KULLANILABİLİRLİĞİ BİYOLOJİ ANABİLİM DALI ADANA, 2008 ÇUKUROVA ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ HALOALKALOFİL BACILLUS SP. İZOLASYONU, AMİLAZ, SELÜLAZ VE KSİLANAZ ENZİMLERİNİN ÜRETİMİ, KARAKTERİZASYONU VE BİYOTEKNOLOJİK UYGULAMALARDA KULLANILABİLİRLİĞİ Ashabil AYGAN DOKTORA TEZİ BİYOLOJİ ANABİLİM DALI Bu tez 04/01/2008 Tarihinde Aşağıdaki Jüri Üyeleri Tarafından Oybirliği İle Kabul Edilmiştir. İmza ………………… İmza …………………. İmza ………………………. Prof. Dr. Burhan ARIKAN Prof. Dr. Ömer ÇOLAK Doç.Dr.Gökhan CORAL DANIŞMAN ÜYE ÜYE İmza .............................. İmza ………………… Doç.Dr. Hatice Kormaz Doç.Dr.Mutlu Nisa Ünaldı Güvenmez Coral ÜYE ÜYE Bu Tez Enstitümüz Biyoloji Anabilim Dalında hazırlanmıştır. Kod No: Prof. Dr Aziz ERTUNÇ Enstitü Müdürü İmza-Mühür Bu çalışma Ç.Ü. Bilimsel Araştırma Projeleri Birimi Tarafından Desteklenmiştir. Proje No: FBE 2003-D-18 Not: Bu tezde kullanılan özgün ve başka kaynaktan yapılan bildirişlerin, çizelge, şekil ve fotoğrafların kaynak gösterilmeden kullanımı, 5846 sayılı Fikir ve Sanat Eserleri Kanunundaki hükümlere tabidir. ÖZ DOKTORA TEZİ HALOALKALOFİL BACILLUS SP. İZOLASYONU, AMİLAZ SELÜLAZ VE KSİLANAZ ENZİMLERİNİN ÜRETİMİ, KARAKTERİZASYONU VE BİYOTEKNOLOJİK UYGULAMALARDA KULLANILABİLİRLİĞİ Ashabil AYGAN ÇUKUROVA ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ BİYOLOJİ ANABİLİM DALI Danışman: Prof. Dr. Burhan ARIKAN Yıl: 2008, Sayfa:186 Jüri:Prof. Dr. Ömer ÇOLAK Doç.Dr. Hatice KORKMAZ GÜVENMEZ Doç.Dr. Gökhan CORAL Doç.Dr. Mutlu Nisa ÜNALDI CORAL Bu çalışmada, Van Gölü topraklarından izole edilen haloalkaloflik Bacillus sp. suşlarından amilaz, selülaz ve ksilanaz enzimi izolasyonu ve karakterizasyonu gerçekleştirilmiştir. Bu amaçla bakterilerin besiyerlerinde üreme ve enzim üretme yetenekleri ve aktivite gösterdiği optimum sıcaklık, pH ve NaCl konsantrasyonu saptanmıştır. Bacillus sp. AB-17 suşundan üretilen amilazın SDS-PAGE analizinde iki band elde edilmiş ve moleküler ağırlıkları 66.25 ve 58 kDa olarak belirlenmiştir. Enzim, optimum aktivitesini 150ºC ve pH 10.5’da gösterirken, 0.5-3.4M NaCl’de ortalama %67’lik aktivite ile halofil özellikte olduğu saptanmıştır. Bacillus sp.C-14 suşundan izole edilen endoglukanaz enzimin molekül ağırlığı 61 kDa olarak belirlenmiş, optimum aktivite ise 50°C’de pH 11.0’de görülmüştür. Maksimum enzim aktivitesi %20 (3.4M) NaCl konsantrasyonunda (%133) elde edilmiştir. Bacillus sp X13 suşundan elde edilen ksilanaz enziminin optimum aktivitesi 40ºC de pH 6.0’da gerçekleşmiştir. Enzim pH 5.0-6.0 aralığında 15 dk süreyle aktivitesini %100 koruyabilmiş, SDS-PAGE analizinde ise moleküler ağırlıkları 108.4, 95.5, 80.6 ve 68.5 kDa olan dört band tespit edilmiştir. Bu sonuçlara göre AB-17 amilaz enziminin Nişastanın sıvılaştırılması ve tekstil endüstrisinde nişastanın uzaklaştırılmasında kullanılabilecek bir enzim olduğu belirlenmiştir. C14 endoglukanaz enziminin tuzlu ortamlarda yüksek düzeyde aktivite göstermesi, halo-stabilitesinin yüksek olması ve deterjanlara dirençli olması bu enzimin tekstil uygulamaları, kağıt endüstrisi ve hayvan yemi üretimi gibi uygulamalarda kullanılabileceğini göstermektedir. X13 Ksilanaz enzimi ise besin endüstrisinde prebiyotik üretiminde ve selülaz ve pektinaz enzimine gerek kalmadan meyve suyu üretiminde kullanılabileceği önerilebilir. Anahtar Kelimeler: Bacillus, amilaz, selülaz, ksilanaz,, haloalkalofil I ABSTRACT Ph.D. THESIS ISOLATION OF HALOALKALOPILIC BACILLUS SP., PRODUCTION, CHARACTERIZATION AND DETERMINATION OF BIOTECHNOLOGICAL APPLICATION OF THEIR AMYLASE, CELLULASE AND XYLANASE Ashabil AYGAN DEPARTMENT OF BIOLOGY INSTITUTE OF NATURAL AND APPLIED SCIENCES UNIVERSITY OF CUKUROVA Supervisor:Prof. Dr. Burhan ARIKAN Year: 2008, Page: 186 Jury: Prof. Dr. Ömer ÇOLAK Doç.Dr. Hatice KORKMAZ GÜVENMEZ Doç.Dr. Gökhan CORAL Doç.Dr. Mutlu Nisa ÜNALDI CORAL In this study, the amylase, cellulase, xylanase enzymes from Bacillus sp. strains isolated from Van lake soil were produced and characterized. The strains were tested for determination of temperature, pH range and NaCl concentration for growth and enzyme production on plate. Molecular weight of the amylase from Bacillus sp.AB-17 was estimated as 66.25 and 58 kDa on SDS-PAGE. The optimum activity was obtained at 150oC, pH 10.5. the enzyme also presented halophilic properties with an activity around 67% between 0.5-3.4M NaCl concentration. Molecular weight of the Endoglucanase from Bacillus sp.C14 was detected as 61kDa on SDS-PAGE, and the optimum enzyme activity was obtained at 50oC, pH 11.0. Maximum endoglucanase activity (%133) was recorded at 20% (3.4M) NaCl. On the other hand the xylanase from Bacillus sp.X13 presented and optimum activity at 40oC and pH 6.0. The enzyme was 100% stable between pH 5.0-6.0 for 15 min. The enzyme was consist of 4 unit as 108.4, 95.5, 80.6 ve 68.5 kDa on SDS-PAGE. The results showed that the amylase AB-17 is a relevant enzyme for desizing processes in textile industries. The increased activity of C14 endoglucanase in high saline conditions, high halo- stability, resistance to detergents makes the enzyme C14 endoglucanase appropriate for textile, paper, animal feed preparation processes. X13 Xylanase enzyme could also be used for prebiotic preparation in food industries and fruit juice production without needing pectinase and cellulase usage. Key Words: Bacillus, amylase, cellulase, xylanase, haloalkalophile II TEŞEKKÜR Çalışmamın her aşamasında bana yardımcı olan, bilgi ve deneyimleriyle yol gösteren danışman hocam sayın Prof. Dr. Burhan ARIKAN’a, Moleküler Biyoloji Anabilim Dalı Başkanı sayın Prof.Dr.Ömer ÇOLAK’a, sayın Prof.Dr.Sadık DİNÇER’e ve Doç.Dr.Hatice KORKMAZ GÜVENMEZ’e çok teşekkür ederim. Bazı suşların dizi analizlerinde yardımlarını esirgemeyen Dr.A.Akın DENİZCİ’ye, bölüm teknisyeni sayın Mustafa TOMAK’a ve bölüm sekreteri sayın Mediha KURTOĞLU’na teşekkür ederim. Ayrıca bütün çalışmalarım süresince manevi desteğini esirgemeyen annem Meliha, eşim Arife ve kızım Irmak Zehra AYGAN’a sonsuz teşekkür ederim. III İÇİNDEKİLER SAYFA NO ÖZ........................................................................................................I ABSTRACT.........................................................................................II TEŞEKKÜR.........................................................................................III İÇİNDEKİLER.....................................................................................IV ÇİZELGELER DİZİNİ.........................................................................XII ŞEKİLLER DİZİNİ ..............................................................................XIV SİMGELER VE KISALTMALAR.......................................................XVI 1.GİRİŞ................................................................................................1 1.1.Enzimler ve Bazı Özellikleri........................................................3 1.2. Enzimlerin Sınıflandırılması .......................................................5 1.2.1. Enzimlerin Numaralandırılması ve Sınıflandırılması........5 1.3.Amilazlar (Amylase)....................................................................6 1.3.1.Amilazların Bazı Uygulama Alanları................................7 1.4. Selülazlar (Endoglukanaz)..........................................................9 1.4.1.Kompleks Olmayan Selülaz Sistemleri.............................10 1.4.2. Kompleks Selülaz sistemleri (Selülozom)........................11 1.4.3. Selülazların Bazı Uygulama Alanları ...............................13 1.4.3.1. Gıda endüstrisinde Selülazlar...............................13 1.4.3.2. İçecek ve Şarap Endüstrisinde Selülazlar.............14 1.4.3.3. Hayvan Yemi Endüstrisinde Selülazlar................14 1.4.3.4. Tekstil Endüstrisinde...........................................14 1.5. Ksilanazlar (Xylanase)................................................................15 1.5.1. Ksilanazların Bazı Uygulama Alanları ....................17 1.6. Ekstremofilik Enzimler (Ekstremozimler)...................................19 1.7. Bacillus Cinsi..............................................................................21 1.8. Elektroforez Uygulamaları..........................................................22 1.8.1. Jel Elektroforezleri..........................................................24 IV 1.8.2. Sodyum Dodesil Sülfat-Poliakrilamid Jel Elektroforezi (SDS-PAGE).................................................................26 1.8.3. Doğal (Native) Jel Elektroforezi......................................28 1.9. Protein İzolasyonu......................................................................28 1.9.1. Çöktürme ile Proteinlerin İzolasyonu...............................31 1.9.1.1. TCA ile Çöktürme...............................................31 1.9.1.2. Nötral tuzlar ile Proteinlerin Çöktürülmesi...........31 1.9.1.3. Organik Çözücülerle Proteinlerin Çöktürülmesi...33 1.9.1.4. Non-İyonik Organik Polimerlerle Çöktürme........33 1.10. Kromatografi............................................................................34 1.10.1. İnce Tabaka Kromatografisi (TLC)........................................35 1.11. Araştırmanın Amacı..................................................................37 2. ÖNCEKİ ÇALIŞMALAR.................................................................39 3. MATERYAL VE METOD...............................................................49
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