Denizel kökenli mikrobiyal suşlardan ürikaz terapötik enziminin üretimi ve karakterizasyonu
Küçük Resim Yok
Dosyalar
Tarih
2021
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Enzimlerin yüksek afinitede, özgül olarak hedeflerine bağlanarak katalitik aktivite göstermeleri onları terapötik amaçlı kullanım için güçlü bir alternatif haline getirmektedir. Ayrıca enzimin mikrobiyal bir kaynaktan üretiminde proses optimizasyonu ile kısa sürede yüksek verimli ve maliyet etkin üretimleri de önemli bir avantaj oluşturmaktadır. Tez çalışmasında, denizel kökenli mikroorganizmaların (aktinomiset, bakteri ve fungus izolatları) ürikaz aktivitesi için taranması, aday üreticilerin proteinaz K dirençlerinin belirlenmesi ve seçilen izolata ait ürikazın saflaştırılarak kısmi karakterizasyonunun yapılması amaçlanmıştır. Bu kapsamda; ilk aşamada, 101 bakteri, 106 aktinomiset ve 94 fungus olmak üzere toplam 301 adet denizel kökenli mikroorganizma ürikaz aktivitesi açısından taranmış ve seçilen iyi üreticiler (MF75, MF77, MF21, 6SM128, 6SM85, 6.6.14, 6.9.14, 6.9.9) ile küçük ölçek fermantasyon çalışmalarına geçilmiştir. Ardından hücre içi ve hücre dışı ürikaz aktiviteleri ve proteinaz K direnç özellikleri incelenmiştir. Elde edilen sonuçlara göre, hücre dışı üretilen, proteinaz K direncine sahip (%90) ve görece yüksek aktivite veren 6SM85 kodlu aktinomiset izolatı ile ileri çalışmalara geçilmiştir. Küçük ölçek fermantasyon çalışmalarında iki farklı substrat (sükroz ve maltoz) kullanılmış ve maltoz ile daha yüksek enzim aktivitesi elde edilmiştir. Enzimin saflaştırılmasında sırasıyla; %70 amonyum sülfat çöktürme, diyaliz, iyon değişim kromatografisi (DEAE-Selüloz) basamakları uygulanmıştır. Enzim, iyon değişim kromatografi kolonundan elüsyon sonucu %1,23 verimlilik ile 3,6 kat saflaştırılmış ve moleküler ağırlığının 14 kDa olduğu belirlenmiştir. Kısmi karakterizasyon çalışmaları ile ürikazın optimum aktivite gösterdiği sıcaklık ve pH değeri 35°C, pH 8,5 olarak belirlenmiştir. Enzimin yarım saat sonunda, 40oC’de ~%46 aktivitesini koruyabildiği ve nötr / bazik pH değerlerinde aktivitesini korumada asidik pH değerlerinden daha iyi olduğu belirlenmiştir. 16S rDNA dizi analizi ile izolatın Streptomyces rochei olduğu belirlenmiştir.
Enzymes show catalytic activity with high affinity, specifically binding to their targets, making them a powerful alternative for therapeutic use. In addition, process optimization in the production of the enzyme from a microbial source and high efficiency and cost-effective production in a short time constitute an important advantage. In the thesis study, it was aimed to screen the marine microorganisms (actinomycetes, bacteria and fungus isolates) for uricase activity, to determine the proteinase K resistance of the candidate producers and to purify and partially characterize the uricase of the selected isolate. In this context, in the first stage, a total of 301 marine-derived microorganisms, 101 bacteria, 106 actinomycetes and 94 fungi, were screened for uricase activity and small-scale fermentation studies were started with selected good producers (MF75, MF77, MF21, 6SM128, 6SM85, 6.6.14, 6.9.14, 6.9.9). Then, intracellular and extracellular uricase activities and proteinase K resistance properties were investigated. According to the results, further studies were started with the actinomycetes isolate (6SM85), which is extracellularly produced, resistant to proteinase K (90%) and giving relatively high activity. In small scale fermentation studies, two different substrates (sucrose and maltose) were used and higher enzyme activity was obtained with maltose. In the purification of the enzyme, respectively; 70% ammonium sulfate precipitation, dialysis, ion exchange chromatography (DEAE-Cellulose) steps were applied. The enzyme was purified 3.6 times with 1.23% efficiency after elution from the ion exchange chromatography column and its molecular weight was determined as 14 kDa. With partial characterization studies, the optimum temperature and pH value of uricase were determined as 35°C and pH 8.5. It was determined that the enzyme was able to maintain ~46% activity at 40C after half an hour and was better at maintaining its activity at neutral / basic pH values than at acidic pH values. The isolate was identified as Streptomyces rochei by 16S rDNA sequencing.
Enzymes show catalytic activity with high affinity, specifically binding to their targets, making them a powerful alternative for therapeutic use. In addition, process optimization in the production of the enzyme from a microbial source and high efficiency and cost-effective production in a short time constitute an important advantage. In the thesis study, it was aimed to screen the marine microorganisms (actinomycetes, bacteria and fungus isolates) for uricase activity, to determine the proteinase K resistance of the candidate producers and to purify and partially characterize the uricase of the selected isolate. In this context, in the first stage, a total of 301 marine-derived microorganisms, 101 bacteria, 106 actinomycetes and 94 fungi, were screened for uricase activity and small-scale fermentation studies were started with selected good producers (MF75, MF77, MF21, 6SM128, 6SM85, 6.6.14, 6.9.14, 6.9.9). Then, intracellular and extracellular uricase activities and proteinase K resistance properties were investigated. According to the results, further studies were started with the actinomycetes isolate (6SM85), which is extracellularly produced, resistant to proteinase K (90%) and giving relatively high activity. In small scale fermentation studies, two different substrates (sucrose and maltose) were used and higher enzyme activity was obtained with maltose. In the purification of the enzyme, respectively; 70% ammonium sulfate precipitation, dialysis, ion exchange chromatography (DEAE-Cellulose) steps were applied. The enzyme was purified 3.6 times with 1.23% efficiency after elution from the ion exchange chromatography column and its molecular weight was determined as 14 kDa. With partial characterization studies, the optimum temperature and pH value of uricase were determined as 35°C and pH 8.5. It was determined that the enzyme was able to maintain ~46% activity at 40C after half an hour and was better at maintaining its activity at neutral / basic pH values than at acidic pH values. The isolate was identified as Streptomyces rochei by 16S rDNA sequencing.
Açıklama
Anahtar Kelimeler
Terapötik Enzim, Denizel Kaynaklı Mikroorganizma, Ürikaz, Streptomyces Rochei, Proteinaz K Direnci, Karakterizasyon, Therapeutic Enzyme, Marine Microorganism, Uricase, Streptomyces Rochei, Resistant to Proteinase K, Characterization
