Mikroakışkan sistemlerde saponin moleküllerinin biyoaktivite destekli enzimatik hidrolizi
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Dosyalar
Tarih
2018
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Küçük hacimlerdeki sıvıların işlenmesine olanak sağlayan mikroakışkan sistemler geleneksel sistemlere kıyasla yüksek ısı ve kütle transferi, düşük reaktif kullanımı, etkin karıştırma gibi avantajlara sahiptir. Bu tez çalışması kapsamında mikroakışkan sistemlerin sahip olduğu bu özellikleri enzimatik reaksiyonların avantajları ile birleştiren enzimatik mikroreaktörlerin geliştirilmesi ve geliştirilen bu sistemde saponin moleküllerinin hidroliz edilmesi amaçlanmıştır. Bu amaç doğrultusunda β-glukosidaz enzimi silika ve aljinat hidrojeller içerisine immobilize edilmiş ve bu hidrojellerin morfoloji, yüzey alanı, por büyüklüğü, hacim değişimi gibi özellikleri karakterize edilerek serbest ve immobilize enzim için kinetik parametreler (Vmax ve KM) belirlenmiştir. Hidrojellerin karakterizasyonunun ardından enzimatik mikroreaktörler hazırlanmış ve model substrat olan pNPG ile saponin molekülleri olan ginsenosit Rb1, soyasaponin Bb ve soyasaponin Aa moleküllerinin hidrolizi gerçekleştirilmiştir. Gerçekleştirilen karakterizasyon çalışmalarında aljinat hidrojellerin sahip olduğu yüksek yüzey alanı (∼304 m2/g) ve gözenekli yapısı ile enzim immobilizasyonu için oldukça elverişli bir matris olduğu belirlenmiştir. Enzimatik mikoreaktörlerde gerçekleştirilen denemelerde kullanılan immobilizasyon yöntemi ile mikroreaktör içerisinde kalış süresinin reaksiyon verimini doğrudan etkilediği ve artan kalış süresi ile birlikte reaksiyon veriminin de arttığı tespit edilmiştir. Aynı kalış süresi için klasik yöntem olan reaksiyon tüpleri içerisinde serbest (%27) ve immobilize enzim (%16) ile gerçekleştirilen hidroliz denemeleri ile karşılaştırıldığında enzimatik mikroreaktörlerde çok daha yüksek substrat dönüşüm oranları (%37.52) elde edilmiştir. Saponin molekülleri asidik, serbest enzim ve enzimatik mikroreaktör ile hidrolize tabi tutulmuş, asidik hidroliz reaksiyonlarında ara ürünlerin yanı sıra istenmeyen yan ürün oluşumu gözlenirken enzimatik hidroliz reaksiyonlarında herhangi bir yan ürün oluşumunun gözlenmemesi enzimlerin kimyasal katalizörlere olan üstünlüğünü göstermiştir. Ginsenosit Rb1 için 15 dk kalış süresinde serbest enzim ile gerçekleştirilen hidroliz denemelerinde tek şeker molekülünün uzaklaştırılması ile elde edilen ginsenosit Rd molekülü tespit edilirken aynı kalış süresi için enzimatik mikroreaktörde gerçekleştirilen hidroliz sonrasında ginsenosit Rd'nin yanı sıra başlangıç molekülünden iki şeker molekülünün uzaklaştırılması ile oluşan ginsenosit F2 molekülüne de rastlanmıştır. Benzer şekilde soyasaponin Bb'nin serbest enzim ile hidrolizinde soyasaponin III ve soyasapogenol B monoglukuronit ara ürünleri elde edilirken mikroreaktörde gerçekleştirilen enzimatik hidroliz ile aglikon olan soyasapogenol B'ye ulaşılmıştır. Elde edilen bu sonuçlar kimyasal katalizörler ile kıyaslandığında enzimlerin yüksek substrat spesifikliğine sahip olduğu ve geleneksel sistemler ile karşılaştırıldığında enzimatik mikroreaktörlerin oldukça etkin sistemler olduğunu göstermektedir. Buna ek olarak saponin ve aglikonlara ait standartların farklı kanser hücre hatları üzerindeki sitotoksik etkisini belirlemek için MTT analizleri yapılmıştır. Elde edilen sonuçlar soyasapogenollerin soyasaponinlerden daha yüksek sitotoksik aktiviteye sahip olduğunu gösterirken compound K'nın tüm hücre hatları üzerinde sitotoksik etki gösterdiği ve bu etkinin apoptoz ile gerçekleştiği belirlenmiştir.
Microfluidics systems which enable the manipulation of liquids in small volumes have various advantages over conventional systems such as enhanced heat and mass transfer, lower reagent consumption and efficient mixing. In this thesis, development of an enzymatic microreactor which combines the advantages of microfluidic systems and enzymatic reactions and the hydrolysis of saponin molecules in the developed system were aimed. In accordance with this purpose, β-glucosidase enzyme was immobilized in silica and alginate hydrogels, these hydrogels were characterized in terms of morpholgy, surface area and shrinkage/swelling properties and the kinetic parameters of free and immobilized enzyme such as Vmax ve KM were determined. After the characterization studies, enzymatic microreactors were prepared and hydrolysis of pNPG (model substrate), ginsenoside Rb1, soyasaponin Bb and soyasaponin Aa were carried out. The characterization studies indicated that the high surface area (∼304 m2/g) and porosity properties of alginate hydrogels made them suitable matrices for the enzyme immobilization. The studies carried out in enzymatic microreactors showed that immobilization method and the residence time had significant effect on the reaction yield and increasing residence time enhanced the conversion yield. Performance of the enzymatic microreactor and free and immobilized enzymes in batch conditions were compared and in microreactor experiments higher conversion yield (37.52%) was obtained than the conventional reaction systems (27% and 16% for free and immobilized enzyme, respectively). Saponin molecules were hydrolyzed by three different approaches. By the acidic hydrolysis, not only the intermediate products but also undesired side products were obtained while no side product formation occured during enzymatic hydrolysis which indicated the main advantage of enzymes over chemical catalysts. The hydrolysis of ginsenoside Rb1 by free enzyme resulted in the formation of ginsenoside Rd, the molecule obtained by the removal of a glucose from ginsenoside Rb1. For the same residence time, ginsenoside Rd and also ginsenoside F2 (the follow up product of ginsenoside Rd) were determined in enzymatic microreactor experiments. In a similar way, only intermediate products (soyasaponin III and soyasapogenol B monoglucuronide) were obtained by the hydrolysis of soyasaponin Bb with free enzyme while enzymatic microreactor was able to produce the aglycone, soyasapogenol B. These results clearly showed the high substrate specifity of enzymes and the efficacy of enzymatic microreactors over conventional systems. In addition, MTT analyses were carried out to determine the cytotoxicity of the saponins and aglycones on different cancer cell lines. Soyasapogenols showed higher cytotoxicity than soyasaponins. The most effective molecule on all tested cell lines was compound K and it was found that compound K induced cell death by apoptosis.
Microfluidics systems which enable the manipulation of liquids in small volumes have various advantages over conventional systems such as enhanced heat and mass transfer, lower reagent consumption and efficient mixing. In this thesis, development of an enzymatic microreactor which combines the advantages of microfluidic systems and enzymatic reactions and the hydrolysis of saponin molecules in the developed system were aimed. In accordance with this purpose, β-glucosidase enzyme was immobilized in silica and alginate hydrogels, these hydrogels were characterized in terms of morpholgy, surface area and shrinkage/swelling properties and the kinetic parameters of free and immobilized enzyme such as Vmax ve KM were determined. After the characterization studies, enzymatic microreactors were prepared and hydrolysis of pNPG (model substrate), ginsenoside Rb1, soyasaponin Bb and soyasaponin Aa were carried out. The characterization studies indicated that the high surface area (∼304 m2/g) and porosity properties of alginate hydrogels made them suitable matrices for the enzyme immobilization. The studies carried out in enzymatic microreactors showed that immobilization method and the residence time had significant effect on the reaction yield and increasing residence time enhanced the conversion yield. Performance of the enzymatic microreactor and free and immobilized enzymes in batch conditions were compared and in microreactor experiments higher conversion yield (37.52%) was obtained than the conventional reaction systems (27% and 16% for free and immobilized enzyme, respectively). Saponin molecules were hydrolyzed by three different approaches. By the acidic hydrolysis, not only the intermediate products but also undesired side products were obtained while no side product formation occured during enzymatic hydrolysis which indicated the main advantage of enzymes over chemical catalysts. The hydrolysis of ginsenoside Rb1 by free enzyme resulted in the formation of ginsenoside Rd, the molecule obtained by the removal of a glucose from ginsenoside Rb1. For the same residence time, ginsenoside Rd and also ginsenoside F2 (the follow up product of ginsenoside Rd) were determined in enzymatic microreactor experiments. In a similar way, only intermediate products (soyasaponin III and soyasapogenol B monoglucuronide) were obtained by the hydrolysis of soyasaponin Bb with free enzyme while enzymatic microreactor was able to produce the aglycone, soyasapogenol B. These results clearly showed the high substrate specifity of enzymes and the efficacy of enzymatic microreactors over conventional systems. In addition, MTT analyses were carried out to determine the cytotoxicity of the saponins and aglycones on different cancer cell lines. Soyasapogenols showed higher cytotoxicity than soyasaponins. The most effective molecule on all tested cell lines was compound K and it was found that compound K induced cell death by apoptosis.
Açıklama
Anahtar Kelimeler
Enzimatik Mikroreaktör, β-glukosidaz, Hidrojel, Ginsenosit Rb1, Soyasaponinler, Sitotoksisite, Enzymatic Microreactor, β-Glucosidase, Hydrogel, Ginsenoside Rb1, Soyasaponins, Cytotoxicity
