Rekombinant aldo/keto redüktaz ve glukoz dehidrogenaz temelli enzimatik kofaktör rejenerasyon sistemlerinin tasarlanması kiral alkollerin eldesine yönelik uygulamalar
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Tarih
2019
Yazarlar
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Yayıncı
Ege Üniversitesi, Fen Bilimleri Fakültesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
İndirgenme reaksiyonlarını katalizleyen ketoredüktazlar (KRED) enantiyoselektif yeşil kimya alanında ilgi uyandırmaktadır. kofaktör bağımlı KRED enzimleri kiral alkollerin sentezinde çok sayıda aromatik ve alifatik prokiral ketonun stereoseçimli olarak indirger. Kiral bileşikler saf kiral bileşiklerin sentezinde başlangıç maddesi olarak değerlidir. KRED enzimlerinin en önemli özelliği NADPH bağımlı olmalarıdır. İndirgenmiş NADPH'ın yüksek maliyeti ve stokiyometrik miktarlarda gerekliliği, enantiyomerik saflıkta ürünlerin elde edilmesi, kofaktör rejenerasyon sistemlerinin birlikte kullanımını zorunlu kılmaktadır. Bu amaçla Glukoz dehidrojenaz (GDH; NAD(P)H bağımlı) enzimatik kofaktör rejenerasyon sistemi olarak endüstriyel sistemlerde kullanılmaktadır. Endüstriyel bakışla immobilize enzimlerin rağbet görmesinin sebebi, verimlilik ve stabilitelerinin serbest enzimlere kıyasla çok daha yüksek olmasıdır. Bu araştırma projesi kapsamında NADPH bağımlı aldo/keto-redüktaz (EC: 1.1.1.2") ve NADPH rejenerasyonu için, glukoz-1-dehidrogenaz (EC: 1.1.1.47") enzimlerine ait gen bölgeleri B. subtilis 168'ten başarıyla klonlanarak E.coli BL21 hücrelerinde üretildi, saflaştırıldı ve kiral katalizlerde karakterize edildi. Endüstiriyel bakış açısıyla immobilize enzimler verimlilikleri ve stabiliteleri nedeniyle serbest enzimlere tercih edilmektedir. Bu tez kapsamında KRED ve GDH enzimlerinin immobilize elde edilebilmesi amacıyla, doğal fonksiyonu silika biyominerilizasyonu katalizlemek olan R5 peptid dizisini taşıyan füzyon enzimleri, KRED-R5 ve GDH-R5 tasarlandı. Böylelikle, R5 peptid dizisinin doğal yeteneği olan biyomimetik silika mineralizasyon, enzimlere başarıyla transfer edildi. R5 füzyon proteinleri kullanılarak, nano yapılı silika matris içerisine yüksek verimlilikle immobilizasyonları gerçekleştirildi. İmmobilize KRED ve GDH enzim sistemi asetofenon ve etil piruvattan (eep>99) saflıkta kiral alkollerin sentezinde etkin biçimde kullanılabildiği gösterildi.
Ketoreductases (KREDs) capable of catalyzing reduction reactions are of special interest across the field of enantioselective green reactions. For the synthesis of chiral alcohols, cofactor dependent KREDs reduce broad range of prochiral aromatic and aliphatic ketones stereoselectively. An important characteristic of KREDs is their dependence on NADPH+H+ as cofactor. Given the high cost of expensive NADPH stoichiometric necessity for reduction reactions, a suitable cofactor regeneration method has to be combined for the production of enantiomerically pure compounds. Whith this purpose Glucose dehydrogenases (GDH; accept NAD(P)H as cofactor) used for the enzymatic regeneration of NAD(P)H cofactors in industry. Within the scope of the thesis, glucose dehydrogenase (EC: 1.1.1.47) as a cofactor recycling enzyme and NADPH dependent aldo/ketoreductase (EC: 1.1.1.2) coding genes were successfully isolated, cloned and expressed in the Escherichia coli BL21 from Bacillus subtilis 168 and coupled for chiral synthesis. From an industrial point of view, immobilized enzymes find approval in contrast with free enzyme due to their efficiency and stability. This doctorate study was also aimed at immobilization of these enzymes, which involved the construction of recombinant fusion proteins between R5 peptide has a natural ability to catalyse silicate. R5 peptide transfers the natural characteristics of biomimetic silica mineralization to a desired enzyme. Furthermore, purified and characterized fusion proteins (KRED-R5 and GDH-R5) and their immobilization in biosilica successfully achieved. Not only the encapsulation but also the activity yield was high ranging over 90% after immobilization with R5 fusion for both enzymes. Immobilized KRED/GDH system exhibited excellent enantioselectivity (>99% ee) in the reduction acetophenon and ethyl pyruvate ester.
Ketoreductases (KREDs) capable of catalyzing reduction reactions are of special interest across the field of enantioselective green reactions. For the synthesis of chiral alcohols, cofactor dependent KREDs reduce broad range of prochiral aromatic and aliphatic ketones stereoselectively. An important characteristic of KREDs is their dependence on NADPH+H+ as cofactor. Given the high cost of expensive NADPH stoichiometric necessity for reduction reactions, a suitable cofactor regeneration method has to be combined for the production of enantiomerically pure compounds. Whith this purpose Glucose dehydrogenases (GDH; accept NAD(P)H as cofactor) used for the enzymatic regeneration of NAD(P)H cofactors in industry. Within the scope of the thesis, glucose dehydrogenase (EC: 1.1.1.47) as a cofactor recycling enzyme and NADPH dependent aldo/ketoreductase (EC: 1.1.1.2) coding genes were successfully isolated, cloned and expressed in the Escherichia coli BL21 from Bacillus subtilis 168 and coupled for chiral synthesis. From an industrial point of view, immobilized enzymes find approval in contrast with free enzyme due to their efficiency and stability. This doctorate study was also aimed at immobilization of these enzymes, which involved the construction of recombinant fusion proteins between R5 peptide has a natural ability to catalyse silicate. R5 peptide transfers the natural characteristics of biomimetic silica mineralization to a desired enzyme. Furthermore, purified and characterized fusion proteins (KRED-R5 and GDH-R5) and their immobilization in biosilica successfully achieved. Not only the encapsulation but also the activity yield was high ranging over 90% after immobilization with R5 fusion for both enzymes. Immobilized KRED/GDH system exhibited excellent enantioselectivity (>99% ee) in the reduction acetophenon and ethyl pyruvate ester.
Açıklama
Anahtar Kelimeler
Ketoredüktaz, Glukoz Dehidrojenaz, Kofaktör Rejenerasyonu, Biyosilika İmmobilizasyon, Kiral Alkoller, Ketoreductase, Glucose Dehydrogenase, Bio-Silisica Encapsulation, Kofactor Regeneration, Chiral Alcohols
