Trametes versicolor lakkaz enziminin çeşitli yöntemlerle nano-immobilizasyonu ve uygulamaları
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Tarih
2019
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Çanakkale Onsekiz Mart Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, son on yılda ciddi biçimde dikkat çekmiş olan üç farklı nanoimmobilizasyon tekniği Trametes versicolor lakkaz enzimi üzerine uygulanmış ve bu tekniklerin fenolik bileşiklerin belirlenmesi ve atık sulardan fenol giderimi uygulamaları açısından potansiyeli incelenmiştir. Lakkaz enzimi "tek enzim nanoparçacıkları", "çapraz bağlı enzim nanoagregatları" ve "elektrospin nanofiberler ile immobilizasyon" yöntemleri kullanılarak immobilize edilmiş ve elde edilen lakkaz preparatlarının karakterizasyonu gerçekleştirilmiştir. En iyi aktivite geri kazanımı değerleri tek enzim nanoparçacıkları biçiminde immobilizasyon sonucunda elde edilenler olup % 66,8 ± 6,5 olarak belirlenmiştir. Öte yandan bu yöntem ile elde edilen preparatlarda ciddi bir kararlılık artışı gözlenmemiştir. Çapraz bağlı enzim nanoagregatları biçiminde immobilizasyon sonucunda elde edilen aktivite geri kazanımı değerleri daha düşük (% 40,5 ± 5,0) olmakla birlikte ısıl kararlılıklarının serbest enzime göre ciddi biçimde daha yüksek (60 oC'de yaklaşık 10–30 kat) olduğu gözlenmiştir. Elektrospin ile immobilizasyon sonucunda ise doğrudan elektron transferi ile ciddi bir sinyal artışı gözlenmemiştir. Elde edilen immobilize enzim preparatlarının boyutlarının, immobilizasyon yöntemine göre ciddi biçimde değiştiği gözlenmiştir. En küçük boyuttaki preparatlar tek enzim nanoparçacıkları yöntemi ile elde edilenler olup bunların yaklaşık çapı 20 nm olarak belirlenmiştir. En büyük boyuttaki preparatlar ise çapraz bağlı enzim nanoagregatları biçiminde elde edilenler olup bunların yaklaşık çapı 800 nm civarı olarak belirlenmiştir. Tek lakkaz nanoparçacıklarının, Kcat/Km değerleri serbest enziminkiyle yaklaşık olarak aynı olduğu halde, çapraz bağlı lakkaz nanoagregatlarının Kcat/Km değerlerinde bir miktar düşüş olduğu gözlenmiştir. Yalnızca çapraz bağlı enzim nanoagregatları biçiminde immobilizasyon ile elde edilen preparatlarda endüstriyel kullanım potansiyeli gözlendiğinden bu preparatların bisfenol A, gallik asit ve zeytin kara suyu çözeltilerinin fenol içeriğinin gideriminde kullanım potansiyeli incelenmiştir. İmmobilize enzimin oda sıcaklığında yirmi dört saatlik süre sonucunda bisfenol A'nın % 43,5; gallik asidin % 80,0 ve zeytin kara suyunun fenol içeriğinin % 80,1'ini giderdiği gözlenmiştir.
In this study, three different nano-immobilization techniques that have drawn serious attention over the past decade were applied on Trametes versicolor laccase enzyme. The potential of these techniques for the determination of phenolic compounds and for the removal of phenolics from wastewaters were examined. Laccase enzyme was immobilized as "single enzyme nanoparticles", "crosslinked enzyme nanoaggregates" and "electrospun nanofibers" and resulting laccase preparations were characterized. The best activity recovery values were obtained for single enzyme nanoparticles and measured as 66.8 % ± 6.5. On the other hand, no significant thermal stability increase was observed for preparations obtained with this method. Although the activity recovery values obtained for crosslinked enzyme nanoaggregates were lower (40.5 % ± 5.0), the thermal stabilities were significantly higher (approximately 10–30 fold at 60 oC) compared to free enzyme. No significant signal increase was observed with direct electron transfer for immobilization with electrospinning. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the immobilization method. The lowest sized products were found to be single enzyme nanoparticles with a diameter of 20 nm. The largest sized products were found to be crosslinked enzyme nanoaggregates with a diameter of 800 nm. Although the Kcat/Km values of single enzyme nanoparticles were approximately the same with the Kcat/Km values of the free enzyme, Kcat/Km values of crosslinked enzyme nanoaggregates were lower. Only crosslinked enzyme nanoaggregates were considered to have industrial application potential, therefore the potentials of crosslinked enzyme nanoaggregates were investigated for the removal of phenolics from bisphenol A solutions, gallic acid solutions and olive mill wastewater. Immobilized enzyme was found to remove 43.5 % of bisphenol A and 80.0 % of gallic acid from their solutions and 80.1 % of phenolics from olive mill wastewater at room temperature within 24 h.
In this study, three different nano-immobilization techniques that have drawn serious attention over the past decade were applied on Trametes versicolor laccase enzyme. The potential of these techniques for the determination of phenolic compounds and for the removal of phenolics from wastewaters were examined. Laccase enzyme was immobilized as "single enzyme nanoparticles", "crosslinked enzyme nanoaggregates" and "electrospun nanofibers" and resulting laccase preparations were characterized. The best activity recovery values were obtained for single enzyme nanoparticles and measured as 66.8 % ± 6.5. On the other hand, no significant thermal stability increase was observed for preparations obtained with this method. Although the activity recovery values obtained for crosslinked enzyme nanoaggregates were lower (40.5 % ± 5.0), the thermal stabilities were significantly higher (approximately 10–30 fold at 60 oC) compared to free enzyme. No significant signal increase was observed with direct electron transfer for immobilization with electrospinning. The sizes of the obtained immobilized enzyme products were found to be greatly variable depending on the immobilization method. The lowest sized products were found to be single enzyme nanoparticles with a diameter of 20 nm. The largest sized products were found to be crosslinked enzyme nanoaggregates with a diameter of 800 nm. Although the Kcat/Km values of single enzyme nanoparticles were approximately the same with the Kcat/Km values of the free enzyme, Kcat/Km values of crosslinked enzyme nanoaggregates were lower. Only crosslinked enzyme nanoaggregates were considered to have industrial application potential, therefore the potentials of crosslinked enzyme nanoaggregates were investigated for the removal of phenolics from bisphenol A solutions, gallic acid solutions and olive mill wastewater. Immobilized enzyme was found to remove 43.5 % of bisphenol A and 80.0 % of gallic acid from their solutions and 80.1 % of phenolics from olive mill wastewater at room temperature within 24 h.
Açıklama
Fen Bilimleri Enstitüsü, Kimya Ana Bilim Dalı
Anahtar Kelimeler
Biyokimya, Biochemistry, Kimya
