Horseradish Peroxidase Immobilized into Organogel-Silica Composite for Transformation of Chlorophenols to Biodegradable Organic Acids

Abstract

Phenol and its derivatives are considered detrimental to environment and need to be removed from environ- ment. Enzymatic oxidation of chlorophenols is considered as viable option but leads to precipitate due to polymerization of phenolic compounds, which require additional step of flocculation towards safe discarding of contaminated waters. In present work, microemulsion organogel from cetyltrimethylammonium bromide reverse microemulsion and gelatin was prepared and used as immobilization matrix for horseradish peroxidase HRP, E.C.1.11.1.7 . The material was further hardened with silica to enhance aqueous solution stability. The composite material was studied for enzymatic kinetics and oxidation of chlorophenols and employed as catalyst in the presence of hydrogen peroxide to oxidize 2-chlorophenol 2-CP , 2,4-dichlorophenol 2,4 - DCP , and 2,4,6-trichlorophenol 2,4,6-TCP in an aqueous media. It was worth noting that phenols get converted to organic acids rather than typical polymerized products. The protective effect of composite material was observed which render the polymerization reaction of phenols. Michaeles-Menten constant and activity of enzyme in free and immobilized system were evaluated using three modified Michaeles-Menten equations and progressive curve experiments, respectively. The immobilized HRP followed Michaeles-Menten kinetics with lower reaction velocity constant Vmax and Michealis constant Km values comparable to free HRP. Further, following parameters were optimized: contact time, pH, hydrogen peroxide concentration, enzyme dose and analyte concentration. Under different optimized condition, the oxidative removal of phenol and their derivatives reaches up to 95-99% in phosphate buffer.

Keywords

• chlorophenols,enzymatic oxidation,HRP,Km Value,Michaeles-Menten Constant,oxidation reaction

Klorofenollerin Biyobozunur Organik Asitlere Dönüşümü İçin Organojel-Silika Kompozit Yaban Turpu Peroksidaz İmmobilizasyonu

Abstract

F enol ve türevleri tehlikeli çevresel atıklardır ve bu nedenle çevreden uzaklaştırılmaları gerekir. Klorofenollerin enzimatik oksidasyonu uygun bir seçenek olarak düşünülür ama fenolik bileşiklerin polimerizasyonu nedeniyle çökmesine yol açar ve kontamine suların güvenli bir şekilde atılması yönünde flokülasyonun ek bir adımını gerektirir. Bu çalışmada, setiltrimetilamonyum bromür ters mikroemülsiyon ile mikroemülsiyon organojel ve jelatin hazırlandı ve yaban turpu peroksidazı HRP, E.C.1.11.1.7 için immobilizasyon matriksi olarak kullanıldı. Bu malzeme sulu çözeltilerin kararlılığını artırmak için silika ile daha fazla sertleştirilmiştir. Bu kompozit malzeme enzimatik kinetiği ve klorofenollerin oksidasyonu için çalışılmış ve sulu ortamlarda 2-klorofenol 2-CP , 2,4-diklorofenol 2,4-DCP ve 2,4,6-triklorofenolü 2,4,6-TCP hidrojen peroksit varlığında okside etmek için katalizör olarak kullanılmıştır. Dikkat edilmesi gereken konu fenoller, tipik polimerize ürünler yerine organik asitlere dönüştürülmüştür. Kompozit malzemelerin koruyucu etkisi fenollerin polimerizasyon tepkimelerini açıklamak için incelenmiştir. Michaelis-Menten sabiti ve serbest ve immobilize sistemdeki enzimin aktivitesi sırasıyla üç modifiye Michaelis-Menten denklemleri ve ilerleme eğrisi deneyleri ile değerlendirilmiştir. İmmobilize HRP, düşük tepkime hız sabiti Vmax ve Michaelis sabiti Km değerlerini serbest HRP ile karşılaştırmak için izlenmiştir. Ayrıca, temas süresi, pH, hidrojen peroksit derişimi, enzim dozu ve analit derişimi parametreleri ile optimize edilmiştir. Farklı optimizasyon koşulları altında, fenollerin ve türevlerinin fosfat tamponunda oksidatif uzaklaştırılması % 95-99 arasında gerçekleştirilmiştir

Keywords

• Klorofenoller,enzimatik oksidasyon,HRP,Km değer,Michaelis-Menten sabiti,oksidasyon tepkimesi

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