Separation of Guaiacol Peroxidase from Onion Roots with Affinity Chromatography

Abstract

Peroxidases are enzymes that catalyze the oxidation of various organic substrates in the presence of hydrogen peroxide and contain heme group in their structure. In particular, plant peroxidases are frequently used in industry, clinical diagnosis, biosensor construction and organic synthesis reactions. Due to their commercial value, the identification and purification of these enzymes in different sources is of great importance. In this study, peroxidase enzyme was purified for the first time by using aminobenzohydrazide-based affinity chromatography technique from onion roots 750-fold with a yield of 37.7. SDS-PAGE was performed to determine the molecular weight of the purified enzyme and a single band was observed at 51.2 kDa. In addition, KM values of the enzyme for guaiacol, ABTS and pyrogallol substrates were calculated as 3.44, 0.46 and 21.27 mM, respectively.

Keywords

• Onion (Allium cepa) root,peroxidase,affinity chromatography,purification

Guaiakol Peroksidazın Soğan Köklerinden Afinite Kromatografisi ile Saflaştırılması

Abstract

Peroksidazlar, hidrojen peroksit varlığında çeşitli organik substratların oksidasyonunu katalizleyen ve yapılarında hem grubu bulunduran enzimlerdir. Özellikle bitki peroksidazları, endüstride, klinik tanıda, biyosensör yapımında ve organik sentez reaksiyonlarında sıklıkla kullanılır. Ticari değerleri nedeniyle bu enzimlerin farklı kaynaklarda tanımlanması ve saflaştırılması büyük öneme sahiptir. Bu çalışmada peroksidaz enzimi ilk kez aminobenzohydrazide tabanlı afinite kromatografi tekniği kullanılarak soğan köklerinden 37.7 verimle 750 kat saflaştırıldı. Saflaştırılan enzimin moleküler ağırlığını belirlemek için SDS-PAGE yapıldı ve 51.2 kDa’da tek bant gözlendi. Ayrıca enzimin guaiakol, ABTS ve pirogallol substratları için KM değerleri sırasıyla 3.44, 0.46 ve 21.27 mM olarak olarak hesaplandı.

Keywords

• Soğan (Allium cepa) kökü,peroksidaz,afinite kromatografisi,saflaştırma

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