SIRTIGÖKÇE MANTARINDAN (Russula Cyanoxantha, L.) POLİFENOL OKSİDAZ ENZİMİNİN ELDE EDİLMESİ VE SAFLAŞTIRILMASI
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2022-09
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info:eu-repo/semantics/openAccess
Özet
Russula cyanoxantha mantarı (Sırtıgökçe) yabani ve yenilebilir bir mantardır ve Karabük civarından elde edilmiştir. Bu mantardan polifenol oksidaz enzimi elde edilmiş, enzim kısmen saflaştırılarak biyokimyasal açıdan araştırılmıştır. Enzimin ekstraksiyonu aşamasında yer alan değişkenler optimize edilmiş daha sonra elde edilen ekstraktın proteinleri amonyum sülfatla çöktürülmüştür. Çökelti diyaliz işlemiyle temizlenmiş ve jel kromatografi kolonuna uygulanarak afinite yöntemiyle kısmen saflaştırılmıştır. Enzim çözeltilerinde protein tayini ve enzim aktivite ölçümleri yapılmış ve verim %11,30, saflaştırma derecesi 9,33 olarak hesaplanmıştır. Russula cyanoxantha polifenol oksidaz enziminin substrat ilişkisini incelemek amacıyla dokuz farklı substrat denenmiş, bu substratlarla yapılan aktivite ölçümleri sonucunda maksimum aktivite katekol ve L-Dopa ile gözlenmiştir. Katekol ve L-Dopa eşliğinde yapılan ölçümlerde maksimum aktivitenin gözlendiği pH ve sıcaklık katekol için sırasıyla 7,0 ve 10 ? şeklinde, L-Dopa için 7,0 ve 30 ? şeklinde bulunmuştur. Katekol substratı ile enzimin ekstraksiyonunda kullanılan optimum pH, pH kararlılığı ve termal kararlılık incelenmiştir. Russula cyanoxantha polifenol oksidaz enzimi kinetik açıdan incelenmiş, Vmax (en yüksek reaksiyon hızı) ve Km (substrat-enzim ilişkisi) araştırılmış, katekol için bu veriler sırasıyla 1,965 ?mol dk-1 ve 1,769 mM şeklinde L-Dopa için 1,369?mol dk-1 ve 15,193 mM şeklinde bulunmuştur. Enzimin –18 ?’de aktivitesini bir yıl boyunca koruyabildiği bulunmuştur.
Russula cyanoxantha mushroom (Sırtıgökçe) is a wild and edible mushroom and was obtained from Karabük countryside. Polyphenol oxidase enzyme was extracted from this mushroom, partially purified and its biochemical characterization was performed. Enzyme extraction variables were optimized, after that, proteins that exist in the extract were precipitated by ammonium sulfate. Precipitate was cleaned by dialysis then, partially purified with affinity method by application to the gel chromatography column. Enzyme solution protein content was determined, activity studies for enzyme in this solution was performed. Yield and purification degree were found as 11,30% and 9,33 respectively. For the purpose of investigation of substrate relationship of Russula cyanoxantha polyphenol oxidase enzyme, nine different substrates were tried, as a result of activity measurements performed with these substrates, highest activities were observed with catechol and L-Dopa. In the measurements performed in the existence of catechol and L-Dopa, optimum measurement pH and optimum temperature were found as 7.0 and 10 ? for catechol and as 7.0 and 30 ? for L-Dopa. Enzyme’s optimum extraction pH, pH and termal stability were observed with catechol substrate. Russula cyanoxantha polyphenol oxidase enzyme’s kinetic parameters, Vmax (maximum reaction rate) and Km (substrate affinity) were searched, these values were found as 1,965 ?mol min-1 and 1,769 mM by catechol and 1,369 ?mol min-1 and 15,193 mM by L-Dopa. It has been found that the enzyme’s activity has not been lost during 12 months at –18 ?."
Russula cyanoxantha mushroom (Sırtıgökçe) is a wild and edible mushroom and was obtained from Karabük countryside. Polyphenol oxidase enzyme was extracted from this mushroom, partially purified and its biochemical characterization was performed. Enzyme extraction variables were optimized, after that, proteins that exist in the extract were precipitated by ammonium sulfate. Precipitate was cleaned by dialysis then, partially purified with affinity method by application to the gel chromatography column. Enzyme solution protein content was determined, activity studies for enzyme in this solution was performed. Yield and purification degree were found as 11,30% and 9,33 respectively. For the purpose of investigation of substrate relationship of Russula cyanoxantha polyphenol oxidase enzyme, nine different substrates were tried, as a result of activity measurements performed with these substrates, highest activities were observed with catechol and L-Dopa. In the measurements performed in the existence of catechol and L-Dopa, optimum measurement pH and optimum temperature were found as 7.0 and 10 ? for catechol and as 7.0 and 30 ? for L-Dopa. Enzyme’s optimum extraction pH, pH and termal stability were observed with catechol substrate. Russula cyanoxantha polyphenol oxidase enzyme’s kinetic parameters, Vmax (maximum reaction rate) and Km (substrate affinity) were searched, these values were found as 1,965 ?mol min-1 and 1,769 mM by catechol and 1,369 ?mol min-1 and 15,193 mM by L-Dopa. It has been found that the enzyme’s activity has not been lost during 12 months at –18 ?."
Açıklama
Anahtar Kelimeler
Polifenol oksidaz, enzim ekstraksiyonu, saflaştırma, Russula cyanoxantha., Polyphenol oxidase, enzyme extraction, purification, Russula cyanoxantha.
