KANLICA MANTARINDAN (Lactarius Salmonicolor L.) POLİFENOL OKSİDAZ ENZİMİNİN EKSTRAKSİYONU VE SAFLAŞTIRILMASI
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2021-05-28
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info:eu-repo/semantics/openAccess
Özet
Yabani ve yenilebilir bir mantar olan Lactarius salmonicolor mantarının polifenol oksidaz enzimi kısmen saflaştırıldı ve biyokimyasal özellikleri karakterize edildi. Enzim ekstraksiyonunun parametreleri optimize edildikten sonra elde edilen homojenata amonyum sülfatla çöktürme ve diyaliz işlemleri uygulandı, elde edilen enzim çözeltisi affinite kolonuna uygulanarak jel kromatografisi ile kısmen saflaştırıldı. Protein tayinleri ve enzim aktivite ölçümleri yapılarak saflaştırma verimi %21.279, saflaştırma derecesi ise 15.821 olarak hesaplandı. Katekol ve L-DOPA substratları varlığında enzimin optimum ölçüm pH ve sıcaklık değerleri sırasıyla 6.00 ve 40 ? olarak saptandı. Katekol substratı kullanılarak optimum ekstraksiyon pH’ı, enzimin pH ve termal kararlılığı araştırıldı. Lactarius salmonicolor enziminin kinetik parametreleri, Vmax (maksimum reaksiyon hızı) ve Km (substratın enzim ilgisi) belirlendi. Bu değerler katekolle yapılan ölçümlerde 7.220 ?mol dak-1 mL-1 ve 2.694 mM olarak L-DOPA ile yapılan ölçümlerde 6.477 ?mol dak-1 mL-1 ve 2.494 mM olarak hesaplanmıştır. Polifenol oksidaz enziminin substrat spesifikliğini araştırmak amacıyla sekiz farklı substratla aktivite ölçümleri yapılmış ve en yüksek sonuçlar katekol ve L-DOPA ile elde edilmiştir. Enzimin –18 ?’da bir sene aktivitesini koruyabildiği saptanmıştır.
Polyphenol oxidase enzyme from edible and wild mushroom Lactarius salmonicolor was partially purified and its biochemical properties were characterized. After optimization of parameters of enzyme extraction, the homogenized enzyme solution was precipitated with ammonium sulfate and dialyzed. Resulting enzyme solution was loaded to the affinity column and partially purified by gel chromatography. Yield and purification factor were calculated as %21.279 and 15.821 after determination of protein contents and enzyme activity measurements. Optimum measurement pH and temperature were obtained as 6.00 and 40 ? in the existence of catechol and L-DOPA. By using catechol substrate, optimum extraction pH, pH and termal stability were obtained. Kinetic parameters of Lactarius salmonicolor enzyme, Vmax (maximum reaction rate) and Km (substrate affinity) were determined as 7.220 ?mol min-1 mL-1 and 2.694 mM by catechol and 6.477 ?mol min-1 mL-1 and 2.494 mM by L-DOPA. To investigate substrate specificity of polyphenol oxidase enzyme, eight different substrates were used for enzyme activity measurements. Highest activities were obtained by catechol and L-DOPA. Enzyme keep its activity for one year at –18 ?."
Polyphenol oxidase enzyme from edible and wild mushroom Lactarius salmonicolor was partially purified and its biochemical properties were characterized. After optimization of parameters of enzyme extraction, the homogenized enzyme solution was precipitated with ammonium sulfate and dialyzed. Resulting enzyme solution was loaded to the affinity column and partially purified by gel chromatography. Yield and purification factor were calculated as %21.279 and 15.821 after determination of protein contents and enzyme activity measurements. Optimum measurement pH and temperature were obtained as 6.00 and 40 ? in the existence of catechol and L-DOPA. By using catechol substrate, optimum extraction pH, pH and termal stability were obtained. Kinetic parameters of Lactarius salmonicolor enzyme, Vmax (maximum reaction rate) and Km (substrate affinity) were determined as 7.220 ?mol min-1 mL-1 and 2.694 mM by catechol and 6.477 ?mol min-1 mL-1 and 2.494 mM by L-DOPA. To investigate substrate specificity of polyphenol oxidase enzyme, eight different substrates were used for enzyme activity measurements. Highest activities were obtained by catechol and L-DOPA. Enzyme keep its activity for one year at –18 ?."
Açıklama
Anahtar Kelimeler
Polyphenol oxidase, enzyme extraction, purification, Lactarius salmonicolor., Polifenol oksidaz, enzim ekstraksiyonu, saflaştırma, Lactarius salmonicolor.
