Özlem BARIŞ, Nazan DEMİR, Yaşar DEMİR, Safinur YILDIRIM ÇELİK, Medine GÜLLÜCE

Agrobacterium radiobacter'den Pektin Liyaz Enziminin Üretilmesi, Saflaştırılması, Karakterize Edilip Meyve Suyu Üretiminde Kullanılması

Ekstraselüler pektin liyaz enzimi (EC 4.2.2.10) Agrobacterium radiobacter'den katı ortam fermantasyonu ile üretilmiş ve DEAE-selüloz iyon değişim kromatografisi kullanılarak 21 kat saflaştırılıp karakterize edilmiştir. Enzimin molekül ağırlığı Sephadex G-100 jel filtrasyon kromatografisi ile 67 kDa olarak bulunmuş ve enzimin saflığı SDS-PAGE ile kontrol edilmiştir. Enzimin optimum pH'sı ve sıcaklığı sırasıyla 8 ve 60°C olarak belirlenmiştir. Enzim aktivitesinin 24 saat boyunca kararlı olduğu en yüksek sıcaklığın 40°C'de olduğu gözlenmiştir. 50°C'de yaklaşık 4 saat boyunca, 60°C'de ise 1 saat boyunca %50 aktivitesini koruduğu bulunmuştur. Saflaştırılan enzimin Vmax ve KM'si Lineweaver-Burk grafiği kullanılarak 4.36 mg/mL and 303 µmol/L.dakika olarak hesaplanmıştır. Pektin liyaz enziminin aktivitesi üzerine metal iyonlarının etkisi incelenmiş ve Mg2+ ve Ca2+ iyonlarının enzimi kısmen, Fe3+, Zn2+, Cu2+ ve Hg2+ iyonlarının enzimi tamamen inhibe ettiği anlaşılmıştır. Co2+ (0,1 mM, 1 mM ve 10 mM) iyonunun ise enzimin az da olsa aktivitesini arttırdığı gözlenmiştir. Saflaştırılıp karakterize edilen enzim meyve suyu üretiminde kullanıldığında kontrole göre meyve suyu veriminde önemli artış görülmüştür.

Production of Pectin Lyase from Agrobacterium radiobacter, Purification, Characterization and Its Use in Fruit Juice Production

Extracellular pectin lyase (EC 4.2.2.10) was produced by Agrobacterium radiobacter in solid state fermentation. Pectin lyase enzyme was purified 21 fold by using DEAE-cellulose anion exchange column chromatography and characterized. Molecular weight of the enzyme was determined as 67 kDa by using Sephadex G-100 gel filtration chromatography. Purification of enzyme was checked by SDS-PAGE. The optimum pH and temperature of enzyme was determined as pH 8.0 and 60°C, respectively. Pectin lyase was mostly stable at 40°C. Its activity deceased by 50% for 1h at 60°C and 40% for 4 h at 50°C. Vmax and KM were calculated as 4.36 mg/mL and 303 µmol/L.min, respectively. Purified pectin lyase was partly inhibited by Mg2+ and Ca2+ and completely inhibited by 10 mM of Fe3+, Zn2+, Cu2+ and Hg2+. A slight activation effect was observed in the presence of Co2+ (10, 0.1 and 0.001 mM) on the purified pectin lyase. The purified pectin lyaseenzyme was used for the production of fruits juices and significant improvement was found in juice yields in comparison to the juice yield of control.

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