Ayçiçeği tohumlarından lipaz enzimi izolasyonu, saflaştırılması ve karakterizasyonu
Küçük Resim Yok
Tarih
2001
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
ÖZET Trakya Bölgesinde bitkisel yağ üretmek amacıyla ayçiçeği tarımı yaygın bir şekilde yapılmaktadır. Bu ayçiçeği tohumları yaklaşık % 20-30 protein ve % 50-60 yağ içermektedir. Türkiye'de ayçiçeği yemeklik yağ üretiminde kullanılmakta ve proses sonrasındaki yan ürün olan küspe yalnızca hayvan yemi olarak değerlendirilmektedir. Proteince zengin olan tohumlardan yeterince yararlanılmamaktadır. Bu nedenle, tohumların yüksek ve değerli protein potansiyelinden öncelikle insan beslenmesinde ve bilimsel amaçlarla faydalanılması gerektiği düşünüldü. Bu çalışmada, ayçiçeği tohumlarından lipaz enziminin izole edilip saflaştırılması ve hidrolitik kataliz özelliklerinin tespit edilmesi amaçlandı. Literatür araştırmalarımızda, ayçiçeği tohumu lipazı ile ilgili saflaştırma ve karakterizasyon çalışmalarına rastlanmadı. Bölgede en çok ekilen ayçiçeği türlerinin tohumları blenderde öğütüldü ve yağı fosfat tamponu (0.1 M, pH 7) ve organik çözücü varlığında uzaklaştırıldı. Ayçiçeği tohumlarından yağları uzaklaştırmak için, beş çözücü kullanıldı ve n-bütanol en uygun çözücü olarak belirlendi. Sonra lipaz enzimi fraksiyonlu amonyum sülfat çöktürmesi ve takiben diyaliz ile kısmen saflaştırıldı. En yüksek enzim aktivitesine sahip olan % 90'lık tuz çökeleğinde üpaz aktivitesi 118 U/mg-protein olarak belirlendi. Diyalizden sonra enzim aktivitesinde azalma gözlendi. Çökelekteki proteinlerin molekül ağırlığı SDS- PAGE jel elektroforezi ile yaklaşık 12.500 Dalton olarak belirlendi. Ayçiçeği lipazının hidrolitik karakterizasyon çalışmasında, substrat spesifitesi ayçiçeği yağı, mısır özü yağı, zeytin yağı, tribütirin, triolein kullanılarak araştırıldı. Zeytin yağı spesifik substrat olarak belirlendi. Optimum pH, sıcaklık ve reaksiyon zamanı araştırıldı ve pH 8-9, sıcaklık 50-60°C ve zaman 5 dakika olarak bulundu. Enzimatik reaksiyonun kinetik sabitleri Lineweaver-Burk grafikleri yardımıyla Km: 0.71, Vmax: 360 U/mg-protein olarak belirlendi. Pankreatik lipaz için önemli bir aktivator olan Ca2+ iyonu, ayçiçeği lipazını yeterince aktive etmedi. Enzimin depo kararlılığı oldukça iyidir, altı aylık bir periyod içinde enzim aktivitesi sadece % 10 kadar azaldı.
SUMMARY The sunflower cultivation has been widely applied to produce vegetable oil in Trakya Region, Turkey. These sunflower seeds contain about 20-30 % protein and 50-60 % oil. Sunflower has been used edible oil production in Turkey and solid residue which is by-product after process has been only used as feed. It is not enough benefited from the sunflower seeds which is rich in protein. Therefore it was considered that it should utilize from high and valuable protein potential of seeds previously in human nutrition and scientific aims. In this study it was aimed to purify lipase enzyme from sunflower seeds and determine its hidrolytic catalytic properties. It was not encountered the purification and characterization studies releated with sunflower seed lipase in our literature researches. Seeds of sunflower species which is most cultivated in the region were ground with blender and its oil was removed in the presence of phosphate buffer (0. 1 M, pH 7) and organic solvent. To remove oil from sunflower seeds, five solvents is used and n-butanol is determined as optimum solvent. Then, lipase enzyme was partially purified by fractional ammonium sulphate precipitation and following dialysis. Lipase activity was determined 118 U/mg- protein in the 90 % salt precipitate which has the highest enzymatic activity. After dialysis, it was observed decreasing in enzymatic activity. Molecular weight of protein in the precipitate was determined as about 12.500 Dalton by SDS-PAGE Gel Electrophoresis. In hidrolytic characterization study of sunflower lipase, substrate specifity was investigated using sunflower oil, corn oil, olive oil, tributyrin, triolein as substrate. Olive oil was determined as specific substrate. Optimum pH, temperature and reaction time were investigated and found 8-9, 50-60°C and 5 min, respectively. The kinetic constans of enzymatic reaction was established as Km=0.71 and Vmax=360 U/mg-protein by using Lineweaver-Burk plots. Ca ion, which is an important activator for pancreatic lipase, was not enough activated sunflower lipase. The storage stability of enzyme against time was rather good, enzymatic activity decreased only about 10 % for six months period. II
SUMMARY The sunflower cultivation has been widely applied to produce vegetable oil in Trakya Region, Turkey. These sunflower seeds contain about 20-30 % protein and 50-60 % oil. Sunflower has been used edible oil production in Turkey and solid residue which is by-product after process has been only used as feed. It is not enough benefited from the sunflower seeds which is rich in protein. Therefore it was considered that it should utilize from high and valuable protein potential of seeds previously in human nutrition and scientific aims. In this study it was aimed to purify lipase enzyme from sunflower seeds and determine its hidrolytic catalytic properties. It was not encountered the purification and characterization studies releated with sunflower seed lipase in our literature researches. Seeds of sunflower species which is most cultivated in the region were ground with blender and its oil was removed in the presence of phosphate buffer (0. 1 M, pH 7) and organic solvent. To remove oil from sunflower seeds, five solvents is used and n-butanol is determined as optimum solvent. Then, lipase enzyme was partially purified by fractional ammonium sulphate precipitation and following dialysis. Lipase activity was determined 118 U/mg- protein in the 90 % salt precipitate which has the highest enzymatic activity. After dialysis, it was observed decreasing in enzymatic activity. Molecular weight of protein in the precipitate was determined as about 12.500 Dalton by SDS-PAGE Gel Electrophoresis. In hidrolytic characterization study of sunflower lipase, substrate specifity was investigated using sunflower oil, corn oil, olive oil, tributyrin, triolein as substrate. Olive oil was determined as specific substrate. Optimum pH, temperature and reaction time were investigated and found 8-9, 50-60°C and 5 min, respectively. The kinetic constans of enzymatic reaction was established as Km=0.71 and Vmax=360 U/mg-protein by using Lineweaver-Burk plots. Ca ion, which is an important activator for pancreatic lipase, was not enough activated sunflower lipase. The storage stability of enzyme against time was rather good, enzymatic activity decreased only about 10 % for six months period. II
Açıklama
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Yüksek Lisans
Yüksek Lisans
Anahtar Kelimeler
Kimya, Chemistry
