Kestane Tohumundan (Castanea sativa) Lipaz Enziminin Saflaştırılması ve Kinetik Özelliklerinin İncelenmesi

Yıl 2018, Cilt: 3 Sayı: 3, 101 - 106, 18.12.2018

Bahar Bilgin Sökmen , Rıdvan İlgün

https://doi.org/10.35229/jaes.416800

Öz

Bu çalışmada, ülkemizde Giresun İli ve çevresinde bol miktarda yetişen kestane (Castanea Sativa) tohumlarından lipaz enzimi ilk defa saflaştırıldı ve kinetik özellikleri incelendi. Petrol eteri ile yağı uzaklaştırılmış kestane tohumları 0,06 M sodyum fosfat tamponu (pH=7,0) ile homojenize edildi. Lipaz esteraz aktivitesi gösteren homojenizatın % 90’lık (NH4)2SO4 kesiti elde edildi. Dializden sonra bu kesit sırasıyla hidroksilapatit ve DEAE-selüloz kolonlara uygulanarak lipaz enzimi 186,87 kez saflaştırıldı. SDS-PAGE elektroforezi sonucunda saflaştırılan enzimin molekül ağırlığının 30 kDa olduğu bulundu. Kestane tohumlarından saflaştırılan lipazın optimum pH’sının 9,0; optimum sıcaklığının 30 °C olduğu bulundu. Enzimin çeşitli substratlara karşı ilgisi incelendiğinde, lipazın ilgisinin en çok p-NFM’a olduğu ve Km ve Vmax değerlerinin sırasıyla 1,017 mM ve 163,874 U olduğu saptandı. 

Anahtar Kelimeler

Kestane (Castanea sativa) tohumu, lipaz, karakterizasyon, saflaştırma

Lipase Purification from the Seeds of chestnut (Castanea sativa) and Its Investigation of Some Kinetic Properties

Öz

In this study, lipase was firstly purified from chestnut (Castanea sativa) seeds, being abundant in Giresun (Turkey) and surroundings and the kinetic properties of the enzyme were investigated. Chestnut seeds, being fat-free with petroleum ether extraction, were homogenized with 0.06 M sodium phosphate buffer (pH=7.0) 90% of (NH4)2SO4 fraction of lipase esterase activity homogenate was obtained. After dialysis, this fraction was applied respectively to hydroxyapatite and DEAE-cellulose columns, and lipase was purified 186.87 times. The
purified enzyme, which showed SDS-PAGE, had a molecular weight of 30 kDa. It was found that the lipase purified from chestnut seeds had an optimum pH value of 9.0 and an optimum temperature of 30C. When affinity against various substrates of the enzyme is investigated, lipase affinite was found that have to p-NPM and its Km and Vmax values were dedected 1.017 mM and 163.874 U, respectively.

Anahtar Kelimeler

Kestane (Castanea sativa) tohumu, lipaz, karakterizasyon, saflaştırma

Kaynakça

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