Alginat+jelatin jel karışımlarında lipaz immobilizasyonu ve immobilize enzimin bazı biyokimyasal özelliklerinin belirlenmesi
No Thumbnail Available
Date
2011
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Trakya Üniversitesi
Access Rights
info:eu-repo/semantics/openAccess
Abstract
Bu çalışmada; Candida rugosa lipazı alginat+jelatin jellere immobilize edildi. Elde edilen immobilize enzimin optimum pH ve sıcaklık, Km ve Vmax kinetik sabitleri, termal ve operasyonel kararlılık, yeniden kullanılabilirlik ve depo kararlılığı gibi bazı biyokimyasal özellikleri belirlendi ve serbest enziminkiyle karşılaştırıldı.Ticari bir mikrobiyal lipaz olan Candida rugosa lipazı alginat+jelatin jellerde tutuklandı. Damlatma çözeltisi olarak CaCl2 kullanıldı. Protein tayinleri Bradford yöntemi uygulanarak gerçekleştirildi. İmmobilizasyon yüzdesi % 60, Serbest enzimin spesifik hidrolitik aktivitesi 22 U/ml protein, immobilize enzimin spesifik hidrolitik aktivitesi ise 36 U/mg protein olarak belirlendi. Hidrolitik enzim aktivitesi titrimetrik yöntemle, substrat olarak zeytinyağı kullanılarak belirlendi. Alginat+jelatin boncuklarda Candida rugosa lipazının immobilizasyon koşullarının optimizasyonunda; optimum alginat konsantrasyonu % 2, optimum jelatin konsantrasyonu % 15, CaCl2 konsantrasyonu % 7.7, optimum yüklenen enzim konsantrasyonu 20 mg/mL, optimum boncuk boyutu 2.9 mm ve optimum boncuk miktarı 1 gr olarak belirlendi.Alginat+jelatin jellerde tutuklanan Candida rugosa lipazı ve serbest lipaz için optimum pH 9.0 olarak belirlendi. Optimum sıcaklık serbest enzim için 45 ? C, immobilize enzim için 50 ? C olarak belirlendi. İmmobilize Candida rugosa lipazının termal kararlığını tutuklama sonrasında da nispeten koruduğu gözlendi. Serbest lipazın ve alginat+jelatin jel boncuklarda tutuklanan lipazın Km ve Vmax kinetik sabitleri sırasıyla, 0.29 g ile 30.3 U/mLdak, 2.5 g ile 83.33U/g boncuk dak olarak bulundu. Yeniden kullanılabilirlik çalışmasında; boncukların 5. döngüde aktivitesinin yaklaşık yarısını koruduğu gözlendi. Substrat spesifitesi için gliseriltributirat, trikaprilin, gliseriltristerat, aspir yağı, soya yağı, defne yağı, rafine fındık yağı ham kanola yağı, ham ayçiçeği yağı, ham zeytinyağı ve ham mısır yağı substrat olarak denendi. Depolama kararlılığı çalışmasında serbest ve immobilize enzimin her ikisinin de aktivitesini 18 gün boyunca koruduğu gözlendi.
In this study, Candida rugosa lipase was immobilized in alginate+gelatine gels. Some properties of obtained immobilized enzyme such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal and operational stability, reuse and storage stability were determined and compared to these of free enzyme.Candida rugosa lipase which is the commercial microbial lipase was entrapped in alginate+gelatine gels. CaCl2 was used as dropping solution. Protein determinations were done by using Bradford method. The immobilization percentage was determined to be 60 %. The specific hydrolytic activity of immobilized enzyme was found to be 36 U/g bead min. By using olive oil as swostrate the optimization of immobilization conditions of Candida rugosa lipase was done, and optimum alginate and gelatine concentrations were found to be 2 % and 15 % (w/v), respectively. Optimum CaCl2 concentration is % 7.7. Optimum bead diameter is 2.9 mm. Optimum bead? amount was determined to be 1 g. The loading enzyme concentration was determined to be 20 mg/mL.The optimum pHs of both free and lipase entrapped in alginate+gelatine gels were determined to be 9.0. Optimum temperatures of free and immobilized lipase in alginate+gelatine gels were found to be 45 ? C and 50 ? C, respectively. It was observed that immobilized lipase preserved relatively its thermal stability. Km and Vmax values were determined to be 0.29 g and 30.3 U/mL min for free enzyme, respectively. These values were 2.5 g and 83,33 U/g beads min for immobilized enzyme in alginate+gelatine beads, respectively. In the reusing study, beads saved about one-half of their activities during 5 cycles. Substrate specifities of both enzymes were carried out by using glyceryl tributyrate, tricaprylin, glyceryl tristearate, safflower oil, soybean oil, laurel oil, refined hazelnut oil, crude canola oil, crude sunflower oil, crude olive oil, crude corn oil, as substrates. It was observed that storage stability study, both enzymes saved their activities during 18 days.
In this study, Candida rugosa lipase was immobilized in alginate+gelatine gels. Some properties of obtained immobilized enzyme such as optimum pH and temperature, kinetic parameters (Km and Vmax), thermal and operational stability, reuse and storage stability were determined and compared to these of free enzyme.Candida rugosa lipase which is the commercial microbial lipase was entrapped in alginate+gelatine gels. CaCl2 was used as dropping solution. Protein determinations were done by using Bradford method. The immobilization percentage was determined to be 60 %. The specific hydrolytic activity of immobilized enzyme was found to be 36 U/g bead min. By using olive oil as swostrate the optimization of immobilization conditions of Candida rugosa lipase was done, and optimum alginate and gelatine concentrations were found to be 2 % and 15 % (w/v), respectively. Optimum CaCl2 concentration is % 7.7. Optimum bead diameter is 2.9 mm. Optimum bead? amount was determined to be 1 g. The loading enzyme concentration was determined to be 20 mg/mL.The optimum pHs of both free and lipase entrapped in alginate+gelatine gels were determined to be 9.0. Optimum temperatures of free and immobilized lipase in alginate+gelatine gels were found to be 45 ? C and 50 ? C, respectively. It was observed that immobilized lipase preserved relatively its thermal stability. Km and Vmax values were determined to be 0.29 g and 30.3 U/mL min for free enzyme, respectively. These values were 2.5 g and 83,33 U/g beads min for immobilized enzyme in alginate+gelatine beads, respectively. In the reusing study, beads saved about one-half of their activities during 5 cycles. Substrate specifities of both enzymes were carried out by using glyceryl tributyrate, tricaprylin, glyceryl tristearate, safflower oil, soybean oil, laurel oil, refined hazelnut oil, crude canola oil, crude sunflower oil, crude olive oil, crude corn oil, as substrates. It was observed that storage stability study, both enzymes saved their activities during 18 days.
Description
Yüksek Lisans
Keywords
Kimya, Chemistry
