Kitosan boncuklara lipaz immobilizasyonu
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Dosyalar
Tarih
2012
Yazarlar
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Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışma kapsamında, defne tohumlarından (Laurus nobilis L.) izole edilen lipaz enzimi kitosan boncuklara immobilize edildi, serbest ve immobilize enzimlerin bazı özellikleri belirlendi ve birbirleriyle karşılaştırıldı. Çimlendirilen defne tohumlarından elde edilen lipaz enzimi kitosan boncuklara iki farklı yöntemle immobilize edildi. İlk yöntemde (K1 yöntemi) damlatma çözeltisi olarak 0.05 M Tris-HCl (pH 7.0) tamponunda hazırlanan pentasodyum trifosfat kullanıldı. Diğer yöntemde (K2 yöntemi) eşit hacimdeki glioksal hidrat ve sodyum pirofosfat tetrabazik karışımından oluşan çözelti kullanıldı ve elde edilen boncuklar glutaraldehitle aktive edildi. İmmobilizasyon verimi K1 yöntemi için % 78, K2 yöntemi için % 64 olarak bulundu. Hidrolitik lipaz aktivitesi serbest enzim, K1 ve K2 boncuklar için sırasıyla; 1.79 U/mg protein, 12.6 U/mg protein ve 16 U/mg protein olarak belirlendi. İmmobilizasyonların optimizasyonlarında, K1 yöntemi için; kitosan miktarı 250 mg, pentasodyum trifosfat konsantrasyonu % 0.4, enzim miktarı 2 mL, boncuk boyutu 2.9 mm, boncuk miktarı 0.25 g olarak bulundu. K2 yöntemi için; kitosan miktarı 1.25 g, glioksal hidrat konsantrasyonu % 3 ve sodyum pirofosfat tetrabazik konsantrasyonu % 4, glutaraldehit konsantrasyonu % 0.2, enzim miktarı 1 mL, boncuk boyutu 2.7 mm, boncuk miktarı 0.5 g olarak belirlendi. Optimum pH; serbest enzim, K1 boncuk ve K2 boncuk için, sırasıyla 7.0, 6.0 ve 8.0, optimum sıcaklık tümü için 40 °C olarak belirlendi. İmmobilizasyon işleminin defne lipazının termal kararlılığını iyileştirdiği görüldü. Km ve Vmax değerleri, serbest enzim, K1 ve K2 boncuk için sırasıyla 16.66 g ile 4.76 U/mg protein, 6.66 g ile 25 U/mg protein ve 1.56 g ile 14.28 U/mg protein olarak belirlendi. Yeniden kullanılabilirlik çalışmasında, K1 ve K2 boncukları 2 ve 3 döngü boyunca aktivitesini koruyabildi. Serbest enzim 16 gün, K1 boncuk 30 gün ve K2 boncuk 35 gün boyunca 4 °C'de aktivitesini korudu.
Abstract
In this study, lipase enzyme isolated from laurel seeds (Laurus nobilis L.) was immobilized onto chitosan beads and some properties of free and immobilized enzymes were determined, and compared with each other. Lipase enzyme isolated from germinated laurel seeds (Laurus nobilis L.) was immobilized onto chitosan beads by using two different methods. In the first method (K1 method), pentasodium triphosphate prepared in 0.05 M Tris-HCl (pH 7.0) buffer solution was used as dropping solution. In the other method (K2 method), the mixture contained equivalent volumes of glyoxal hydrate and sodium pyrophosphate tetrabasic was used as dropping solution and the obtained beads were activated with glutaraldehyde. Immobilization yields were found to be 78 % and 64 % for K1 and K2 methods, respectively. Hydrolytic lipase activities were determined as 1.79 U/mg protein, 12.6 U/mg protein and 16 U/mg protein for free enzyme, K1 and K2 beads, respectively. In the optimization of immobilizations, for K1 method, chitosan amount was 250 mg, pentasodium triphosphate concentration was 0.4 %, enzyme amount was 2 mL, bead size was 2.9 mm, bead amount was 0.25 g. For K2 method, chitosan amount was 1.25 g, glyoxal hydrate concentration was 3 %, sodium pyrophosphate tetrabasic concentration was 4 %, glutaraldehyde concentration was 0.2 %, enzyme amount was 1 mL, bead size was 2.7 mm, bead amount was 0.5 g. Optimum pH?s were determined as 7.0, 6.0 and 8.0 for free enzyme, K1 and K2 beads, respectively. Optimum temperatures of all enzyme samples were found to be 40 °C. Thermal stability of laurel lipase was improved by both immobilization treatments. Km-Vmax values were 16.66 g-4.76 U/mg protein, 6.66 g-25 U/mg protein and 1.56 g-14.28 U/mg protein for free enzyme, K1 and K2 beads, respectively. In the reusability assay, K1 and K2 beads saved their activities during 2 and 3 cycles, respectively. At 4 °C, free enzyme saved its activity for 16 days while K1 and K2 beads saved 30 and 35 days, respectively.
Abstract
In this study, lipase enzyme isolated from laurel seeds (Laurus nobilis L.) was immobilized onto chitosan beads and some properties of free and immobilized enzymes were determined, and compared with each other. Lipase enzyme isolated from germinated laurel seeds (Laurus nobilis L.) was immobilized onto chitosan beads by using two different methods. In the first method (K1 method), pentasodium triphosphate prepared in 0.05 M Tris-HCl (pH 7.0) buffer solution was used as dropping solution. In the other method (K2 method), the mixture contained equivalent volumes of glyoxal hydrate and sodium pyrophosphate tetrabasic was used as dropping solution and the obtained beads were activated with glutaraldehyde. Immobilization yields were found to be 78 % and 64 % for K1 and K2 methods, respectively. Hydrolytic lipase activities were determined as 1.79 U/mg protein, 12.6 U/mg protein and 16 U/mg protein for free enzyme, K1 and K2 beads, respectively. In the optimization of immobilizations, for K1 method, chitosan amount was 250 mg, pentasodium triphosphate concentration was 0.4 %, enzyme amount was 2 mL, bead size was 2.9 mm, bead amount was 0.25 g. For K2 method, chitosan amount was 1.25 g, glyoxal hydrate concentration was 3 %, sodium pyrophosphate tetrabasic concentration was 4 %, glutaraldehyde concentration was 0.2 %, enzyme amount was 1 mL, bead size was 2.7 mm, bead amount was 0.5 g. Optimum pH?s were determined as 7.0, 6.0 and 8.0 for free enzyme, K1 and K2 beads, respectively. Optimum temperatures of all enzyme samples were found to be 40 °C. Thermal stability of laurel lipase was improved by both immobilization treatments. Km-Vmax values were 16.66 g-4.76 U/mg protein, 6.66 g-25 U/mg protein and 1.56 g-14.28 U/mg protein for free enzyme, K1 and K2 beads, respectively. In the reusability assay, K1 and K2 beads saved their activities during 2 and 3 cycles, respectively. At 4 °C, free enzyme saved its activity for 16 days while K1 and K2 beads saved 30 and 35 days, respectively.
Açıklama
Yüksek Lisans Tezi
Anahtar Kelimeler
Defne, Sodyum Pirofosfat Tetrabazik, Pentasodyum Trifosfat, Enzim İmmobilizasyonu, Kitosan, Lipaz, Enzyme Immobilization, Sodium Pyrophosphate Tetrabasic, Pentasodium Triphosphate, Laurel Seeds, Lipase, Chitosan
