Katı substrat fermentasyonu ile ham nişastayı parçalayan yeni bir fungal amilaz üretimi saflaştırılması ve biyokimyasal özelliklerinin belirlenmesi
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Dosyalar
Tarih
2008
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Trakya Üniversitesi Fen Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/openAccess
Özet
Bu çalışmada, yeni bir ham nişasta hidrolizleyen fungal amilazın SSF yöntemi ile üretilmesi, saflaştırılması ve bazı biyokimyasal özelliklerinin belirlenmesi amaçlanmıştır. Ham nişastayı hidrolizleyen amilaz taraması sonucunda en iyi aktiviteye Penicillium brevicompactum' un sahip olduğu bulundu ve amilaz kaynağı olarak kullanıldı. Buğday kepeği, pirinç kabuğu ve ayçiçeği küspesi en iyi substratı belirlemek için test edildi. En iyi katı substratın buğday kepeği olduğu belirlendi. Amilaz üretimi için katı substrat fermentasyon şartları optimize edildi. Optimum fermentasyon şartları, başlangıç nem içeriği % 55, nemlendirici ajan 0.1 M sodyum fosfat tamponu (pH 5.0), üretim süresi 7 gün, aşı miktarı 2.5 mL ve üretim sıcaklığı 30 oC olarak saptandı. Penicillium brevicompactum amilazı nişasta afinite metodu kullanılarak 45.98 kez saflaştırıldı. Saflaştırılan enzimin çözünür nişasta için Km değeri 5.71 mg/ml, Vmax değeri ise 666.6 U/ml olarak hesaplandı. Enzim 30-50 oC arasında ve pH 5.0 de maksimum aktivite gösterdi. 30 oC' de 45 dk. inkübasyondan sonra başlangıç aktivitesini %100 korudu. pH 4.0-5.0 arasında kararlı idi. Mn+2, Cu+2 and Na+ iyonları enzim aktivitesini arttırırken Mg+2, K+, Fe+3 ve EDTA enzim aktivitesini inhibe etti. P. brevicompactum ?-amilazının molekül ağırlığı SDS-PAGE ile 32.5 kDa olarak tespit edildi. Anahtar Kelimeler: ?-amilaz, katı substrat fermentasyon, ham nişasta sindirimi, saflaştırma, Penicillium brevicompactum.
In this study, it was intended to the production of new a fungal amylase with solid state fermentation, purification and also to determine its some biochemical properties. It was found that Penicillium brevicompactum had the best enzyme activity according to raw starch degrading amylase screening methods, and P. brevicompactum was selected as amylase source. Wheat bran, rice husk and sunflower oil meal were tested to determine the best solid substrate. Wheat bran was determined as the best solid substrate. The fermentation contitions were optimized for the production of amylase. The optimum fermentation conditions were found to be initial moisture level of solid substrate of 55 %, moistening agent of 0.1 M sodium phosphate buffer (pH 5.0), incubation period of 7 days, inoculum concentration of 2.5 mL and incubation temperature at 30 oC. P. brevicompactum ?-amylase was purified 45.98 times by using starch affinity method. The Km and Vmax values of ?-amylase for soluble starch were 5.71 mg/ml, 666.6 U/ml respectively. This amylase showed maximum activity at between 30-50 oC and pH 5.0. Initial enzyme activity was kept to be 100% after incubation at 30 oC for 45 min. Enzyme was stable in the pH range of 4.0-5.0. This enzyme was activated by Mn+2, Cu+2 and Na+ ions, and inhibited by Mg+2, K+, Fe+3 and EDTA. The molecular weight of P. brevicompactum ?-amylase was found as 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Key words: ?-amylase, solid substrat fermentation, raw starch digesting, purification, Penicillium brevicompactum
In this study, it was intended to the production of new a fungal amylase with solid state fermentation, purification and also to determine its some biochemical properties. It was found that Penicillium brevicompactum had the best enzyme activity according to raw starch degrading amylase screening methods, and P. brevicompactum was selected as amylase source. Wheat bran, rice husk and sunflower oil meal were tested to determine the best solid substrate. Wheat bran was determined as the best solid substrate. The fermentation contitions were optimized for the production of amylase. The optimum fermentation conditions were found to be initial moisture level of solid substrate of 55 %, moistening agent of 0.1 M sodium phosphate buffer (pH 5.0), incubation period of 7 days, inoculum concentration of 2.5 mL and incubation temperature at 30 oC. P. brevicompactum ?-amylase was purified 45.98 times by using starch affinity method. The Km and Vmax values of ?-amylase for soluble starch were 5.71 mg/ml, 666.6 U/ml respectively. This amylase showed maximum activity at between 30-50 oC and pH 5.0. Initial enzyme activity was kept to be 100% after incubation at 30 oC for 45 min. Enzyme was stable in the pH range of 4.0-5.0. This enzyme was activated by Mn+2, Cu+2 and Na+ ions, and inhibited by Mg+2, K+, Fe+3 and EDTA. The molecular weight of P. brevicompactum ?-amylase was found as 32.5 kDa by SDS-polyacrylamide gel electrophoresis. Key words: ?-amylase, solid substrat fermentation, raw starch digesting, purification, Penicillium brevicompactum
Açıklama
Doktora
Anahtar Kelimeler
Biyoloji, Biology
