Alginat kapsüllerinde tutuklanan Bacillus amyloliquefaciens α-amilaz enziminin farklı nişasta kaynaklarını hidrolizleme yeteneğinin araştırılması
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Date
2009
Authors
Dinçbaş, Serhan
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Publisher
Uludağ Üniversitesi
Abstract
Bu çalışmada Bacillus amyloliquefaciens kaynaklı -amilaz enzimi kalsiyum alginat kapsüllerinde immobilize edilmiştir. Serbest (immobilize edilmemiş) ve immobilize enzimin farklı nişasta kaynakları (çözünür (Merck) ve patates, mısır, buğday, pirinç gibi lokal marketlerden satın alınan ticari nişastalar) üzerindeki hidroliz yetenekleri araştırılmıştır. Enzim immobilizasyonu %2 alginat ve %5 CaCI2 varlığında gerçekleştirilmiştir. İmmobilizasyon verimi yaklaşık %89 olarak saptanmıştır. Her iki enzim formunun kullanılan nişasta kaynaklarını benzer bir tarzda parçaladıkları görüldü. İmmobilize enzimin sırasıyla ticari patates>çözünür patates> tic. buğday>tic. mısır>tic. pirinç şeklinde hidroliz ettikleri, serbest enzimin ise sırasıyla ticari patates>çözünür patates>tic. mısır>tic. buğday>tic. pirinç nişastalarını en iyi parçaladıkları saptanmıştır. Nişastaların düşük ve yüksek konsantrasyonlarında fazla bir aktivite saptanmamıştır. -amilazın immobilize ve serbest formunun sırasıyla %8 ve 10, %1 pirinç nişastası varlığında etki göstermediği belirlenmiştir. Serbest ve immobilize α-amilaz enzimlerinin sırasıyla, 50-600 C’ler arasındaki sıcaklıklarda ve 6.0-6.8 pH değerleri arasında optimum aktiviteye sahip oldukları tespit edilmiştir. İmmobilize enzim yüksek sıcaklıklarda serbest enzimden daha stabildi. α-amilaz enzim aktivitesi metal iyonlarına bağlı olduğundan, farklı metal iyonları kullanılarak bunların enzim aktivitesi üzerindeki etkileri araştırılmıştır. Hiçbir metal iyonunun göreceli olarak fazla bir aktivite artışı göstermemelerine rağmen, göreceli olarak bir değerlendirme yapıldığında Mg+2 ‘un her iki enzim formu üzerinde stimülatör etki yaptığı, buna karşın Cu+2’nun inhibitör etkisi saptanmıştır. Diğer yandan, serbest enzimin Li+2, Ba+2, Mn+2 ve Ca+2 varlığında immobilize enzime kıyasla daha aktif olduğu belirlenmiştir. Her iki formdaki enzimlerin subtsrat parçalanma ürünlerini belirlemek üzere yapılan ince tabaka kromatografisi sonuçlarına göre, enzimlerin nişastaları az miktarda glukoza, büyük miktarda da maltoz olmak üzere maltooligosakkaritlere ayrıştırdığı tespit edilmiştir. Serbest ve immobilize enzimlerin nişasta parçalanma ürünleri sırasıyla, G2=G3>G1 ve G2=G3>G1>G4 olarak görülmüştür.
In this study, α-amylase enzyme from Bacillus amyloliquefaciens was immobilized in calcium alginate beads. The ability of hydrolysis on various starch sources (soluble potato (Merck) and raw starches such as potato, maize, wheat, rice purchased from the local markets) of free (un-immobilized) and immobilized forms of α-amylase were investigated. Enzyme immobilization was performed in the solution of %2 alginate and %5 CaCI2. The efficiency of immobilization was determined approximately %89. It was indicated that both of enzyme forms hydrolysed using various starch sources on the similar pattern. Hydrolysis by immobilized enzyme was raw potato>soluble potato>raw wheat>raw maize>raw rice, respectively, while it was raw potato>soluble potato>raw maize>raw wheat>raw rice by free enzyme. At low and high concentrations of starches, the activity was not significant. Immobilized and free forms of α-amylase did not show any effect in the presence of 8 and 10%, 1% rice starch, respectively. It was determined that both the free and immobilized α-amylase had optimum activity at 50 and 600 C, at pH 6 and 6.8, respectively. The immobilized enzyme was more stabile than free enzyme in high temperature. Since -amylase activity is depended on metal ions, various metal ions for their effects on the enzyme activity were tested. Though an appearent stimulatory effect of any metal ions were not detected, a stimulator effect was observed for Mg+2 on both forms of enzyme, however, an inhibitor effect of Cu+2 was determined. On the other hand, free enzyme in the presence of Li+2, Ba+2, Mn+2 ve Ca+2 had a greater effect compared with immobilized enzyme. In according to the results of thin layer chromatography method that was performed in order to determine the split-products of starches hydrolysed by both enzymes, it was found that the main product was maltose within maltooligosaccharides and a little amount of glucose was also determined. The split-products of starches hydrolysed by free and immobilized enzymes were observed as G2=G3>G1 and G2=G3>G1>G4, respectively.
In this study, α-amylase enzyme from Bacillus amyloliquefaciens was immobilized in calcium alginate beads. The ability of hydrolysis on various starch sources (soluble potato (Merck) and raw starches such as potato, maize, wheat, rice purchased from the local markets) of free (un-immobilized) and immobilized forms of α-amylase were investigated. Enzyme immobilization was performed in the solution of %2 alginate and %5 CaCI2. The efficiency of immobilization was determined approximately %89. It was indicated that both of enzyme forms hydrolysed using various starch sources on the similar pattern. Hydrolysis by immobilized enzyme was raw potato>soluble potato>raw wheat>raw maize>raw rice, respectively, while it was raw potato>soluble potato>raw maize>raw wheat>raw rice by free enzyme. At low and high concentrations of starches, the activity was not significant. Immobilized and free forms of α-amylase did not show any effect in the presence of 8 and 10%, 1% rice starch, respectively. It was determined that both the free and immobilized α-amylase had optimum activity at 50 and 600 C, at pH 6 and 6.8, respectively. The immobilized enzyme was more stabile than free enzyme in high temperature. Since -amylase activity is depended on metal ions, various metal ions for their effects on the enzyme activity were tested. Though an appearent stimulatory effect of any metal ions were not detected, a stimulator effect was observed for Mg+2 on both forms of enzyme, however, an inhibitor effect of Cu+2 was determined. On the other hand, free enzyme in the presence of Li+2, Ba+2, Mn+2 ve Ca+2 had a greater effect compared with immobilized enzyme. In according to the results of thin layer chromatography method that was performed in order to determine the split-products of starches hydrolysed by both enzymes, it was found that the main product was maltose within maltooligosaccharides and a little amount of glucose was also determined. The split-products of starches hydrolysed by free and immobilized enzymes were observed as G2=G3>G1 and G2=G3>G1>G4, respectively.
Description
Keywords
B. amyloliquefaciens, α-amilaz, İmmobilizasyon, Kalsiyum alginat, Nişasta hidrolizi, İnce tabaka kromatografisi, α-amylase, Immobilization, Calcium alginate, Starch hydrolysis, Thin layer chromatography
Citation
Dinçbaş, S. (2009). Alginat kapsüllerinde tutuklanan Bacillus amyloliquefaciens α-amilaz enziminin farklı nişasta kaynaklarını hidrolizleme yeteneğinin araştırılması. Yayınlanmamış yüksek lisans tezi. Uludağ Üniversitesi Fen Bilimleri Enstitüsü.