Cross-Linked Glucose Oxidase Aggregates: Synthesis and Characterization

Abstract

The aim of the submitted study is to realize carrier-free immobilization as cross-linking enzyme aggregates of Aspergillus niger Glucose Oxidase enzyme, to determinate the optimum immobilization conditions, and to investigate the properties of immobilized enzyme sets. Carrier-free enzyme immobilization was realised by forming cross-linked glucose oxidase enzyme aggregates CLEA’s . Different precipitants were used in different media conditions during forming CLEA’s. The “immobilized enzyme activity” was measured by activity determination method with the use of glucose substrate. The optimum conditions which were determined in the light of the experiments for the cross-linked enzyme aggregates can be summarized as; the initial enzyme concentration, 0.05 mg/ml; the optimum temperature, 25ºC; the precipitant type, BSA; the precipitant concentration, 5 mg/ml; the concentration of glutaraldehyde, 2% v/v . After determining the optimum conditions, glucose solutions at different concentrations 1.0-4.0 mg/dl were prepared for investigating the performance of the glucose oxidase aggregates. The kinetic parameters were calculated by Lineweaver-Burk plots such as; Km =0.0115 mM; Vm =1.206 mM.min-1 for native enzyme and Km = 0.025 mM; Vm =0.593 mM.min-1 for cross-linking enzyme aggregates.

Keywords

• Glucose oxidase,carrier-freeenzyme Immobilization,cross-linked enzyme aggregates,CLEA

Çapraz Bağlı Glukoz Oksidaz Agregatları: Sentez ve Karakterizasyonu

Abstract

S unulan çalışmanın amacı Aspergillus niger Glukoz Oksidaz enziminin taşıyıcısız immobilizasyonunu çapraz bağlı enzim agregatları olarak gerçekleştirmek, en uygun immobilizasyon koşullarını bulmak ve immobilize enzim kümelerinin özelliklerini belirlemektir. Taşıyıcısız enzim immobilizasyonu çapraz bağlı glukoz oksidaz enzim agregatları ÇBEA oluşturarak uygulanmıştır. ÇBEA’nın oluşumu sırasında farklı çöktürücüler yine farklı ortamlarda kullanılmıştır. “İmmobilize enzim aktivitesi” aktivite tayin yöntemine göre glukoz substratı kullanılarak ölçülmüştür. Deneylerin sonunda çapraz bağlı enzim agregatları için bulunan ve en yüksek enzim aktivitesini veren en uygun koşullar: başlangıç enzim derişimi, 0.5 mg/ml; en uygun sıcaklık, 25oC; çöktürücü türü, Bovin Serum Albümin BSA ; çöktürücü derişimi, 5 mg/ml; ve glutaraldehit derişimi, % 2 v/v . En uygun koşulların belirlenmesinden sonra glukoz oksidaz agregatlarının performanslarını belirlemek üzere farklı derişimlere sahip glukoz çözeltileri hazırlandı 1.0 - 4.0 mg/dl . Verilerin Lineweaver-Burk doğrusu olarak çizimi ile kinetik parametreler Km= 0.0115 mM ve Vm=1.206 mM.min-1 serbest enzim için ve ÇBEA için Km= 0.025 mM; V=0.593 mM.min-1 olarak hesaplanmıştır. m

Keywords

• Glukoz oksidaz,taşıyıcısız enzim immobilizasyonu,çapraz bağlı enzim agregatları,ÇBEA

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