Optimization of the Immobilization Conditions of Horseradish Peroxidase on TiO2-COOH nanoparticles by Box-Behnken Design

Abstract

In this study, TiO₂ nanoparticles were prepared and -COOH functionalized with 3-(3,4-dihydroxyphenyl) propionic acid. The characterization of nanoparticles was performed by FTIR, TEM, EDS and XRD. HRP was immobilized on those nanoparticles by EDC/NHS coupling reaction. The immobilization conditions of HRP including A: enzyme concentration (0.5-1.5 mg/mL), B: immobilization pH (4.0-8.0), C: immobilization temperature (4-50°C), D: immobilization time (1-20 h) were optimized by response surface methodology and Box-Behnken design. The optimized immobilization conditions were identified as 0.5 mg/mL HRP, at pH 5.5, 40 °C for 8 h for activity of immobilized HRP, 1.5 mg/mL HRP, at pH 4 and 18°C for 20 h for protein binding yield (%). At these optimum conditions, the experimental value for the activity of immobilized HRP was 80.39 U ± 1.06; protein binding yield was 94.25 ± 3.58%. Moreover, the optimum temperature and pH of free and immobilized enzyme were determined as 50°C and 4.0; 50°C and 3.5, respectively. The activity of the immobilized HRP sustained 52% of its initial activity after 10 days storage at 4°C. Furthermore, the immobilized HRP sustained 48% of its initial activity after 6 consecutive reactions. 

Keywords

• Immobilization,Horseradish peroxidase,TiO2 nanoparticles,EDC/NHS coupling,Carboxyl group functionalization

Horseradish Peroksidaz Enziminin TiO2-COOH Nanopartiküller Üzerine İmmobilizasyon Koşullarının Box-Behnken Metodu ile Optimize Edilmesi

Abstract

Bu çalışmada, TiO₂ nanopartiküller hazırlandı ve 3-(3,4-dihidroksifenil) propiyonik asit ile –COOH fonksiyonelleştirildi. FTIR, TEM, EDS ve XRD ile nanopartiküllerin karakterizasyonu gerçekleştirildi. HRP elde edilen nanopartiküllerin üzerine EDC/NHS bağlama yolu ile immobilize edildi. A: enzim konsantrasyonu (0.5-1.5 mg/mL), B: immobilizasyon pH’sı (4.0-8.0), C: immobilizasyon sıcaklığı (4-50°C), D: immobilizasyon süresi (1-20 h)’ni içeren HRP’nin immobilizasyon koşulları yanıt yüzey yöntemi ve Box-Behnken dizayn kullanılarak optimize edildi. Optimize edilen immobilizasyon koşulları, immobilize HRP aktivitesi için 0.5 mg/mL HRP, pH 5.5, 40 °C ve 8 saat, protein bağlama verimi (%) için 1.5 mg/mL HRP, pH 4, 18°C ve 20 saat olarak belirlendi. Bu immobilizasyon koşullarında immobilize HRP için elde edilen deneysel değer 80.39 U ± 1.06 iken, protein bağlama verimi 94.25 ± 3.58%’dir. Bunun dışında serbest ve immobilize enzimin optimum sıcaklık ve pH değeri sırasıyla 50°C ve 4.0; 50°C ve 3.5 olarak belirlendi. 4°C’de 10 gün saklama sonunda immobilize HRP’nin başlangıç aktivitesinin %52’si kalmıştır. Ayrıca immobilize HRP 6 kez arka arakaya kullanım sonucunda başlangıç aktivitesinin %48’ini sürdürmüştür. 

Keywords

• İmmobilizasyon,Horseradish peroksidaz,TiO2 nanopartiküller,EDC/NHS bağlama,Karboksil grubu fonksiyonelleştirmesi

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