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

Yıl 2019, Cilt: 23 Sayı: 3, 904 - 916, 25.12.2019

Selmihan Şahin

https://doi.org/10.19113/sdufenbed.557021

Cited By: 4

Öz

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. 

Anahtar Kelimeler

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

Öz

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. 

Anahtar Kelimeler

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

Kaynakça

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