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.

Optimization of immobilized Lactobacillus pentosus cell fermentation for lactic acid production

“…Box-Behnken design are applied in most studies for optimization, while Doehlert design and superlative box design have also been used in researches to obtain a credible result (Huang et al 2019;Wahla et al 2019;Al-Gheethi et al 2019;Sahin 2019;Chollom et al 2019).…”

Support Materials of Organic and Inorganic Origin as Platforms for Horseradish Peroxidase Immobilization: Comparison Study for High Stability and Activity Recovery

“…Titanium dioxide nanoparticles exhibiting UV resistance and photocatalytic properties have also been successfully used in enzyme immobilization. Sahin [16], for instance, used 3-(3,4-dihydroxyphenyl) propionic acid-functionalized TiO 2 nanoparticles as matrices for HRP immobilization. Given their abundant availability and polymeric nature, the use of non-metallic nanoparticles, including biodegradable chitosan or thermally stable silica, has also gained popularity [17].…”