▎ 摘　　要

The covalent bonding process was applied to immobilize horseradish peroxidase (HRP) onto a functionalized reduced graphene oxide with size of 60 nm through glutaraldehyde as a cross-linker. The catalytic constant, k(cat), and the catalytic efficiency, k(cat)/K-m, increased 6.5 and 8.5 times, respectively, after immobilization. The circular dichroism analysis revealed that the alpha-helical content decreased from 18% to 10% after immobilization. The reusability of HRP was improved by immobilization and 70% of initial activity retained after 10 cycles. Due to the buffering effect, the immobilized HRP was less sensitive to pH changes than the free HRP. At 40 degrees C, the immobilized HRP retained 90% of the initial activity while 60% initial activity remained for the free HRP after 120 minutes. After 35-day storage, the activity reached 97% of initial activity for the immobilized HRP. The removal efficiency for high phenol concentration (2500 mg/L) was 100% and 55% for the immobilized HRP and free HRP, respectively. (C) 2017 Elsevier B.V. All rights reserved.