Action Mechanism of Secondary Bond Forces on Chemical Immobilization of Horseradish Peroxidase

Abstract

Glycidyl methacrylate (GMA) was graft-polymerized onto micron-sized silica gel particles. Ethylenediamine (EDA) was bonded to the surfaces of PGMA/SiO2 particles by the ring-opening reaction of epoxy groups resulting in the difunctional composite carrier, EDA-PGMA/SiO2, which was used for enzyme immobilization. We immobilized horseradish peroxidase (HRP) using the chemical bonding method. In this work, the effects and action mechanisms of two secondary bond forces, electrostatic interaction and hydrophobic interaction, on the enzyme immobilization were investigated. The experimental results show that the protonated amino groups on the EDA-PGMA/SiO2 particles enable the carrier particles to be positively charged and the zeta potential of the carrier particles are positive over a wider range of pH values. At a pH value of 8.5 for the medium, which is higher than the isoelectric point of HRP, the strong electrostatic interaction between the enzyme protein and the carrier significantly promotes the immobilization of HRP. For the carrier with an EDA bonding rate of about 30%, the strongest electrostatic interaction was observed between the enzyme protein and the carrier while the immobilized enzyme has the highest coupling rate and specific activity. Hydrophobic interaction between the enzyme protein and the carrier also affects HRP immobilization greatly. As the grafted particles PGMA/SiO2 are used as the carrier, the addition of NaCl electrolyte will facilitate the hydrophobic interaction between the enzyme protein and the carrier and it will result in an increase in the coupling rate and specific activity of the immobilized enzyme. [Article] www.whxb.pku.edu.cn 物理化学学报(Wuli Huaxue Xuebao) Acta Phys. -Chim. Sin. 2011, 27 (11), 2697-2704 November Received: June 27, 2011; Revised: September 1, 2011; Published on Web: September 13, 2011. ∗Corresponding author. Email: gaobaojiao@126.com; Tel: +86-351-3921414. The project was supported by the Natural Science Foundation of Shanxi Province, China (201002100843). 山西省自然科学基金(201002100843)资助项目 C Editorial office of Acta Physico-Chimica Sinica 2697 Acta Phys. -Chim. Sin. 2011 Vol.27