物理化学学报 >> 2011, Vol. 27 >> Issue (11): 2697-2704.doi: 10.3866/PKU.WHXB20111122

生物物理化学 上一篇    下一篇

次价键力在化学键合法固定辣根过氧化酶过程中的作用机理

雷青娟, 高保娇, 张正国   

  1. 中北大学化学工程系, 太原 030051
  • 收稿日期:2011-06-27 修回日期:2011-09-01 发布日期:2011-10-27
  • 通讯作者: 高保娇 E-mail:gaobaojiao@126.com
  • 基金资助:

    山西省自然科学基金(201002100843)资助项目

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

LEI Qing-Juan, GAO Bao-Jiao, ZHANG Zheng-Guo   

  1. Department of Chemical Engineering, North University of China, Taiyuan 030051, P. R. China
  • Received:2011-06-27 Revised:2011-09-01 Published:2011-10-27
  • Contact: GAO Bao-Jiao E-mail:gaobaojiao@126.com
  • Supported by:

    The project was supported by the Natural Science Foundation of Shanxi Province, China (201002100843).

摘要: 将甲基丙烯酸缩水甘油酯(GMA)接枝聚合在微米级硅胶微粒表面, 又通过环氧基团的开环反应将乙二胺(EDA)键合在接枝微粒表面, 制备了双功能复合载体EDA-PGMA/SiO2, 通过共价偶联法实施了辣根过氧化酶(HRP)的固定化. 本文重点考察静电相互作用与疏水相互作用两种次价键力在共价键合法固定HRP过程中的作用, 探讨作用机理. 结果表明, 在水介质中, 复合载体EDA-PGMA/SiO2表面胺基的质子化作用, 使载体微粒的ζ电位在较大的pH范围内保持正值; 当介质的pH=8.5, 大于HRP的等电点时, 酶蛋白与载体之间所产生的强静电相互作用会显著促进HRP的固定化; EDA的键合率在30%附近的载体, 静电相互作用对固酶的促进作用最强, 固定化酶的偶联率与比活力具有最高值. 疏水相互作用对化学法固定辣根过氧化酶也会产生明显的作用, 当以接枝微粒PGMA/SiO2为载体时, 增大NaCl浓度, 可有效促进酶蛋白与载体之间的疏水相互作用, 提高固定化酶的偶联率与比活力.

关键词: 辣根过氧化酶, 固定化酶, 甲基丙烯酸缩水甘油酯, 复合载体, 静电相互作用, 疏水相互作用

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.

Key words: Horseradish peroxidase, Immobilized enzyme, Glycidyl methacrylate, Composite carrier, Electrostatic interaction, Hydrophobic interaction

MSC2000: 

  • O641