A new strategy for oriented immobilization of glucose oxidase

• Main Authors: Guang-ze Chen, 陳光澤
• Other Authors: Ruoh-chyu Ruaan
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/45221163838591659863

碩士 === 國立中央大學 === 化學工程與材料工程研究所 === 98 === In the past decades, protein chips are an important tool for applying in the examinations of bioassay and disease, but the examination efficiency of protein chips usually decrease as immobilized protein adopting random orientation. In this study, a new strategy for oriented immobilization of proteins was proposed. The strategy contains two steps. The first step is to search for a docking site away from the active site on the protein surface. The second step is trying to find a ligand that is able to grasp the targeted site of the protein. To avoid ligand binding to the active site of protein, the targeted docking site is selected to own opposite charges to those near the active site. To enhance the ligand-protein binding, both hydrophobic and electrostatic interactions need to be included. The targeted docking site should therefore contain hydrophobic amino acids. The ligand is then selected through the help of molecular docking simulations. The enzyme glucose oxidase (GOX) derived from Aspergillus niger was taken as an example for oriented immobilization. The active site of GOX is surrounded by hydrophilic amino acids. All the possible hydrophobic sites on the surface of GOX were evaluated by the free energy estimation through naphthalene docking. A hydrophobic site on the opposite side of GOX’s active site was found to be positive in net charges. A possible peptide ligand, LLGEG, was found to catch GOX by the designated docking site. Then, the LLGEG molecules were grafted onto silica gels and measured the affinity of GOX adsorption and the specific activity of thereby immobilized enzymes. It was found that GOX had a dissociation constant as low as 1.69×10-6 M toward the ligand LLGEG on silica gel. The decrease in ionic strength has little effect on desorption of GOX, which indicated the existence of hydrophobic and electrostatic interactions between ligands and proteins. The specific activity of the III immobilized GOX was compared with the randomly adsorbed GOX on primary amine containing silica gel. It was found that the orderly immobilized GOX owns a specific activity with about five-folds as high as the one randomly adsorbed by ionic interaction. Consequently, this new strategy for protein oriented immobilization by designing the proper peptide ligand through the help of molecular docking is executable.