Silicification of β-glucosidase for its immobilization on magnetic nanoparticle surface.

• Main Authors: Ming-Fen Chang, 張名棻
• Other Authors: Cheng-Kang Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/28701115747368470964

碩士 === 國立臺灣科技大學 === 化學工程系 === 101 === β-Glucosidase, from Aspergillus niger, was used as a model enzyme to be immobilized by silicification on magnetic nanoparticles. The enzyme catalysts the reaction of converting the colorless substrate p-nitrophenyl-β-D- glucopyranoside (p-NPG) to yellowish production p-nitrophenol. Enzymes immobilization by encapsulation in silica have been studied for some times but usually carried out by solvent containing sol-gel process. Recently, polycationic peptides isolated from diatom cell walls such as R5 have been used to induce the precipitation of silicic acid in the presence of enzyme for the purpose of enzyme immobilization in silica. In addition to polycationic peptides, polycationic compound such as polyethyleneimine (PEI) was found to be a good biomimicking catalyst can also induce the polycondensation of silicic acid. In this thesis, silicic acids from hydrolyzed tetramethyl orthosilicate (TMOS) and neutralized sodium silicate were employed to be precipitated by PEI coated on β-glucosidase immobilized magnetic nanoparticles (MPBP) which was prepared by immobilizing β-glucosidase on PEI coated magnetic nanoparticles (MP) via electrostatic interactions. The size of silicificated β-glucosidase magnetic particles is approximately 350 nm with β-glucosidase content about 4.3 mg/gram of dry MP. As compared with free enzyme, the relative activity of silicificated β-glucosidase is 91.84% with half-life about 4 times longer at 60℃. Moreover, immobilized β-glucosidase can be reused for 5 times still maintains 70% of its initial activity. The facile enzyme immobilization method demonstrated that silica encapsulation can protect the β-glucosidase from leakage and denaturation, and the β-glucosidase activity can be easily retrieved by applying a magnetic field.