Applications of Nano-sized Magnetic Carriers in Biocatalysis and Bioseparation

• Main Authors: Min-Hung Liao, 廖敏宏
• Other Authors: Dong-Hwang Chen
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/rg9eh3

博士 === 國立成功大學 === 化學工程學系碩博士班 === 91 === 　　This dissertation concerns the applications of nano-sized magnetic carries in biocatalysis and bioseparation. In the former, yeast alcohol dehydrogenase (YADH) was immobilized on Fe3O4 magnetic nanoparticles. The preparation conditions, product properties, and the performances in both the water and microemulsion systems were investigated. In addition, the stability and activity of YADH in the mixed reverse micelles was also studied. In the latter, polyacrylic acid (PAA) was covalently bound onto Fe3O4 magnetic nanoparticles to be a novel nano-adsorbent. The preparation conditions, product properties, and the application in the adsorption of lysozyme in aqueous solution were investigated. 　　The stability and activity of YADH in the mixed reverse micelles were studied by adding Brij30 to the AOT reverse micelles. By the investigation on the hydrodynamic diameter of mixed reverse micelles and its distribution via dynamic light scattering, it was suggested that the structure of mixed reverse micelles and the stability of YADH were determined by four important factors, including the surface charge density, bound water, reverse micellar size, and the entrapment of water by hydrophilic-hydrophilic interaction of AOT and Brij30. The effects of these four factors on the stability of YADH at various Wo values and Brij30 concentration have been discussed. When they were decreased, the stability of YADH might be improved. In addition, it was found that the activity of YADH in AOT/Brij30 mixed reverse micelles might be enhanced at appropriate Brij30 concentrations and ω0 values. According to the hydrodynamic diameter of mixed reverse micelles and its distribution, three main factors were suggested. They were the hydrophobic and electrostatic interactions between enzyme and surfactants, the reverse micellar size, and the bound degree of water molecules. An optimal reverse micellar size and the decreases of other two factors would lead to the enhancement of enzyme activity. 　　YADH was covalently bound onto Fe3O4 magnetic nanoparticles via carbodiimide activation. The magnetic nanoparticles with a mean diameter of 10.6 nm were prepared by co-precipitating Fe2+ and Fe3+ ions in an ammonia solution and treating under hydrothermal conditions. From the analyses of Transmission electron microscopy (TEM), X-ray diffraction (XRD) and magnetism, the magnetic nanoparticles showed no changes in size, structure and superparamagnetic characteristics after binding YADH. The analysis of Fourier transform infrared (FTIR) spectroscopy confirmed the binding of YADH to magnetic nanoparticles and suggested a possible binding mechanism. The bound YADH retained 62% of its original activity and exhibited improved stability. The kinetic behavior of bound YADH was also determined in aqueous solution. In addition, the performance of YADH-bound magnetic nanoparticles in the NADH-containing water-in-oil microemulsions of water/AOT/isooctane was examined. Both water and NADH were present in the aqueous phase of microemulsion solution and on particle surface. The thickness of aqueous film on particle surface increased with increasing the Wo value. At a constant NADH amount, the concentration of NADH in the aqueous phase of microemulsion solution was not significantly affected by the Wo value. The specific activity of bound YADH in the microemulsion system was 40% of that in aqueous solution. The bound YADH showed excellent storage stability and good thermal stability in the microemulsion system. The kinetic behavior of bound YADH in the microemulsion system was also determined. 　　PAA was covalently bound onto Fe3O4 magnetic nanoparticles via carbodiimide activation. The magnetic nanoparticles with a mean diameter of 13.2 nm were prepared by co-precipitating Fe2+ and Fe3+ ions in an ammonia solution and treating under hydrothermal conditions. From the analyses of TEM, XRD and magnetism, the magnetic nanoparticles showed no change in size, structure and superparamagnetic characteristics after binding PAA. The analyses of FTIR, thermogravimetric analysis (TGA), differential thermal analysis (DTA) and X-ray photoelectron spectroscopy (XPS) confirmed the binding of PAA to magnetic nanoparticles and suggested the binding mechanism of PAA. The ionic exchange capacity of the resultant magnetic nano-adsorbents was estimated to be 1.64 meq/g, much higher than those of the commercial ionic exchange resins. When the magnetic nano-adsorbents were used for the recovery of lysozyme, it was found that the adsorption/desotption of lysozyme was achievable within 1 min due to the absence of pore-diffusion resistance and the specific activity of recovered lysozyme retained 95% of its original activity. In addition, the adsorption behavior followed the Langmuir adsorption isotherm.