聚甲基丙烯酸二羥基乙酯固定化金屬親和吸附材於蛋白質純化之應用

• Main Author: 劉哲良
• Other Authors: 林松池
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/24746261523489076892

碩士 === 國立中興大學 === 化學工程學系 === 91 === In this study, polyhydroxymethacrylate ( pHEMA ) microbeads were prepared by a suspension polymerization technique and modified by IDA( iminodiacetic acid ), loading with Ni2+, and used as adsorbent to purify a target protein (D-hydantoinase , DHTase) from the large volumes of liquid culture medium. In the text, the such are factors that influence the adsorption of DHTase are discussed, for example: category of metal ion, crosslinking ratio (EGDMA to HEMA) , pH ionic,salt composition, and NaCl conc. in the buffer. The results indicate that the optimum adsorbent crosslinking ratio is 2.0/30 and water content is 46%. The optimum purification procedure condition is 4℃、0.5M NaCl、pH 6.0、50mM Tris-HCl buffer for loading, and 4℃、300mM NaCl、300mM Imidazole、pH 8.0、50mM phosphate buffer for elution, which results in a total protein adsorption incrase from 3.9 mg g-1 (under non-optimal condition) to 7.8 mg g-1, a total protein elution increase from 2.39 mg g-1 to 4.33 mg g-1, and a purification of 91%. After seven repeated uses, metals ion was loss about 30%, and total protein elution also declines to 50%. The adsorption phenomena appeared to follow a typical Langmuir isotherm. The maximum capacity (Qm) of the Ni2+ -complexed beads for D-hydantoniase is 10.17 mg g-1, and the dissociation constant (kd) is 1.12*10-2 g ml-1.