Renaturation of fructosyl amino acid oxidase inclusion bodies

• Main Authors: Po-Sheng Kang, 康博勝
• Other Authors: Sung-Chyr Lin
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/ayv2ra

碩士 === 國立中興大學 === 化學工程學系所 === 107 === Recombinant fructosyl amino acid oxidase was over-expressed in Escherichia coli, but most of them formed insoluble and inactive inclusion bodies (IBs). In this thesis, we used urea as solubilizing agent to denature inclusion bodies and obtain functional protein from it. We denatured the inclusion bodies with different concentrations of urea and solubilizing temperature, and compared the effect of urea concentration on total protein recovery by direct dilution method (DDM). It has been shown that 5M Urea 100 mM phosphate, pH=8.0 would be the centerpiece of solubilization system at 4 °C. In order to prevent low yield protein recovery from folding-intermediate aggregation, we investigated the pH of the refolding environment and the effect of different chemical additives on the refolding yield. We determined 100 mM Tris pH=9.0 as the refolding buffer in this study and the soluble protein recovery was 57.12% by DDM. Compared with non-additives, the recovery yield of refolding buffer which was accompanied by 0.8 M arginine, 40% glycerol, 1% Triton X-100 is higher. It has been shown the highest recovery of soluble protein is 90.92% by the addition of 40% glycerol. In addition, the effect of different additive combinations was investigated. The highest recovery of soluble protein is 93.04% with 0.8M arginine and 40% glycerol as co-solute in the refolding buffer. However, there was a limit on the recovery yield when the combination of Triton X-100 and other additives.