Expression and purification of affinity glutathione S-transferase tag PHA polymerase in recombinant E. coli

• Main Authors: Ying-Li Lin, 林霙利
• Other Authors: 藍祺偉
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/00492229959856494421

碩士 === 元智大學 === 生物科技與工程研究所 === 98 === Polyhydroxyalkanoate(PHAs) is a bio-polymer, which can be accumulated by microbes. The mechanical properties of PHAs is similar to those traditional polymer including polyethylene(PE) and polypropylene(PP) . Most PHAs produced in vivo display the disadvantages of high fermentation and recovery cost, controlling of molecular weight and low yield. Hence, synthesis in vitro of PHAs has drawn much attention for both academia and industry. However, the synthase enzyme plays the key role in success of in vitro synthesis. In the present study, phaC gene derived from R.Eutropha H16 was constructed in recombinant E.coli with labeled GST-tag. The expressed GST-phaC was directly introduced to custom-made adsorbent for investigation of pH and temperature effect.The recovery, purity and behaviour of affinity adsorption were examined and discussed. Then the enzyme-particle was applied to synthase PHA in vitro. The results demonstrated that total protein concentration of 2.50 mg per mL under transferring fresh LB at 37℃ after preculture 6 hours can be achieved and highest specificity activity of 29.28 mU per mg under transferring fresh LB afterpreculture 6 hrs at 30℃ was obtained. Glutathione ligand solution reacted with STREAMLINETM and BBEL adsorbent can acheive 79 and 37% of glutathione bound after 48 hours. The recovery of GST-phaC STREAMLINETM adsorbent and GST Fusion Protein Purification Kit, reached 10.10 and 17.32% under PBS buffer at pH=6 and 25℃ conditions. Finally, we try to synthesize PHA using enzyme-particle.