On-column refolding and characterization of an alkalinemetalloprotease from Pseudomonas aeruginosa NCCU expressed in Escherichia coli

• Main Authors: Chung Hsin, 林忠信
• Other Authors: Min Jen Tseng
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/29701774051284729536

碩士 === 國立中正大學 === 分子生物研究所 === 95 === Pseudomonas aeruginosa, an air-borne microorganism, secreted multiple proteases that played important roles in clinical infection and industrial enzyme application. In this thesis, the alkaline metalloprotease gene from Pseudomonas aeruginosa NCCU was cloned into a pET expression vector. Overexpression of C terminal 6xHis-tagged Pseudomonas alkaline proteases by IPTG induction in E. coli BL21 CondonPlus(DE3)-RIL resulted in the formation of inactive inclusion bodies. The renaturing ability in protease zymography was evaluated and showed a clear zone that located at the molecualr weight of 51 kDa. Therefore, the on Ni-column refolding approach was used to renature the inclusion bodies of this protease to obtain native enzyme for physical and chemical analysis. The inclusion bodies was first solublized in denaturant condition (8 M urea) and applied onto Ni-column. The refolding were performed with a gradient of decreasing concentration of urea (8 M → 0 M) and eluted the refolded enzyme with 0.5 M imidazole. The refolded alkaline metalloprotease show the optimal pH and temperature of 7 and 55℃ on the azocasein hydrolysis, respectively. The thermostability of the alkaline metalloprotease at 55 ℃ showed a half-life of 80 min. This enzyme was slightly inhibited with 1 mM of Co2+, Ni2+, Mn2+, EGTA, and EDTA at 55 ℃. The Km and Vmax are 0.057 mM and 3.3 × 1000 U/min•μmol, respectively, toward azocasein at 55 ℃ and pH 7。This study demonstrated the inclusion bodies of alkaline metalloproteinase from Pseudomonas aeruginosa NCCU can be refolded into active enzyme, however, the yield of recovry was quite low (~10%). We need to improve the refolding technique to increase the recovery yields.