Protein engineering of the D-amino acid oxidase

• Main Authors: Tung-Lin Wu, 吳東嶺
• Other Authors: I-Ching Kuan
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/34030596457897385977

碩士 === 大同大學 === 生物工程研究所 === 91 === Yeast Rhodosporidium toruloides (CCRC 20306) DAO cDNA gene was overexpressed in the heterologous host E. coli strain BL-21(DE3). The DAO expression induced by IPTG at lower temperature was better than those induced at 37℃. The highest intracellular DAO activity obtained was ~5000 U/l from cultures induced 5 hours at 30℃. The effects of pH and temperature on the activity and stability of heterologous expressed R. toruloides and Trigonopsis variabilis (CCRC 21509) DAO were also examined. The optimal pH for catalysis was 8.5~9 for both DAO, but the former had better pH stability. The optimal catalytic temperatures for R. toruloides and T. variabilis were 40~45℃and 45~50℃, respectively. The latter also had better thermostability. The presence of D-alanine could dramatically increase their pH stability and thermostability. The presence and position of His˙tag in DAOs showed little influence on their enzymatic characteristics. R. toruloides and T. variabilis DAO cDNA genes were together subject to DNA shuffling to generate mutant proteins. Only 4 active E. coli clones with DAO activity were obtained after extensive screening and showed similar DAO activity to the original proteins.