HETEROLOGOUS EXPRESSION OF ZEBRA FISHD-AMINO ACID OXIDASE IN ESCHERICHIA COLI

• Main Authors: Jhen-Yang Chi, 蔡鎮陽
• Other Authors: I-Ching Kuan
• Format: Others
• Language: zh-TW
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/73075677355720114099

碩士 === 大同大學 === 生物工程學系(所) === 97 === D-amino acid oxidase (DAO), a flavoenzyme, using flavin adenine dinucleotide (FAD) as its prosthetic group can catalyze the oxidative deamination of D-amino acids to produce the corresponding keto acids, NH3 and H2O2. It is ubiquitously present in eukaryotes and plays important roles in metabolism, immunity and neurotransmission. In this study, we overexpressed zebra fish DAO (ZDAO) cDNA gene in E. coli and purify the recombinant protein by taking advantage of His-tag. When overexpressed in TB and TB containing sorbitol and betaine at 37 oC, the DAO activities reached the maximum 8 and 24hr after IPTG induction, respectively. The latter was 2000 U/L, two times more than the former. SDS PAGE analysis showed that the molecular mass for each subunit approximately was 40 kDa, however the native PAGE analysis suggested that ZDAO expressed with a His-tag existed as a heptamer. The optimal temperature and Tm were 60 oC and 43 oC, respectively. The pH optimum was at pH 10 while the best pH stability was at pH 9. The half-lives for the oxidative resistance to 10 、20 and 30 mM H2O2 were 90, 55 and 45 min, respectively. The best substrate was D-Phe with a kcat/KM value of 9.4 s-1 mM-1.