斑馬魚D型胺基酸氧化酶於大腸桿菌中的異源表現

HETEROLOGOUS EXPRESSION OF ZEBRA FISH D-AMINO ACID OXIDASE IN ESCHERICHIA COLI

蔡鎮陽(Jhen-Yang Chi)

指導教授 ： 官宜靜

大同大學/工程學院/生物工程學系所/碩士(2009年)

摘要

D型胺基酸氧化酶 (D-amino acid oxidase; DAO)是一種以FAD為
輔酶的flavoenzyme，可催化D型胺基酸產生α-keto acid、NH3及H2O2，
普遍的存在於真核生物體中，於代謝、免疫及神經傳導上皆扮演了重
要的角色。本研究將斑馬魚DAO基因置於E. coli中進行大量表現，再
利用表現蛋白具His-tag的特性來進行純化，以利後續分析。於37 oC
下分別以TB及含sorbitol與betaine的TB進行培養，經IPTG誘導後，分
別在第8小時及第24小時可得活性高峰。約為1150及2000 U/L；經純
化後，以SDS PAGE分析確認每個次單元分子量約為40 kDa；Native
PAGE分析則發現含His-tag的斑馬魚DAO是以7個次單元聚集的形式
存在；最適反應溫度為60oC，Tm為43 oC，最適反應pH為10，熱穩定
性於pH 9時最佳；對10 、20及30 mM H2O2 處理的半衰期分別約為
90、55及45分鐘；20種D型胺基酸中，D-Phe為最佳受質，其kcat/KM
值為9.4 s-1 mM-1。

關鍵字

斑馬魚 ； D型胺基酸

並列摘要

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.

並列關鍵字

DAO ； D-amino acid oxidase ； Zebra fish

參考文獻

王詩瑞，2008，Rhodosporidium toruloides與Trigonopsis variabilis D型胺基酸氧化酶以基因融合與固定化增加穩定性，博士論文，生物工程研究所，大同大學，臺北。

李文華，2005，D型胺基酸氧化酵素在大腸桿菌異源表現的最適化，碩士論文，生物工程研究所，大同大學，臺北。

黃美韻，2006，斑馬魚D 型胺基酸氧化酶（D-Amino Acid Oxidase）之基因表現與酵素活性分析，碩士論文，生命科學研究所，淡江大學，臺北。

Abe, H., Yoshikawa, N., Sarower, M.G. and Okada, S. 2005. Physiological function and metabolism of free D-alanine in aquatic animals. Biol. Pharm. Bull. 28：1571 – 1577.

Blackwell, J.R. and Horgan, R.1991, A novel strategy for production of a highly expressed recombinant protein in an active form. FEBS Lett. 295：10-2.