大腸桿菌表現綠竹苯丙胺酸脫氨裂解酶及功能鑑定

Expression and Characterization of Bamboo(Bambusa oldhamii) Phenylalanine Ammonia Lyase in Escherichia coli

潘宏記(Hung-Chi Pan)

指導教授 ： 李 平 篤

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.10053

摘要

中文摘要
苯丙胺酸脫氨裂解酶 (phenylalanine ammonia lyase, EC 4.3.1.5，簡稱 PAL) 為連結一次代謝與二次代謝的關鍵酵素，其催化 L-phenylalanine 進行非氧化性脫氨產生 trans-cinnamic acid ，為 phenylpropanoid 衍生物生合成途徑上第一個反應。
將綠竹 PAL 1 與 PAL 2 cDNA 片段經由 PCR 反應放大，選殖進入載體 pTrcHisA 建構出表現載體 pTrcHisA/PAL 1 pTrcHisA/PAL 1。兩個表現載體以限制酶分析酶切圖譜，及定序方法分析核酸序列的正確性，再轉形進入至大腸桿菌 Top 10。兩種表現系統產生 N 端帶 (His)6-tag 的融合蛋白質 PAL1 與 PAL 2，可經鎳離子螯合的膠體純化得到。
以膠體過濾層析法測得表現蛋白質 PAL 1 與 PAL 2的原態分子量分別約為320 kD與 330 kD，而 SDS-PAGE 測得兩者單元體分子量約為 79 kD ，推測兩者為同質四元體酵素。表現蛋白質 PAL 1 與 PAL 2 對基質 Phe 之 Km 值分別為 200 μM 與 250 μM。酵素最適反應溫度為 50℃，最適反應 pH 為 9.0。PAL 1 與 PAL 2活化能分別為9.3 kcal/mol 與 10.1 kcal/mol；酵素活性會受到鈣、錳、汞離子的抑制。二級代謝產物，如 p-coumaric acid及 tannic acid，具有顯著產物回饋抑制現象。酵素之seryl、tyrosyl、histidyl 基團為 PAL 1與 PAL 2 催化作用所必需。在本研究中，我們認為綠竹 PAL 1 與 PAL 2 經大腸桿菌表現後，與綠竹原態 PAL 具有相似的生化性質，但是對於基質具有較佳親和力與催化能力。

關鍵字

苯丙胺酸裂解酶二次代謝 ； 綠竹

並列摘要

Abstract
Phenylalanine ammonia lyase (EC 4.3.1.5) plays a key role in linking primary metabolism to phenyl- propanoid metabolism. The enzyme catalyzes non-oxidative deamination of L-phenylalanine to produce trans-cinnamic acid, which is used for biosynthesis of phenylpropanoid derived products in plant.
Bamboo (Bambusa oldhamii) PAL1 and PAL 2 cDNA were amplified by polymerase chain reaction and subcloned into the expression vector pTrcHisA. Two expression constructs, pTrcHisAPAL1 and pTrcHisAPAL 2, were analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence, and then transformated into E.coli (Top10 strain). Two constructs produced fusion protein PAL 1 and PAL 2 containing (histidine)6-tag in N-terminal. The expressed products were purified with a resin containing Ni2+ that retains proteins with polyhistidine fragments.
. By using gel filtration chromatography, the molecular mass of expressed PAL 1 and PAL 2 were estimated to be 320 and 330 kD, respectively.The subunit mass of PAL1 and PAL2 were determined to be about 79 kD by SDS-PAGE, and both proteins were estimated to be homotetrameric enzymes. The Km values for phenylalanine were 200

並列關鍵字

secondary metabolism ； phenylalanine aammonia lyase

參考文獻

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