綠竹筍苯丙胺酸脫氨裂解酶在酵母菌中之表現與檢定

Abstract

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。 酵母菌 Pichia pastoris具有高等真核表現系統之許多優點，如蛋白質之加工、折疊以及後轉譯修飾作用，並有如 E. coli一般容易操作。故在本研究中，我們用Pichia pastoris 蛋白質表現系統進行 PAL 的表現。將綠竹筍 Bopal 1 與 Bopal 2 cDNA 片段經由 PCR 反應放大，先建構保存型質體 pGEM®-T/Bopal 1與 yT&A/Bopal 2，再選殖進入表現載體 pPICZ A，建構出表現質體 pPICZ A/Bopal 1 與 pPICZ A/Bopal 2。兩個表現質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 Pichia pastoris (X-33)。 兩種表現系統產生 C 端帶 (His)6-tag 的融合蛋白質 BoPAL 1 與 BoPAL 2，可經鎳離子螯合的膠體純化得到。經 SDS-PAGE 測得表現蛋白質 BoPAL 1 與 BoPAL 2，兩者單元體分子量約為 80 kD；以膠體過濾層析法測得表現蛋白質 BoPAL 1 與 BoPAL 2 原態分子量約為 323 kD 與 331 kD，推測兩者為同質四元體酵素。表現蛋白質 BoPAL 1 與 BoPAL 2 對基質 L-Phe 之 Km 質分別為 1.01 mM 與0.33 mM。表現蛋白質 BoPAL 1 與 BoPAL 2 最適反應溫度為 50℃，最適反應 pH 為 9.0。表現蛋白質 BoPAL 1 與 BoPAL 2 活化能分別為 10.4 kcal/mol 與 12.8 kcal/mol；酵素活性會受到汞、鎳、鈷、鐵離子的抑制。二次代謝產物，如 trans-cinnamic acid 及 tannic acid，具有產物回饋抑制現象。酵素之 seryl、tyrosyl、histidyl 基團為表現蛋白質 BoPAL 1 與 BoPAL 2 催化作用所必需。表現蛋白質 BoPAL 2 具有 TAL 的活性。在本實驗中，我們認為 BoPAL 1 與 BoPAL 2 經酵母菌表現後，與綠竹各部位 PAL 具有相似的生化性質，但是表現蛋白質 BoPAL 1 對於基質具有較差的親和力。與大腸桿菌表現蛋白質 BoPAL 1 與 BoPAL 2 比較，經由轉譯後修飾，催化活性雖低於大腸桿菌表現蛋白質，但酵素構形卻較為穩定。

Phenylalanine ammonia lyase (EC 4.3.1.5) plays a key role in linking primary metabolism to phenylpropanoid 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. Pichia pastoris is a better expression system than E.coli and Saccharomyces for the production of heterologous proteins, and is relatively easy to manipulate like E.coli. Unlike bacterial expression systems, P. pastoris has the ability to perform many of the post-translational modifications usually performed in higher eukaryotes, e.g. correct folding, disulphide bond formation, O- and N- linked glycosylation and processing of signal sequences. Therefore we chose P. pastoris to express PAL in this thesis. Bamboo shoot (Bambusa oldhamii) Bopal 1 and Bopal 2 cDNA were amplified by polymerase chain reaction and cloned into the T vector pGEM®-T and yT&A to construct pGEM®-T/Bopal 1 and yT&A/Bopal 2, then to subclone into the expression vector pPICZ A . Two expression constructs, pPICZ A/Bopal 1 and pPICZ A/Bopal 2, were analyzed by restriction enzyme mapping and by sequencing confirming the correct orientation and nucleotide sequence, and then transformated into P. pastoris. Two constructs produced fusion protein BoPAL 1 and BoPAL 2 containing (histidine)6-tag in C-terminal. The expressed products were purified with a resin containing Ni2+ that retains proteins with polyhistidine fragments. The subunit mass of expressed BoPAL1 and BoPAL2 were determined to be about 80 kD by SDS-PAGE. By using gel filtration chromatography, and the molecular mass of expressed BoPAL 1 and BoPAL 2 were estimated to be 323 and 331 kD, respectively. And both proteins were estimated to be homotetrameric enzymes. The Km values for phenylalanine were 1.01 mM for expressed BoPAL 1 and 0.33 mM for expressed BoPAL 2. The optimum temperature and pH for both expressed BoPAL activity were 50℃ and 9.0, respectively. The activation energy was 10.4 kcal/mol for expressed BoPAL 1 and 12.8 kcal/mol for expressed BoPAL 2. Ni2+, Fe2+, Co2+ and Hg2+ inhibited both expressed BoPAL activity. Plant secondary metabolites, such as trans-cinnamic acid and tannic acid , had feedback inhibition to expressed enzymes. The seryl, tyrosyl and histidyl groups in enzyme were found to be essential residues for expressed BoPAL 1 and BoPAL 2 catalysis. Expressed BoPAL 2 has tyrosine ammonia lyase activity. In this study, we concluded that expressed BoPAL 1 and BoPAL 2 have quite similar biochemical properties compared with bamboo native PAL, but expressed BoPAL 1 has less affinity toward substrate. Comparing with E. coli expressed BoPAL 1 and BoPAL 2, although the catalytic activity of yeast expressed BoPAL 1 and BoPAL 2 both are less active, the conformation of two yeast expressed enzymes are more stable.