Biochemical Studies of Phenylalanine Ammonia-Lyase from Water Bamboo (Zizania latifolia)

苯丙胺酸裂解 (phenylalanine ammonia-lyase, PAL EC 4.3.1.5) 是植物 phenylpropanoids 生合成之第一個酵素。此酵素催化苯丙胺酸 (phenylalanine) 經過非氧化型脫氨反應產生肉桂酸 (trans-cinnamic acid)。這個反應導致植物二級代謝產物及大量的 phenylpropanoid 衍生物生合成，包括木質素 (lignin)、黃酮素 (flavonoid) 等。論文以筊白筍為材料；經緩衝液粗抽、硫酸銨分劃、膠體過濾法 (Sepharacyl S-300)、疏水性作用層析法 (Phenyl-Sepharose) 及快速蛋白質液相層析法 (FPLC) 後，得到純化倍率為 110.5 倍，比活性為 53.3 nkat/mg protein。以膠體過濾法 (Superose 6, Superose 12) 測得之 PAL 原態分子量介於 290-300 kD，其次單元體分子量由 SDS-PAGE 測得約為 75 kD，因此推測筊白筍 PAL 可能為同質四元體結構。藉綠竹之 PAL 抗體進行西方點墨法顯示純化所得確實為 PAL，並將所得之 PAL 進行相關生化學研究 筊白筍與其他禾本科植物之 PAL 具有類似生化性質。苯丙胺酸裂解酶對基質 Phe 之 Km 值為筊白筍 516 μM。酵素最適反應溫度為 45℃。活化能為16.1 kcal/mol；最適反應 pH 為 8.5。鈣、鎂、錳在低濃度事會活化 PAL 活性；鈷、汞離子則會抑制。二級代謝產物，包括 trans-cinnamic acid、p-coumaric acid、ferulic acid、p-amino-benzonic acid 以及 caffeic acid，有產物回饋抑制現象。PAL 對基質 L-Phe 有專一性。酵素催化作用之活化中心具有 serine、tyrosine、histidine 等胺酸。Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first enzyme of the phenylpropanoid biosynthetic pathway. The enzyme catalyzes the non-oxidative deamination of phenylalanine to trans-cinnamic acid. This reaction leads to bio- synthesis of many phenylpropanoid-derived secondary metabolic products in plants, such as flavonoids and lignin.n this study, PAL was purified from water bamboo by buffer extraction, ammonium sulfate precipitation, Sephacryl S-300, Phenyl- Sepharose, and Fast Protein Liquid Chromatography (FPLC). The purified PAL with 110.5-fold purification and specific activity of 53.3 nkat/mg protein obtained from water bamboo. Using Superose 6 and Superose 12 column (FPLC), the molecular weight of native form PAL was estimated to be 290-300 kD, the molecular weight of subunit form was determined to be 75 kD by SDS-PAGE.Using Western blot by antibody of bamboo PAL, and the results indicated that after the purification procedure the enzyme PAL had been purified. The pure enzyme was used to test biochemical properties.AL from water bamboo is similar with Gramineae plants in biochemical properties. The Km values for L-Phe were 516.The optimum temperature and pH were 45℃ and 8.5, respectively. The activation energy was 16.1 kcal/mol. Ca2+, Mg2+, Mn2+ actived PAL activity in low concentration; Co2+, Hg2+ inhibited PAL activity. Secondary metabolites including trans-cinnamic acid, p-coumaric acid, ferulic acid, p-amino-benzonic acid, and caffeic acid with phenomena of feedback inhibition. PAL has high substrate specificity to L-phenylalanine, and the presence of seryl, tyrosyl and histidyl groups was found to be essential for enzyme catalysis.目錄……………………………………………………………………………………a寫表………………………………………………………………………………….i文摘要………………………………………………………………………………ii文摘要……………………………………………………………………………...iii一章 緒論…………………………………………………………………………..1.1 筊白筍……………………………………..….……………………………...1.2 植物的二級代謝………………………………..……………………..……..2.3 類苯丙胺酸路徑.……………………….…...……………………………….4.4 苯丙胺酸裂解酶…………..………….…..………..…………………..6.5 苯丙胺酸裂解酶催化機制……….………..……….………...……………...7.6 單子葉植物 PAL 可能具有 TAL 活性………….……………………..9.7 轉殖菸草中 PAL 之相關研究……………………….…………………10.8 苯丙胺酸裂解酶同功酶……………………………………………………12.9 苯丙胺酸脫氨裂解酶之調控...……………….……………………………12 1.9.1 環境與發育因子在轉錄層次上之調控……………………………12.9.2 磷酸化與後轉譯修飾………………………..………………………13.9.3 產物調控……………………………………………………………..14.10 實驗緣起…………………………………………………………………..14二章 材料與方法…………………………………………………………………15 2.1材料、藥品與儀器………………….……………………………………….15 2.1.1 材料…………………..…..…..……………………………………..15 2.1.2 藥品與儀器…………………..…………...………...………………15 2.2 蛋白質定量法………..……………….……………………………………16 2.3苯丙胺酸裂解酶活性分析………………….……………………………...17 2.4 電泳檢定系統……………………………………………………………...18 2.4.1 原態膠體電泳 (native-PAGE)……..…………………….………….19 2.4.2 SDS 膠體電泳 (SDS-PAGE)………………………………………..21 2.4.3 膠體染色法…………………………………………………………..23 2.4.3.1 CBR 染色法…………………………………………………23 2.4.3.2膠片乾燥法………………………………….……………..24 2.5 苯丙胺酸裂解酶的純化…………………………………………………...25 2.5.1 粗抽與硫酸銨分劃…………………………………………………..25 2.5.2 膠體過濾管柱層析…………………………………………………..27 2.5.3疏水性作用管柱層析………………………………………………...27 2.5.4快速蛋白質液相管柱 (FPLC) 層析….……………….…………….30 2.6蛋白質電泳轉印及相關應用…………….………………………...………32 2.6.1蛋白質轉印法………………………………………………...………32 2.6.2酵素免疫染色…………………………………………………..…..33 2.7 分子量測定……………….…..……………………………………………33 2.7.1 Superose 6 和 Superose 12 (FPLC) 膠體過濾層析……………..…33 2.7.2 SDS-PAGE……………………………………………..………….….35 2.8 酵素生化性質……………………………………………………………...35 2.8.1 酵素反應最適 pH 值…………………………………………….…35 2.8.2 最適反應溫度………………………………………………………..35 2.8.3活化能 (Activation energy, Ea)……..………………………………..35 2.8.4 熱安定性……………………………………………………………..36 2.8.5 Km 值…………………………………………………………….…..36 2.8.6 金屬離子對活性之影響……………………...……………………...36 2.8.7 二級代謝物對活性影響………...……………………………….…..36 2.8.8化學修飾劑對活性影響………………………………………….…..36 2.8.9 基質專一性………...…………………………………………….…..36 2.8.10酪胺酸脫氨裂解酶 (TAL) 活性分析法…………………………...37三章 結果與討論…………………………………………………………………38 3.1筊白筍苯丙胺酸裂解酶純化………………………………………..…...38 3.1.1純化步驟之流程…………………………………………………..….38 3.1.2活性測定…………………………………………………….………..39 3.1.3硫酸銨分劃…………………………………………………….....…..39 3.1.4 Sephacryl S-300 (Gel filtration)………………………………………39 3.1.5 Phenyl-Sepharose……………………………………………..……....40 3.1.6 Mono Q HR5/5 (FPLC)…...……………………………………..……40 3.1.7各純化步驟電泳分析比較………………………………………...…41 3.1.8純化表…………………………………………….…………..…….41 3.1.9 原態分子量測定……………………………………………………..41 3.2 PAL 生化性質………………………………………………………..……42 3.2.1 最適反應溫度與活化能……………………………………………42 3.2.2 熱安定性…………………………………………………………..…43 3.2.3 最適反應 pH…………………………………………………….…44 3.2.4 Km 值………………………………………………………………...44 3.2.5 金屬離子影響……………………………………………......………44 3.2.6 二級代謝物對 PAL 活性的影響……………………………….….45 3.2.7 基質專一性…………….……………...……...…………………..….45 3.2.8 化學修飾劑對活性影響………...………….….………..………..….46四章 結果與展望…………………………………………………………………47 4.1 筊白筍苯丙胺酸裂解酶純化………………………….…………..……....47 4.2苯丙胺酸裂解分子形式……………………….……………..…………….48 4.3筊白筍與綠竹之苯丙胺酸裂解酶生化性質比較……………….….……..48 4.4 未來展望…………………………………………………...………………50考文獻……………………………..……………………………….…………...…7