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研究生:張明暉
研究生(外文):Ming-Hui Chang
論文名稱:植酸酵素phyA基因在PichiapastorisX33重組菌株之表現與應用
論文名稱(外文):Expression and application of recombinant Pichia pastoris X33 constructed by phytase gene phyA
指導教授:楊秋忠楊秋忠引用關係
指導教授(外文):Chiu-Chung Young
學位類別:博士
校院名稱:國立中興大學
系所名稱:土壤環境科學系所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
畢業學年度:96
語文別:中文
論文頁數:105
中文關鍵詞:植酸酵素Pichia pastoris X33pPICZαApGAPZαASDS-PAGE米糠大豆粕油菜籽粕葵花籽粕
外文關鍵詞:phytasePichia pastoris X33pPICZαApGAPZαASDS-PAGEphosphorousrice bransoybean mealrapeseed mealsunflower meal
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為增進人類、豬及家禽等單胃的動物和植物難以吸收、代謝之植酸磷的有效性,本研究乃自可分泌植酸酵素之Aspergillus ficuum選殖出植酸酵素基因phyA,分別構築於pPICZαA和pGAPZαA表現載體,建構P. pastoris X33(pPICZαA)和Pichia pastoris X33(pGAPZαA)兩株重組菌株,產出胞外植酸酵素。並分別應用於水解米糠和大豆粕、油菜籽粕及葵花籽粕植酸磷以釋出磷素。構築之重組菌株皆可生長於含有 100 μg mL-1 Zeocin抗生素之YPDS(Yeast Extract Peptone Dextrose Sorbitol)篩選培養基,且重組載體和染色體DNA經由PCR與限制酵素確認,另主要核酸片段大小之電泳分析均與phyA符合。
P. pastoris X33(pPICZαA)重組菌株以含1%(v v-1)甘油之BMGY生長培養基培養2日之後,再以含1% 甲醇之新鮮BMMY培養基置換 BMGY,並以1% 甲醇追加、誘導培養至第9日,酵素活性可達200 U mL-1 。另pH 5.5 ± 0.1之去離子水可以替代pH 5.5的0.1 M醋酸鈉緩衝液作為粗酵素液水解米糠之反應溶液,且反應24小時之後磷濃度達1.31%,相當於81% 無機磷由米糠之植酸磷水解釋出。以不同稀釋濃度之粗酵素液分別與米糠反應24小時,結果顯示粗酵素液水解米糠釋出水溶性磷的濃度與100、50 U mL-1 無顯著差異,但與20、5及0 U mL-1 則差異顯著;萵苣盆栽試驗的結果顯示採收後地上部乾重,施用含有植酸酵素活性與米糠反應後濾液之Hoagland solution皆略高於酵素先失活之對照處理,且稀釋倍數較高之處理差異比較明顯。
P. pastoris X33(pGAPZαA)重組菌株不需甲醇誘導與培養基置換,即可分泌胞外植酸酵素,以YPD液體培養基於30℃與200 rpm條件下培養4日後,粗酵素液之植酸酵素活性達16.5 U mL-1,而非重組菌株P. pastoris X33則明顯無活性表現。SDS-PAGE 分析亦顯現重組菌株明顯有酵素活性表現而非重組菌株則無。酵素特性分析顯示該酵素具有兩個最適化pH值,分別為pH 5.5和pH 2.5,最適化溫度為50℃。熱穩定性比源自A. niger之商品化植酸酵素高。由酵素動力學分析Km = 0.115 mM和Vmax = 1.4 μmole min-1,顯示該植酸酵素與植酸之間的親和力較A. niger弱,但比P. pastoris Pholp強。P. pastoris X33(pGAPZαA)重組菌株可以代謝利用葡萄糖、果糖和甘油,但無法有效利用木糖和雙糖(蔗糖、麥芽糖、乳糖)以及多醣(澱粉、纖維素)。含有不同濃度葡萄糖之YPD液體培養基培養6日後之酵素活性依序為 6% > 4% > 3% > 2% > 1% ,而且含有2% 葡萄糖之糖蜜可以取代對照YPD液體培養基中之等濃度葡萄糖,降低植酸酵素生產成本;粗酵素液與大豆粕、油籽粕及葵花籽粕於pH 5.5之0.1 M 醋酸鈉緩衝液中,在37℃反應24小時,皆明顯有無機磷釋出,尤其以葵花籽粕最為明顯,比初始無機磷濃度約增加7倍,而油菜籽粕與大豆粕亦可分別約增加5倍與3倍。
Attempts were made to enhance the availability of phytate-phosphate, poorly metabolized by monogastric animals, such as human, swine, poultry, and plants. The Aspergillus ficuum phytase gene phyA was cloned and constructed by pPICZαA and pGAPZαA, expressed in Pichia pastoris X33 (pPICZαA) and Pichia pastoris X33 (pPGAPZαA). The crude phytase extracts were applied to recover phosphorus from the fermented liquids of rice bran and soybean meal, rapeseed meal and sunflower meal, separately. The recombinant P. pastoris X33 were confirmed by a screening medium containing 100 μg mL-1 Zeocin, PCR and restriction enzyme. In addition, through electrophoresis the size of major fragment coincided with phyA.
The phytase was overexpressed in P. pastoris X33 (pPICZαA) after 2 days incubation with BMGY medium containing 1% (v v-1) glycerol then replaced with fresh BMMY medium containing 1% methanol (on daily basis) as carbon sources. The phytase activity increased evidently with time and reached 200 U mL-1 after 9 days induction. Also a practical way of releasing phosphorus from rice bran was found when 0.1 M sodium acetate buffer replaced with de-ionised water (pH 5.5 ± 0.1). Phosphorus recovery increased with time and reached 1.31% after 24 h incubation contributing to 81% inorganic P released from rice bran phytate. Hydrolysis of rice bran phytate adding phytase of the recombinant yeast showed no significant effect on hydrolysis rates at different activities of 200, 100, 50 U mL-1. However, hydrolysis rates differed significantly with 20, 5, 0 U mL-1. The dry weight of lettuce shoot after harvest treated with phytase was slightly higher than that treated with inactived phytase.
The phytase was successfully expressed in P. pastoris X33 (pPGAPZαA) without methanol induction and replacing culture medium over incubation period. The activity of extracellular crude phytase extract reached 16.5 U mL-1 after 4 days incubation at 30℃. Notably, all the non-recombinant P. pastoris X33 showed no enzyme activities. The SDS-PAGE analyses also presented the same result. The enzyme characteristics indicated two pH (5.5 and 2.5), and 50℃ were optimal for phytase. The thermostabilities of phytase appeared higher than a commercial enzyme (from A. niger). The Km 0.115 mM and Vmax 1.4 μmole min-1 from enzyme kinetics indicated the affinity for phytate of the phytase was higher than A. niger phytase, but lower than P. pastoris Pholp. P. pastoris X33 (pPGAPZαA) was able to metabolize glucose, fructose and glycerol, but unable to effectively metabolize xylose, disaccharides (sucrose, maltose, lactose) and polysaccharides (starch, cellulose). After 6 days incubating with various glucose concentration in YPD liquid media, the phytase activity was 6% > 4% > 3% > 2% > 1%. Moreover the molasses with 2% glucose could substitute glucose in YPD liquid media to produce phytase for reducing cost. Comparing phosphorus releasing effect of phytase, it was the highest when applied to sunflower meal with 7 fold increased of the initial concentration, then followed by appling to rapeseed meal and soybean meal with 5 fold and 3 fold increased, respectively.
中文摘要…………………………………………………………………Ⅰ
英文摘要…………………………………………………………………Ⅲ
目錄………………………………………………………………………Ⅴ
表次…………………………………………………………………….XⅠ
圖次…………………………………………………………………….XⅡ
壹、前言………………………………………………………………….1
貳、前人研究…………………….............................................................3
一、植酸………………………………………………………….3
二、植酸酵素之重要性及應用….…………………………….8
三、Pichia pastoris之表現系統….………………………….16
參、材料與方法………………………………………………………...24
一、菌株、載體及引子……………………………………….24
(一)菌株……………………………………………………….24
(二)載體……………………………………………………….24
(三)引子……………………………………………………….24
二、培養基、化學藥品及試驗材料…………………………26
(一)培養基……………………………………………………..26
(二)化學藥品與試驗材料……………………………………..27
(三)儀器設備…………………………………………………..29
三、實驗方法…………………………………………………..30
(一)P. pastoris X33重組菌株之構築與確認…………………30
1. Aspergillus ficuum染色體DNA之抽取…………………...30
2. Aspergillus ficuum植酸酵素phyA基因之選殖……...........30
3. phyA-pPICZαA和phyA-pGAPZαA重組載體之構築….....31
4. P. pastoris X33勝任細胞之製備………………..…………31
5. P. pastoris X33之轉形作用……………………………..…32
6. P. pastoris X33重組菌株之確認……………………..……33
(1)P. pastoris X33(pPICZαA)重組菌株之確認.............33
(2)P. pastoris X33(pGAPZαA)重組菌株之確認……...33
(二)P. pastoris X33重組菌株植酸酵素之表現…..…………33
1. P. pastoris X33(pPICZαA)重組菌株植酸酵素之表現...33
(1)三角瓶規模甲醇誘導時機之建立……………….……33
(2)三角瓶規模不同甲醇濃度之誘導表現之比較…….....34
(3)三角瓶置換培養基之後,甲醇誘導與否對植酸酵素活性表現之比較…………………………………………...34
(4)三角瓶規模置換培養基及甲醇誘導對酵素活性表現之影響…………………………………………………… ..35
①三角瓶規模置換培養基及追加1% 甲醇之誘導表現..…..35
②胞外植酸酵素粗酵素液SDS-PAGE之分析……………….. 35
2. P. pastoris X33(pGAPZαA)重組菌株植酸酵素之表現...35
(1)植酸酵素粗酵素液之製備及表現分析………………...35
(2)胞外植酸酵素粗酵素液SDS-PAGE之分析…………..37
(三)植酸酵素活性測定………………………………………..37
(四)P. pastoris X33(pPICZαA)植酸酵素粗酵素液水解米 糠植酸磷之探討…………………………………………..38
1. 以去離子水作為米糠水解反應溶液之可行性評估………38
2. 以去離子水作為米糠水解反應溶液,無機磷隨時間釋出 之變化………………...…………………………………….38
3. 米糠植酸磷水解反應,最適植酸酵素活性之探討………39
4. 盆栽試驗…..………………………………………………..39
(五)P. pastoris X33(pGAPZαA)重組菌株植酸酵素之最適 化條件和酵素動力學之探討……………………………..41
1. 植酸酵素最適化pH値……………………………..……...41
2. 植酸酵素最適化溫度 ……………………………………..41
3. 植酸酵素之熱穩定性分析……………………………..…..41
4. 酵素動力學之探討……..…………………………………..41
(六)P. pastoris X33(pGAPZαA)重組菌株液體培養基中較 適碳源之探討……………………………………………..42
1. 碳源對於植酸酵素產量之探…..…………………………..42
(1)不同碳源種類對菌數濃度、酵素活性表現之比較…...42
(2)較適碳源之不同濃度對菌數濃度、酵素活性表現之
比較...…………………………………………………..42
2. 以糖蜜取代YPD液體培養機中葡萄糖之可行性評估…..43
3. 葡萄糖濃度之定量分析..…………………………………..43
(七)P. pastoris X33(pGAPZαA)表現之粗酵素液應用於增 進大豆粕、油菜籽粕、葵花籽粕水溶性磷釋出之探43..43
肆、結果與討論………………………………………………………...45
一、植酸酵素phyA基因構築於pPICZαA及其在Pichia pastoris X33重組菌株之表現與應用…...…………….45
(一)P. pastoris X33(pPICZαA)重組菌株之構築與確認…..45
(二)P. pastoris X33(pPICZαA)重組菌株植酸酵素之表現..49
1. 三角瓶規模甲醇誘導時機之建..…………………………..49
2. 三角瓶規模不同甲醇濃度之誘導表現之比較..…………..49
3. 三角瓶置換培養基之後,甲醇誘導與否對植酸酵素活性 表現之比較…………………………………………………49
4. 三角瓶規模置換培養基及甲醇誘導對酵素活性表現之影
響……………………………………………………………49
5. 植酸酵素粗酵素液應用於增進米糠植酸磷水解之探討....53
(1)去離子水和0.1 M醋酸鈉緩衝液(pH 5.5)作為粗酵 素液水解米糠之反應溶液之比較……………………...53
(2)植酸酵素水解米糠植酸磷反應之最適酵素活性
之探討……………………………………………………57
6. 盆栽試驗…………………………..………………………..57
二、植酸酵素phyA基因構築於pGAPZαA及其在Pichia pastoris X33之表現與應用......................................65
(一)P. pastoris X33(pGAPZαA)重組菌株之構築與確認…65
(二)P. pastoris X33(pGAPZαA)重組菌株植酸酵素之表現…………………………………………………………..69
(三)P. pastoris X33重組菌株植酸酵素最適化條件和酵素動 力學之探討………………………………………………..72
1. 植酸酵素最適化pH値…..………………………………...72
2. 植酸酵素最適化溫度..……………………………………..74
3. 植酸酵素之熱穩定性分析..………………………………..74
4. 酵素動力學之探討..………………………………………..77
(四)碳源對於植酸酵素產量影響之探討……………………..81
1. 以不同碳源培養對植酸酵素產量影響之比較……………81
2. 不同葡萄糖濃度對植酸酵素產量影響之比較……..……..87
3. 以糖蜜取代YPD液體培養基中葡萄糖之可行性評估..…90
(五)植酸酵素水解生質柴油生產後之油粕副產物釋出無機 磷之比較…………………………………………………..93
伍、結論…………………………………………………………………95
陸、參考文獻……………………………………………………………97
柒、附錄………………………………………………………………..105
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