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研究生:陳鐿壬
研究生(外文):Yi-Ren Chen
論文名稱:建構酵母菌表面呈現系統用於表現脂肪酶及β-葡聚醣酶
論文名稱(外文):Construction of yeast surface-displayed system for expression of lipase and β-glucanase
指導教授:劉宏仁劉宏仁引用關係
口試委員:陳怡寧范洪春
口試日期:2021-07-26
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2021
畢業學年度:109
語文別:中文
論文頁數:83
中文關鍵詞:脂肪酶β-葡聚醣酶酵素活性飼料添加酵母菌表面呈現
外文關鍵詞:lipaseβ-glucanaseenzyme activityfeed additiveyeast surface display
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酵素發展至今已有一百多年歷史,種類多元且大幅降低化學反應所需時間,因此極具商業應用價值。為增加離乳仔豬對飼料營養成分之吸收,防止因抗營養因子消化不良所引起之下痢,透過飼料添加酵素可改善此現象。經由基因工程蛋白表現系統量產目標酵素為目前研究努力方向。本研究探討之脂肪酶及 β-葡聚醣酶是具商業價值之酵素,廣泛應用於食品、清潔劑、生質能源及家畜飼料之添加等產業。本實驗室已成功利用大腸桿菌表現系統表現脂肪酶,證實利用於腸胃道尚未發育完全之小豬,其營養吸收具更佳之效果,透過飼料添加酵素分解後提高營養吸收率並加速其成長。本研究研發酵母菌表面呈現系統 (yeast surface-displayed system)將酵素經由酵母細胞壁鑲嵌蛋白 pir1 呈現於酵母菌表面,因此酵母菌表現之酵素可直接添加於飼料。結合本實驗室先前研發之低濃度 0.005% 葡萄糖誘導啟動子 (GTH1 promoter),改善傳統嗜甲醇巴斯德畢赤酵母菌 (Pichia pastoris)需利用昂貴及高危險性之甲醇作為誘導劑之缺點,可減少誘導期間所需碳源及誘導劑,達降低生產成本之目的。此外,為提升脂肪酶與 β-葡聚醣酶之表現量,將目標基因序列優化及建構兩套及四套之多套表面呈現之載體,以提升酵素產量及符合產業化需求。本研究先以表現 eGFP 確認系統可行性後,再將脂肪酶與 β-葡聚醣酶之優化基因構築於酵母菌表面多套呈現載體 (pA4GTH pir1 His)。線性質體 DNA 電穿孔於 P. pastoris GS115,以 2000 μg /ml 之高濃度 ZeocinTM 挑選高度同源重組菌株,經 0.005% 葡萄糖誘導 72小時後,以免疫螢光染色法及西方墨點法確認酵素表現。在活性分析結果發現以四套表現載體之轉殖株較單套提升2至4倍之活性,證實套數與酵素活性成正向關係。脂肪酶及 β-葡聚醣酶之生化活性分析結果顯示,兩者之最佳反應溫度分別為 85℃ 及 75℃,最適反應 pH 環境則為 pH 7.0 與 pH 6.0,而在55℃ 至 85℃ 之熱穩定性及 pH 3.0 至 pH 8.0耐受性處理後,脂肪酶及 β-葡聚醣酶仍具有活性;於腸胃道環境模擬測試,在 0.5% pepsin 及 0.5% trypsin作用後,皆仍維持高活性。因此本研究建立之 P. pastoris 表面呈現系統,不僅大幅減少蛋白量產步驟降低生產成本,且脂肪酶及 β-葡聚醣酶皆於高溫環境具高活性及產業化價值。
Enzymes have been developed for hundred years in many different types of regions and reduce the chemical reaction time dramatically. They are commonly supplemented in animal’s diet not only for feed efficiency improvement but also for avoiding intestinal disorders, e.g., diarrhea that often occurred in early-weaned piglets. A functional enzyme with specificity now can be produced through genetic engineering approach and, by adopting protein expression system, a cost-effective amount can be obtained. The lipase and β-glucanase are commercially extracted and widely used in the sectors of the food industry, detergent, biomass energy, and livestock feed. Lipase supplementation has been proven effective to promote the growth performance of piglets by digestibility improving. In this study, the yeast surface-displayed system was used to present enzymes on the surface of yeast via the anchor protein pir1, therefore the enzymes could be added to the animal feed directly. To minimize the production costs, P. pastoris surface-displayed system, combined with GTH1 promoter, was induced by a low glucose concentration of 0.005% to reduce the shortcomings of using methanol as an inducer. According to the analysis of gene codon usage, the target genes of lipase and β-glucanase were optimized and multiple expression cassettes of cell surface-displayed vectors were constructed to increase the production of enzymes in P. pastoris for the demand of commercialization. In this study, eGFP was first used to confirm the feasibility of the system and the multiple expression cassettes of optimized lipase and β-glucanase were transformed to P. pastoris GS115, then selected by 2000 μg /ml high Zeocin concentration directly. After induced with 0.005% glucose for 72 hours, the enzyme performance was confirmed by immunofluorescence assay (IFA) and Western blot. The results of the enzyme activity test showed that four cassettes resulted in an increase by 2 to 4 times compared with the single cassette, proving that the number of cassettes exerted a positive relationship with the activity. In addition, the results of the biochemical activity analysis of lipase and β-glucanase in P. pastoris cell surface-displayed system revealed that the optimal reaction temperature of the two were 85℃ and 75℃, and the optimal reaction pH was pH 7.0 and pH 6.0, respectively. Both maintained the activity in the tests of thermal stability between 55℃ and 85℃ and the tolerance from pH 3.0 to pH 8.0. Also, the two maintained the high activity in the environmental simulation test under the influence of gastrointestinal protease, 0.5% pepsin and 0.5% trypsin. Therefore, the P. pastoris cell surface-displayed system could not only greatly reduce the cost of production, but also maintain a greater activity of lipase and β-glucanase in higher temperature environments. Results obtained have a high commercial value for the industry.
摘要 i
Abstract ii
目錄 iii
圖目錄 vii
表目錄 ix
第一章 前言 1
第二章 文獻回顧 2
2.1 酵素與工業 2
2.2 脂肪酶 2
2.3 脂肪酶之應用 3
2.3.1 食品工業 4
2.3.2 清潔劑工業 4
2.3.3 紡織工業 4
2.3.4 皮革工業 5
2.3.5 紙漿及造紙工業 5
2.3.6 生物柴油之生產 5
2.3.7 有機合成工業 5
2.3.8 醫療與藥物應用 5
2.3.9 化妝保養品應用 6
2.3.10 飼料添加劑 6
2.4 葡聚醣酶 6
2.4.1 β-葡聚醣酶 (β-glucanase) 8
2.4.2 β-1,3-1,4-glucanases (Lichenase) 9
2.4.2.1 β-1,3-1,4-glucanases (Lichenase)之應用 10
2.4.2.1.1 動物飼料 10
2.4.2.1.2 啤酒工業 10
2.4.2.1.3 生質燃料 11
2.4.2.1.4 食品添加 (寡醣) 11
2.5 巴斯德畢赤酵母菌蛋白表現系統 (Pichia pastoris expression system) 11
2.5.1 酵母菌表面呈現 (Yeast surface display, YSD) 12
2.5.2 酵母菌細胞壁鑲嵌蛋白 12
2.5.3 高親和性葡萄糖轉運蛋白啟動子 (High-affinity glucose transporter 1, GTH1) 12
2.5.4 酵母菌飼料添加物 13
第三章 材料與方法 14
3.1表現載體之構築 14
3.1.1 目標基因序列優化 14
3.1.2 引子設計 14
3.1.3 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 14
3.1.4 DNA 膠體純化 14
3.1.5 限制酶反應 14
3.1.6 DNA 接合反應 15
3.1.7 大腸桿菌 (E. coli)勝任細胞之製備 15
3.1.8 大腸桿菌 (E. coli)勝任細胞轉型 (transformation)試驗 15
3.1.9 重組質體 DNA 萃取 15
3.1.10 酵母菌 genomic DNA 之萃取 16
3.1.11 Sanger定序 16
3.2酵母菌表面蛋白表現載體之構築 16
3.2.1 表現載體 pAGTH pir1 A 及 pAGTH-pir1-eGFP-His 之構築 16
3.2.2 多套 expression cassettes 表現載體之構築 16
3.2.2.1 pir1-His 片段置換 α factor 之構築 16
3.2.2.2 雙套表現載體之製備 17
3.2.2.2.1 pA2GTH pir1 His 雙套expression cassettes載體之建構 17
3.2.2.2.2 pO2GTH pir1 His 雙套expression cassettes載體之建構 17
3.2.2.3 四套重組表現質體之製備 17
3.2.2.3.1目標基因置入雙套表現載體 17
3.2.2.3.2 pA4GTH-pir1-His-lipase及pA4GTH-pir1-His-β-glucanase 四套重組表現質體之構築 17
3.3 酵母菌表現系統 18
3.3.1 P. pastoris GS115表現系統之基因重組 18
3.3.2 P. pastoris GS115勝任細胞之製備 18
3.3.3 酵母菌電穿孔轉型作用 18
3.3.4 P. pastoris重組蛋白之誘導表達 19
3.4 重組蛋白質分析 19
3.4.1 重組蛋白濃度定量 19
3.4.2 免疫螢光染色法 (Immunofluorescence assay, IFA) 19
3.4.3 SDS PAGE 電泳 19
3.4.4 西方墨點法分析 (Western blot assay) 20
3.5 脂肪酶之活性分析 20
3.5.1 脂肪酶之活性標準曲線 21
3.5.2 脂肪酶活性測定方法 21
3.5.3 脂肪酶最適反應溫度 22
3.5.4 脂肪酶之熱穩定性分析 22
3.5.5 脂肪酶最適反應pH值 22
3.5.6 脂肪酶不同 pH 值之穩定性分析 22
3.5.7 脂肪酶腸胃道環境模擬之耐受性分析 22
3.6 β-葡聚醣酶之活性分析 23
3.6.1 β-葡聚醣酶之活性標準曲線 23
3.6.2 β-葡聚醣酶活性測定方法 23
3.6.3 β-葡聚醣酶最適反應溫度 24
3.6.4 β-葡聚醣酶最適反應pH值 24
3.6.5 β-葡聚醣酶之熱穩定性分析 24
3.6.6 β-葡聚醣酶不同 pH 值之穩定性分析 24
3.6.7 β-葡聚醣酶腸胃道環境模擬之耐受性分析 24
第四章 結果 25
4.1 P. pastoris 表面呈現載體之建構 25
4.1.1 一套 pAGTH pir1 A表現載體之構築 25
4.1.2 重組質體 pAGTH-pir1-A-eGFP-His 之構築 25
4.1.3 綠螢光蛋白之表現確認 25
4.1.4 多套expression cassettes表現載體之構築 25
4.1.4.1 pir1-His 片段置換 α factor 之過渡質體構築 25
4.1.4.2 pA2GTH pir1 His 雙套expression cassettes載體之建構 26
4.1.4.3 pO2GTH pir1 His 雙套expression cassettes載體之建構 26
4.1.4.4 四套表現載體之製備 26
4.1.4.4.1 目標基因置入雙套表現載體 26
4.1.4.4.2 pA4GTH-pir1-His-lipase及pA4GTH-pir1-His-β-glucanase 四套重組表現質體之構築 26
4.2 脂肪酶之蛋白表現 27
4.2.1 脂肪酶高套數重組菌株篩選 27
4.2.2 脂肪酶單套、雙套及四套重組菌株之生長曲線 27
4.2.3 脂肪酶重組蛋白檢測 27
4.2.4 脂肪酶生化活性分析 27
4.2.4.1 脂肪酶單套、雙套及四套表現質體轉殖酵母菌株之活性比較 27
4.2.4.2 脂肪酶之最適反應溫度 28
4.2.4.3 脂肪酶之熱穩定性分析 28
4.2.4.4 脂肪酶之最適反應 pH 值 28
4.2.4.5 脂肪酶不同 pH 值之穩定性分析 28
4.2.4.6 脂肪酶腸胃道環境模擬之耐受性分析 28
4.3 β-葡聚醣酶之蛋白表現 29
4.3.1 β-葡聚醣酶高套數重組菌株篩選 29
4.3.2 β-葡聚醣酶單套、雙套及四套重組菌株之生長曲線 29
4.3.3 β-葡聚醣酶重組蛋白檢測 29
4.3.4 β-葡聚醣酶生化活性分析 30
4.3.4.1 β-葡聚醣酶單套、雙套及四套表現質體轉殖酵母菌株之活性比較 30
4.3.4.2 β-葡聚醣酶之最適反應溫度 30
4.3.4.3 β-葡聚醣酶之熱穩定性分析 30
4.3.4.4 β-葡聚醣酶最適反應pH值 30
4.3.4.5 β-葡聚醣酶不同 pH 值之穩定性分析 31
4.3.4.6 β-葡聚醣酶腸胃道環境模擬之耐受性分析 31
第五章 討論 32
附錄 74
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作者簡介 83
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