| Summary: | 脂肪分解酶(Lipase)為一種存在於自然界多種生物體內的水解酵素。因其對於許多基質都具有專一性及選擇性，近年來已被廣泛使用在生物技術和工業應用上，例如：食品加工、特用化學品合成、環境工程、醫學檢測等。由於產率高、成本低且表現量穩定等特性，使大腸桿菌(Escherichia coli , E. coli) 成為生產重組蛋白質重要的宿主細胞，但其純化過程較為複雜，也有可能於蛋白質表現時產生沒有活性的包涵體(inclusion body)，而使得生產難度增加。 本實驗運用表面表現系統生產目標蛋白，藉由冰核蛋白(ice nucleation protein, INP)之C端結合目標蛋白(Candida antarctica lipase A ,CALA)重組成融合蛋白而使其表現於細胞表面上。其誘導條件為：將含有表現質體的E. coli JM109 (DE3)於37℃，200 rpm下培養，待OD600約為0.8時加入0.1mM的IPTG，於15℃誘導表現24小時，之後進行酵素活性測定。經由電泳分析可以看出CALA有和INP結合並大量表現，但可能因蛋白質錯誤摺疊產生包涵體使活性降低，總活性和比活性分別約為0.285 U/ml和0.289 U/mg。為了提升蛋白質酵素活性，我們分別選殖fkpA、ppiA和skp三種攜伴蛋白，送入大腸桿菌中和CALA共同表現。實驗中發現當選殖攜伴蛋白基因ppiA與CALA共同表現時，添加0.1 mM IPTG和 5 g/L的誘導劑L-arabinose時有較佳的酵素總活性和比活性，約為0.64 U/ml 和0.392 U/mg，相較於未使用攜伴蛋白時分別提升了2.25倍和1.4倍。 Lipases are ubiquitous in nature and produced by several plants, animals, and microorganisms. Lipases are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Owing to the benefits of protein expression system in Escherichia coli, such as high production yield, low manufacturing cost, and well-established expression system, E. coli was used as a popular host to produce a wide variety of recombinant proteins. However, the drawbacks of the E. coli protein expression system were the complexity of protein purification and the ease of forming inclusion bodies. In this study, the surface display system via the ice nucleation protein: Xanthomonas campestris INP was used. Candida antarctica lipase A (CALA) was chosen as the target protein fusing to the C terminus of the INP segment. Lipase A was produced on E. coli cell’s surface. E. coli JM109 (DE3) harboring plasmid pINP-CALA was incubated at 37℃ and 200 rpm until OD600 reached about 0.8, followed by the addition of 0.1mM IPTG. The induction was carried out at 15℃ and 200 rpm for 24 h. The INP-CALA activity and its specific activity were obtained as 0.285 U/ml and 0.289 U/mg. In order to improve enzyme activity, three chaperone genes, such as fkpA, ppiA, and skp, were cloned into CALA surface display system, respectively, and coexpressed to produce CALA. The results showed the best activity and specific activity of CALA coexpressed with ppiA gene as 0.64 U/ml and 0.392 U/mg when induced by 0.1mM IPTG and 5 g/L L-arabinose . The CALA activity and its specific activity were increased by 2.25-fold and 1.4-fold, respectively. 中文摘要 I Abstract II 目 錄 III 圖目錄 VI 表目錄 VII 縮寫字對照表 VIII 第一章、緒 論 1 1.1 前 言 1 1.2 研究動機 3 第二章、文獻回顧 4 2.1 脂肪分解酶 (Lipase) 4 2.1.1 簡介 4 2.2.1 Candida antarctica lipase A 7 2.2 細胞表面表現系統 (Cell surface display system) 8 2.3 冰核蛋白 (Ice nucleation protein) 11 2.4 攜伴蛋白 (Chaperone) 13 第三章、實驗材料與方法 14 3.1 實驗材料 14 3.1.1 實驗儀器設備 14 3.1.2 實驗藥品 16 3.1.3 實驗用試劑 18 3.2 實驗流程 23 3.3 實驗方法 24 3.3.1 菌株與質體 24 3.3.2 大腸桿菌質體DNA抽取 26 3.3.3 DNA電泳分析(Agarose gel electrophoresis ) 27 3.3.4 剪切反應 (Digestion) 27 3.3.5 DNA純化回收 28 3.3.6 黏接反應(Ligation) 28 3.3.7 Heat shock 轉型作用 (Transformation) 29 3.3.8 電穿孔作用 (Electroporation) 30 3.3.9 重組質體建構方法 31 3.3.10 菌種保存與培養方法 39 3.3.11 蛋白分離方法 40 3.3.12 三丁酸甘油脂平板試驗(Tributyrin agar plate test) 40 3.3.13 CALA酵素活性分析 (Activity assay) 41 3.3.14 蛋白質電泳(SDS-PAGE)分析 43 第四章、結果與討論 45 4.1 重組質體pINP-CALA之建構 45 4.2 IPTG誘導pINP-CALA重組質體之表現 49 4.2.1 不同培養基之比較 49 4.2.2 不同誘導溫度與IPTG濃度之影響 50 4.2.3 不同反應溫度對CALA之影響 53 4.3 表面表現系統pINP-CALA與胞內表現系統pET22b-CALA生產酵素CALA之比較 55 4.4 攜伴蛋白pBAD-skp、pBAD-fkpA、pBAD-ppiA之建構 59 4.5 IPTG及 L-Arabinose 誘導CALA之表現 63 4.5.1 L-Arabinose誘導濃度之影響 63 4.5.2 CALA與攜伴蛋白共同表現之結果 65 第五章、結論與展望 67 5.1 結論 67 5.2 未來展望 68 參考文獻 69
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