臺灣博碩士論文加值系統

使用中央研究院植物暨微生物學研究所蕭介夫教授研究室所構築之 Pichia pastoris GS115/CRL3，以 YPD 為基礎培養基，探討Candida rugosa 脂肪酶第三型 (CRL3) 之最適生產條件。三角瓶培養結果，菌體量 (OD600) 由 50 提升至 170，CRL3酵素活性由4.5 U ml-1 提升至180 U ml-1。培養基之起始 pH 值會影響菌體生長與酵素表現。利用五公升桌上型發酵槽之批次培養時， pH 9雖可得到較高濃度的菌體量及總蛋白表現量，但 CRL3 所佔的比例較低。控制於 pH 6 時可得最高酵素表現量，活性 140 U ml-1，酵素蛋白質 83.2 mg l-1，為 pH 8 時之六倍。最後進行饋料批次培養結果酵素活性提升至 210 U ml-1，酵素蛋白質為 103.48 mg l-1。

Pichia pastoris GS115/CRL3, constructed by Prof. Shaw’s lab, Institute of Plant and Microbial Biology, Academia Sinica, was used to produce Candida rugosa lipase 3 (CRL3). Optimal conditions for the enzyme production were investigated in this study. Under the optimal conditions developed, biomass concentrations expressed as OD600 were increased from 50 to 170 and enzyme activities of CRL3 were increased from 4.5 U ml-1 to 180 U ml-1, respectively, after 5 days cultivation in shaking flasks and were shown to be influenced by initial pH of the medium. Using a 5-liter benchtop fermentor, production process of batch-type cultivation was further studied under pH controlled at 5, 6, 7 and 8. Increasing the pH of control resulted in increase of the amounts of biomass and total protein production. However amounts of CRL3 obtained were low. The enzyme activity of 140 U ml-1, with a 6-fold higher than that controlled at pH 8 was achieved under the condition of pH controlled at 6. The yield was 83.2 mg l-1 of growth media. The enzyme activity of 210 U ml-1 at fed-batch culture. The yield was 103.48 mg l-1 of growth media.

第一章 前 言 11
第一節 脂肪酶 11
第二節 脂肪酶在工業上之應用 18
2.1 脂肪酶在清潔劑工業之應用 18
2.2 脂肪酶在食品工業之應用 20
2.3 脂肪酶在纖維及造紙工業之應用 22
2.4 脂肪酶在製藥工業之應用 23
2.5 脂肪酶在生物感應器之應用 23
2.6 脂肪酶在環保工業之應用 23
2.7 脂肪酶在化妝品與香水工業之應用 23
2.8 脂肪酶新機能之開發 24
第三節 Candida rugosa lip3 (CRL3) 24
第四節 酵母菌異源蛋白之表現 25
第五節 Pichia pastoris 32
第六節 醱酵槽之培養模式與控制原理 37
第七節 研究目的 38
第二章 材料與方法 41
第一節 使用菌株與菌株保存方法 41
第二節 藥品與試劑 41
第三節 Hinton’s 三角瓶之培養、表現及各種探討方法 44
3.1 菌種活化及種培養 44
3.2 P. pastoris 之生長曲線 44
3.3 最適碳源 44
3.4 最適氮源 45
3.5 最適起始 pH 值 45
3.6 最適甘油濃度 45
3.7 最適Peptone 濃度 46
3.8 最適Yeast extract 濃度 46
3.9 最適培養溫度 46
第四節 五公升醱酵槽之培養方法 47
4.1 批次培養 47
4.2饋料批次培養 47
第五節 分析方法 48
5.1 菌體生長量 48
5.2 葡萄糖濃度之測定 48
5.3 酵素活性 49
5.4 蛋白質定量 54
5.5 蛋白質膠體電泳 54
5.6 甲基藍染色法 58
第六節 儀器設備 58
第三章 結果與討論 60
1. 酵素活性測定穩定性 60
2. 轉形株於 YPD medium 之生長曲線 60
3. 碳源的探討 64
4. 氮源的探討 64
5. Hinton’s 三角瓶中最適起始 pH 值的探討 64
6. Glycerol 濃度之影響 68
7. Peptone 濃度之探討 68
8. Yeast extract 濃度之探討 68
9. 溫度之探討 72
10. 金屬離子之探討 72
11. Hinton’s 三角瓶最適化之生長曲線及其分析 72
12. 批次培養之 pH 值探討 72
13. 饋料批次培養 78
第四章 結論 86
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