利用巨大芽孢桿菌轉化魚廢和蔗渣為Alcalase之綠色循環模組

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葉芳宜(Fang-I Yeh) 查詢紙本館藏

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環境工程研究所

論文名稱

利用巨大芽孢桿菌轉化魚廢和蔗渣為Alcalase之綠色循環模組
(Valorization of fish waste and sugarcane bagasse for Alcalase production by Bacillus megaterium via a circular bioeconomy model)

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摘要(中)

“責任消費與生產”是聯合國2030年前可持續發展目標 (Sustainable development goals, SDGs) 17個主要目標中之一。在臺灣，循環經濟也是5+2產業創新計畫的重點。魚內臟、蔗渣和豆渣是常見的有機固體廢棄物，通常被再利用為動物飼料及肥料，只能有限度提升價值。Alcalase是一種耐熱的鹼性蛋白酶，目前廣泛應用於食品加工、紡織和清潔劑等領域，是一種具有價值之酵素。為減少有機固體廢棄物對環境的傷害，及提升其再利用的價值。依據循環經濟的概念，本研究擬建立一個可利用有機固體廢棄物，來生產有價值酵素的綠色循環模組。利用分子選殖 (Molecular cloning)，將分別帶有可產生綠螢光蛋白 (Green fluorescent protein, GFP) 的gfp和產生Alcalase (原稱subtilisin Carlsberg) 的subC的質體轉形至Bacillus megaterium YYBM1中 (分別簡稱為YYBM1- gfp和YYBM1- subC) 。利用GFP易於定量和定性之特性，優化異源蛋白表達的條件，並應用於YYBM1- subC表達Alcalase之上。在含0–1% (w/v) Xylose的Lysogeny broth中，YYBM1-gfp的生長曲線並未出現太大的差異，說明YYBM1-gfp不具有可降解Xylose的能力或影響細菌之生長。當Xylose的濃度越高，所誘導產生的GFP濃度越高。在同樣為1% Xylose的誘導下， BD-xylose誘導YYBM1-gfp所產生的GFP量高於商業Xylose兩倍。其結果也與誘導YYBM1-subC異源蛋白表達相同。在pH 9.0和60 ℃時，Alcalase水解魚內臟蛋白的Vmax最高，其約為0.2 protein mg/mL/min，以及酵素飽和濃度為 0.1 mg/mL。取得最佳酵素水解條件後，將經誘導的YYBM1-subC細胞加入魚內臟和豆渣溶液中，在pH 9.0和60 ℃的環境下加熱兩小時，細菌細胞破裂的同時，胞內酵素釋出，確實可將魚內臟和豆渣進行水解。使用魚廢培養基和BD-xylose產生Alcalase的綠色循環模組與商業版之生產成本相差約19倍，說明了此綠色循環模組具有經濟效益，且也符合SDGs中的第12項與5+2產業創新計畫中的循環經濟。

摘要(英)

"Responsible consumption and production" is one of the United Nations′ Sustainable Development Goals (SDGs) in 2030. In Taiwan, Circular Economy is also a focus of the 5+2 Industries Initiatives. Fish viscera, bagasse, and okara are common organic solid wastes. They are often reused as animal feed and fertilizer with limited values. Alcalase is a heat-tolerant alkaline protease (< 65 ℃) widely used as food softener, textile, and detergent. To reduce the negative effects of organic solid waste on the environment and to further enhance its value, this thesis aims to establish a sustainable recycling system for producing Alcalase using fish viscera and bagasse as the substrates for microbial growth. We constructed two plasmids for heterologous protein expression in B. megaterium strain YYBM1 that harbor gfp encoding green fluorescent protein (GFP) and subC encoding Alcalase respectively (YYBM1-gfp and YYBM1-subC). Taking the advantage of the ease in real-time measurements of GFP, we optimized the conditions for heterologous protein expression in strain YYBM1. Later, we applied the optimized conditions to the express recombinant Alcalase in YYBM1-subC. The growth yield of YYBM1-gfp cultures cultivated in LB containing 0–1% (w/v) xylose are highly similar, indicating that YYBM1-gfp does not have the capability to degrade xylose. The yield of GFP is enhanced with the increase in xylose concentrations (from 0–1%). Moreover, the use of bagasse xylose (1%) for inducing the protein overexpression resulted in a two-fold GFP/Alcalase yield than using commercial xylose (1%) in strain YYBM1. The recombinant Alcalase revealed a Vmax of 30 U per milligram at 60 ℃ and pH 9.0 as well as a saturation centration of 0.1 mg Alcalase per mL for fish visceral protein hydrolysis. The induced YYBM1-subC cells were added to the fish viscera and okara, respectively, and the pH of the suspensions were adjusted to pH 9.0 and were incubated at 60°C for two hours so that the bacterial cells were autolyzed and the intracellular enzymes were released. In addition to fish viscera, the recombinant Alcalase also hydrolyzed okara into smaller polypeptides and amino acids. Therefore, the cost of using protein waste and BD-xylose to manufacture Alcalase based on the circular bioeconomy model established in this thesis is 20 times less than the commercial medium, buttressing the SDGs and 5+2 Industries Initiative.

關鍵字(中)

★ 巨大芽孢桿菌
★ 豆渣
★ 異源蛋白質表達
★ 魚內臟
★ 循環經濟
★ 綠螢光蛋白
★ 蔗渣
★ 鹼性蛋白酶

關鍵字(英)

★ Alcalase
★ Bacillus megaterium
★ Bagasse
★ Circular economy
★ Fish viscera
★ Green fluorescent protein
★ Heterologous protein expression
★ Okara

論文目次

摘要 i
Abstract i
謝誌 iii
目錄 iv
圖目錄 vii
表目錄 ix
名詞縮寫說明 x
第一章前言 1
1.1. 研究動機 1
1.2. 研究目的 2
第二章文獻回顧 4
2.1 相關環境永續策略 4
2.1.1 聯合國永續發展目標 4
2.1.2 5+2產業創新計畫 6
2.2 細菌 7
2.2.1 細菌細胞結構 7
2.2.2 內孢子 8
2.2.3 內毒素 9
2.2.4 巨大芽孢桿菌 (Bacillus megaterium ) 9
2.3 酵素 10
2.3.1 鹼性蛋白酶簡介 10
2.3.2 Alcalase 15
2.3.3 酵素動力學 15
2.4 操作組 17
2.5 綠色螢光蛋白 (Green fluorescent protein, GFP) 19
2.6 魚內臟蛋白水解 19
2.6.1 化學水解 20
2.6.2 酵素水解 20
2.7 蔗渣酸水解 21
2.8 豆渣 23
第三章材料與方法 25
3.1 實驗架構 25
3.2 實驗材料 26
3.3 實驗設備 28
3.4 菌種保存 29
3.4.1 -80 ℃保存 29
3.4.2 平板培養基保存 29
3.5 菌種培養 29
3.6 基因操作 ( DNA manipulation) 29
3.6.1 限制酶切割 (Restriction digestion) 29
3.6.2 洋菜凝膠電泳 (Agarose gel electrophoresis) 30
3.6.3 基因體DNA萃取 (Genomic DNA extraction) 30
3.6.4 質體DNA萃取 (Plasmid DNA extraction) 31
3.6.5 膠體純化 (Gel extraction) 32
3.6.6 聚合酶連鎖反應 (Polymerase chain reaction, PCR) 32
3.7 分子選殖 (Molecular cloning) 33
3.7.1 接合作用 (Ligation) 33
3.7.2 轉形作用 (Transformation) 34
3.8 異源蛋白表達 (Heterologous protein expression) 35
3.9 異源蛋白的萃取和純化 (Heterologous protein expression extraction and purification) 36
3.9.1 異源蛋白萃取 36
3.9.2 異源蛋白純化 36
3.10 蛋白質之定性和定量 37
3.10.1 布拉德福蛋白質定量法 (Bradford protein assay) 37
3.10.2 十二烷基硫酸鈉聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 37
3.11 魚廢培養基製作 38
3.12 最佳化Alcalase水解魚內臟蛋白的條件 38
3.12.1 不同pH下水解魚內臟試驗 38
3.12.2 酵素飽和濃度測定 38
3.12.3 胺基酸定量 39
3.13 YYBM1- subC細胞水解液水解蛋白質 39
3.13.1 水解BSA 39
3.13.2 水解魚內臟 40
3.13.3 水解豆渣 40
第四章結果與討論 41
4.1 重組質體之轉形 41
4.2 異源蛋白表達和純化 45
4.3 優化誘導YYBM1- gfp異源表達之木糖濃度 47
4.4 商業Xylose和BD-xylose誘導YYBM1異源表達之比較 49
4.5 最佳化Alcalase水解魚內臟蛋白的條件 50
4.6 綠色循環模組之建立 53
4.6.1 水解魚內臟 54
4.6.2 水解豆渣 57
第五章結論與建議 60
5.1 結論 60
5.2 建議 60
參考文獻 61
附錄A 69
附錄B 75

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指導教授

王柏翔(Po-Hsiang Wang)

審核日期

2022-12-26

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