利用枯草芽孢桿菌轉化魚內臟之亮胺酸為酮異己酸

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顏振升(Jhen-Sheng Yan) 查詢紙本館藏

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環境工程研究所

論文名稱

利用枯草芽孢桿菌轉化魚內臟之亮胺酸為酮異己酸
(Biosynthesis of α-ketoisocaproate from fish waste by Bacillus subtilis whole-cell biocatalyst)

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摘要(中)

伴隨著工業化與科技技術的進步下，促使農業糧食生產效率更勝以往，有效滿足人類對於環境資源的需求，卻也導致大量的固體有機廢棄物產生。以台灣農業為例每年約有二十萬公噸的魚類廢棄物和十三萬公噸的蔗渣等副產物產生，過往其回收再利用，如: 青貯、堆肥和做為動物飼料中的高蛋白粉等方式，所增加的經濟價值十分有限，無法賦予更高的價值，此外，這些農業廢棄物置於環境中易因細菌發酵而產生有毒硫化氫氣體或脂肪酸經氧化反應而所散發出的油耗味。為了解決此農業廢棄物後續處理問題並達到升值再利用化，因此本篇以魚廢和蔗渣作為模式固體廢棄物，以微生物催化的方式，用以製造高價值的酮異己酸 (α-Ketoisocaproic acid, KIC)。在實驗設計中，藉由優化Bacillus subtilis分泌胞外酵素subtilisin將魚廢蛋白水解為胺基酸，可為B. subtilis提供氮源外，其中所含亮胺酸 (Leucine)佔魚廢總胺基酸的10.1%，為KIC的前驅物，本研究藉由剔除KIC後續代謝基因bkdAB累積 KIC，並透過異源表達方式誘導 L-氨基酸脫氨酶（LAAD）表達以增加KIC之產量。此外，本研究所使用的木糖來源為酸解蔗渣，相較於過往文獻，此方法能從蔗渣中獲取高濃度的木糖 (26 g/L)，並運用於誘導蛋白表達。實驗結果顯示 B. subtilis在pH 8.0、40℃下具有最佳蛋白降解速率及轉化KIC速率，且B. subtilis突變株∆bkdAB+LAAD具有最高KIC的產量，相較於B. subtilis突變株∆bkdAB多約1.75倍(3.37 mM KIC)。此外，在代謝過程中，B. subtilis突變株∆bkdAB+LAAD不僅具較高的氨基酸利用率，相較於野生株B. subtilis亦減少近半的CO2產量 (43.3%)。因此，本文成功利用微生物轉化固體有機廢棄物為無毒增值產物，不僅符合聯合國提出的Sustainable Development Goals (SDGs)中的第12項¬(有責任之消費與生產)，更與政府提出的5+2產業創新計劃中的循環經濟、低排碳相互呼應。

摘要(英)

With the advances of industrial technology, the efficiency of agricultural production has been greatly improved. While satisfying human’s needs, the agriculture also comes along with large amounts of solid organic waste. For example, Taiwan′s agriculture produces 200,000 tons of fish waste and 130,000 tons of bagasse annually, respectively. In the past, the economic value added by organic waste recycling is low (such as compost), reducing the public’s will for organic waste recycling. Therefore, this study aims to valorize fish waste and bagasse to produce highly value-added products, and thereby enhancing the public’s will for organic waste recycling. α-Ketoisocaproic acid (KIC) is a naturally occurring metabolite of leucine that is capable of boosting animal muscle growth. In this study, we managed to develop a Bacillus-based fermentation system to produce KIC using fish waste and bagasse as the substrates. We selected Bacillus subtilis as the model organism for the KIC bioproduction because B. subtilis readily secrets extracellular protease subtilisin to the environments, which can hydrolyze fish waste protein into free amino acids. The leucine released would be subsequently converted into KIC by B. subtilis. We observed the optimal protein degradation rate in the wild-type B. subtilis cultures at pH 8.0 and at 40℃, and thus performing all the KIC bioproduction trials under such conditions. Later, we found that deletion of the bkdAB, which is responsible for KIC degradation, led to the KIC accumulation. In addition, heterologous expression of L-amino acid deaminase (LAAD) in B. subtilis increased the KIC yield up to 26 g/kg of fish waste. The results showed that the B. subtilis ∆bkdAB + LAAD cultures produced a 1.75 times higher yield of KIC (3.37 mmol per L of culture) than the yield of the B. subtilis ∆bkdAB culture. Moreover, we observed a higher amino acid utilization rate and a lower CO2 emission in B. subtilis ∆bkdAB + LAAD cultures compared to that in wild-type B. subtilis cultures. Altogether, this thesis demonstrated a successful microbial fermentation system capable of converting solid organic waste into the non-toxic, value-added products. This system represents an addition to circular bioeconomy, buttressing the 12th goal (responsible consumption and production) of the Sustainable Development Goals (SDGs) and facilitating the net-zero CO2 emission.

關鍵字(中)

★ 循環經濟
★ 轉化有機廢棄物
★ 增值產物
★ 減少碳排

關鍵字(英)

★ Circular economy
★ Transforming organic wastes
★ Value-added products
★ Reduce carbon emissions

論文目次

摘要 ii
謝誌 iv
目錄 v
一、研究緣起與目的 1
1.1 研究緣起 1
1.2 研究目的 2
1.3 實驗架構 2
二、文獻回顧 3
2.1 5+2產業創新計畫之「循環經濟」 3
2.2 現況與市場分析 3
2.3 環境污染 7
2.3.1 魚類廢棄物 7
2.3.2 處理蔗渣衍生汙染 7
2.4 枯草芽孢桿菌B. subtilis 8
2.4.1 枯草芽孢桿菌應用之特點 9
2.5 微生物生長營養需求 (培養基) 9
2.5.1 氮源 9
2.5.2 碳源 10
2.6 魚蛋白水解物 (Fish Protein Hydrolysate, FPH) 10
2.6.1 化學水解 11
2.6.2 酵素水解 12
1.內源性蛋白酶 12
2.外源性蛋白酶 12
2.7 蔗渣組成 13
2.7.1 半纖維素水解 14
2.8 胺基酸之亮胺酸代謝途徑 15
2.10.1 亮胺酸 15
2.8.2 α-酮異己酸 (α-Ketoisocaproic acid, KIC) 16
2.9 微生物合成酮異己酸 17
2.9.1 L-胺基酸脫胺酶（LAAD） 20
2.10 木糖誘導LAAD在B. subtilis 168中異源表達 21
2.11 質體剔除 (Plasmid curing) 22
2.12 優化B. subtilis分泌蛋白參數變因 22
2.13 胺基酸衍生化 23
三、材料與方法 24
3.1 實驗材料 24
3.1.1 培養基 25
3.1.2 菌株來源與保存 26
3.2 預處理 26
3.2.1 移除魚廢之脂質及內源性蛋白酶 26
3.2.2 魚廢培養基製作 27
3.2.3 水解蔗渣 27
3.2.4 異源蛋白表達 29
3.3 特性分析 29
3.3.1 定量蛋白質 29
3.3.2定量胺基酸 30
3.3.3 定量亮胺酸 30
3.3.4 定量酮異己酸 31
3.3.5 定量木醣 31
3.4 優化B. subtilis 分泌subtilisin 水解蛋白質 32
3.5 聚丙烯醯胺凝膠電泳 (Sodium dodecyl sulfate–polyacrylamide gel electrophoresis, SDS-PAGE electrophoresis) 32
3.6 定量二氧化碳 33
四、結果與討論 35
4.1 標準方法之建立 35
4.1.1 蛋白質檢量線 35
4.1.2 胺基酸檢量線 36
4.1.3 Leucine檢量線 36
4.1.4 KIC檢量線 38
4.1.5 Xylose檢量線 39
4.1.6 CO2檢量線 39
4.2 魚廢培養基製作基本特性分析 40
4.2.1 魚廢中蛋白質、胺基酸和KIC濃度 41
4.3 蔗渣基本特性分析 43
4.4 B. subtilis 168菌株異源蛋白表達的優化 43
4.4.1 不同pH值、溫度下分泌subtilisin與KIC速率 43
4.5 環境與經濟效益 50
五、結論與建議 51
5.1 結論 51
5.2 建議 51
參考文獻 53
附錄 60

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指導教授

王柏翔(Po-Hsiang Wang)

審核日期

2022-9-20

推文