臺灣博碩士論文加值系統

近來消費者的喜好傾向具高品種硫醇含量的葡萄酒和啤酒，其特徵包含新鮮、柑橘味和果香。主要被研究的品種硫醇是4-mercapto-4-methylpentan-2-one (4MMP) 讓人想起黃楊木和黑醋栗的味道以及3-mercaptohexan-1-ol (3MH) 讓人想起百香果和葡萄柚的香氣。先前文獻指出可透過重組半胱胺酸-S-共軛β-裂解酶 (CBL) 從前驅物釋放揮發性硫醇。本研究主要以來自Photobacterium damselae之重組CBL MetC從半胱胺酸化前驅物釋放揮發性硫醇之能力，並探討其特性分析，以及對不同前驅物之基質特異性及選擇性。首先表現與純化MetC，利用尺寸排除層析法確定具活性酵素以四聚體形式存在。接著以Ellman's reagent測定酵素在30℃和pH 9的條件下具有最佳活性。而由酵素動力學結果顯示MetC對L-Cystathionine具最佳親和力和催化效率 (kcat/Km為1.08×〖10〗^6 M-1s-1)，並對於揮發性香氣化合物之前驅物Cys-4MMP也有較高的催化效率 (kcat/Km為4.60×〖10〗^4 M-1s-1)。透過競爭試驗也顯示此酵素對基質L-Cystathionine有強烈偏好。接著進一步以同源蛋白序列比對和電腦分子對接模擬探討酵素與基質交互作用的胺基酸序列及位置。最後規模化製備4MMP，在有機相萃取得到的回收率為44.35%，顯示來自Ph. damselae之MetC在規模化生產方面可有效率地釋放揮發性硫醇，也具有廣泛的基質特異性，相信對於往後香料與食品工業有很好的發展潛力。

Recently, consumer preferences have shown a tendency towards wines and beers with high varietal thiol content, characterized by fresh, citrusy, and fruity notes. The primary varietal thiols studied are 4-mercapto-4-methylpentan-2-one (4MMP), reminiscent of boxwood and blackcurrant, and 3-mercaptohexan-1-ol (3MH), evocative of passionfruit and grapefruit aromas. Previous researches indicate that volatile thiols can be released from precursors via the enzyme cysteine-S-conjugate β-lyase (CBL). This study focuses on the ability of recombinant CBL MetC from Photobacterium damselae to release volatile thiols from cysteine-conjugated precursors, investigating its characterization, substrate specificity, and selectivity towards different precursors. Firstly, MetC was expressed and purified, and size exclusion chromatography confirmed that the active enzyme exists as a tetramer. The optimal activity of enzyme was determined using Ellman's reagent under conditions of 30°C and pH 9. Enzyme kinetics revealed that MetC has the highest affinity and catalytic efficiency for L-Cystathionine (kcat/Km of 1.08×106 M-1s-1), and also a relatively high catalytic efficiency for the volatile aroma precursor Cys-4MMP (kcat/Km of 4.60×104 M-1s-1). Competitive assays demonstrated a strong preference of the enzyme for the substrate L-Cystathionine. Furthermore, homology protein sequence alignment and computational molecular docking simulations were performed to explore the amino acid sequences and positions involved in the enzyme-substrate interactions. Finally, scale-up preparation of 4MMP resulted in a recovery rate of 44.35% in the organic phase, indicating that MetC from Ph. damselae can efficiently release volatile thiols on a large scale and possesses broad substrate specificity. This suggests great potential for future applications in the flavor and food industries.

摘要 I
目錄 III
壹、 前言 1
貳、 文獻整理 2
2.1. 香氣化合物 2
2.1.1. 葡萄酒中的香氣化合物 2
2.1.1.1.萜 (Terpenes) 2
2.1.1.2. 醛 (Aldehydes) 2
2.1.1.3. 酯 (Esters) 3
2.1.1.4. 甲氧基吡嗪類 (Methoxypyrazines, MPs) 3
2.1.1.5. 揮發性硫醇香氣化合物 4
2.2. 製造香氣化合物 5
2.2.1. 香氣化合物的生產方法 5
2.2.1.1. 萃取 5
2.2.1.2. 酵素 5
2.2.2. 以酵素方法生產香氣化合物 6
2.3. 半胱胺酸-S-共軛β-裂解酶 (Cysteine-S-conjugate β-lyase, CBL) 6
2.3.1. 酵素特性 7
2.3.2. 酵素CBL來源 7
2.3.2.1. IRC7 7
2.3.2.2. STR3 8
2.3.2.3. Cys3 8
2.3.2.4. metC 8
2.3.2.5. tnaA 9
2.3.3. CBL之作用機制 9
2.4. 酵素基質的特異性與選擇性 10
2.4.1. 酵素基質特異性 10
2.4.2. 酵素動力學分析 11
2.4.3. 模擬酵素基質對接 11
2.4.4. 前驅物基質簡介 12
參、 實驗設計 14
14
肆、 材料與方法 15
4.1. 實驗材料 15
4.1.1. 實驗菌株 15
4.1.2. 培養基 15
4.1.3. 抗生素 15
4.1.4. 化學藥品 15
4.1.5. 試劑套組 16
4.1.6. 酵素 17
4.1.7. 實驗儀器與耗材 17
4.2. 實驗方法 18
4.2.1. 菌株保存 18
4.2.2. 菌株活化 18
4.2.3. 蛋白質表現 18
4.2.3.1. 蛋白質表現 18
4.2.3.2. 蛋白質萃取 19
4.2.3.3. 親和性管柱層析法 19
4.2.4. 蛋白質分析 20
4.2.4.1. 蛋白質定性 20
4.2.4.2. 蛋白質濃度測定 21
4.2.4.3. 酵素構型分析 22
4.2.5. 酵素特性分析 22
4.2.5.1. 酵素活性測定 22
4.2.5.2. 酵素最適反應條件 23
4.2.5.3. 酵素安定性 23
4.2.6. 基質特異性分析 23
4.2.6.1. 酵素動力學 23
4.2.6.2. 以競爭法確定CBL活性位點的規則 24
4.2.7. CBL之活性位點 24
4.2.7.1. 同源蛋白之序列比對 24
4.2.7.2. 同源建模型及對接模擬 24
4.2.8. 以小規模製備4MMP 24
4.2.9.統計分析 25
伍、 結果與討論 26
5.1. Ph. damselae MetC重組CBL之純化與特性分析 26
5.1.1. 蛋白質表現及純化 26
5.1.2. 蛋白質電泳 26
5.1.3. 蛋白質濃度鑑定 26
5.1.4. 蛋白質之聚體型態 26
5.1.5. MetC之最佳反應條件 27
5.1.6. MetC之酵素安定性 27
5.2. MetC之活性及特異性分析 28
5.2.1. MetC對不同基質之酵素動力學 28
5.2.1.1. L-Cystathionine動力學 28
5.2.1.2. L-Cystine動力學 29
5.2.1.4. Cys-4MMP動力學 29
5.2.1.5. Cys-4MMPOH動力學 30
5.2.1.6. Cys-4NP 動力學 30
5.2.2. 以競爭法確定CBL活性位點的規則 30
5.3. MetC之活性位點 31
5.3.1. 同源蛋白之序列比較 32
5.3.2. 以分子對接模擬酵素與基質對接情形 32
5.4. 以小規模製備4MMP 33
陸、 結論 34
柒、 參考文獻 35
捌、 圖表 45

 

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劉郁玟。(2022)。海洋菌Shewanella putrefaciens之半胱胺酸-S-共軛β-裂解酶釋放硫醇香氣化合物3-巰機-1-己醇。國立臺灣海洋大學食品科學系碩士學位論文。基隆。臺灣
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