臺灣博碩士論文加值系統

本實驗提高Clonostachys compactiuscula生產lovastatin酯解酶，以及改善純化流程，建立大量純化系統，增加總體純化酵素的產量，並定量純化各步驟的酵素產率，以利進行定序lovastatin酯解酶。實驗測試酵素在不同pH下最佳的活性和穩定性，以及有無tween 20存在環境下的酵素動力學，估算酵素的動力學參數Vmax、Km、kcat和kcat / Km，來了解酵素的催化特性。
實驗改良菌種培養條件，由原先加入6 ml 61.8 mM lovastatin溶液誘導6天的培養條件更改為加藥0.3 ml誘導8小時，酵素的生產速率提升為原先的30.44倍，且誘導菌種使用藥量降低為原先的1/20倍。此外實驗還改良純化過程，將大量菌絲體經破碎提取粗酵素液後，經陰離子管柱層析以及Bis-Tris native-PAGE純化；其樣品進行SDS-PAGE電泳，再利用影像分析軟體，可知每5g菌絲體可生產8.10 µg的lovastatin酯解酶；另外，在Bis-Tris native-PAGE電泳後進行電萃，分析純化過程，可知其總產率為65.38 %，酵素的總比活性為264.57 U/µg，總純度提高387.10倍。
在生化特性方面，酵素在pH 9下有最佳催化活性；在pH 6至10的溶液下酵素活性殘留80%以上。在酵素動力分析方面，酵素在pH 10下添加0.1% Tween 20其kcat/Km，是未添加Tween 20的3.297倍，水解受質的速率增加，而Km未因添加Tween 20改變，所以酵素對受質的親和性不受影響。比較在0.1% Tween 20存在下不同pH環境中酵素的活性，酵素在pH 6的Km (0.214 mM )略大於在pH 10的Km (0.159 mM )，所以pH 6中酵素對受質的親和性降低；而酵素在pH 10下kcat(184.36 sec-1)為在pH 6下kcat (42.63 sec-1)的4.32倍，顯示酵素在pH 10下水解受質的速率為pH 6的4.32倍；酵素在含0.1% Tween 20的pH10環境下為最佳催化條件，其Km為0.159 mM，kcat/Km為1160.993 mM -1/sec。

This experiment improves the production of lovastatin esterase from Clonostachys compactiuscula, and the process of the enzyme purification, that established a scaled-up purification system, to increase the total yield and quantify the yield in each step of the enzyme purification, which is convenient for sequencing the lovastatin esterase.
The experiment tested the optimal activity and stability of the enzyme at different pH, and the enzyme kinetics in the presence or absence of tween 20, estimate the kinetic parameters of the enzymes Vmax, Km, kcat, and kcat/Km to understand the catalytic properties of the enzyme.
Experiment was improved the cultivation conditions of strains, that changed from inducing 6 day with 6ml of medicine added in the original culture condition to the 8 hour with 0.3ml of medicine added, the rate of enzyme production was increased to 30.44 times of the original, and the dosage induced of the fungus was reduced to 1/20 times of the original.
In addition, the experiment also improved the process of the purification. After crushing a large amount of mycelium to extract the crude enzyme solution, it was purified by anion column chromatography and Bis-Tris native-PAGE. This samples purified by SDS-PAGE electrophoresis, and using image analysis software, it showed that every 5g of mycelium can produce 8.10µg of lovastatin esterase. In addition, Gel Eluting after Bis-Tris native-PAGE electrophoresis purified, and the purification process was analyzed, it showed that the total yield is 65.38 %, and the total specific enzyme activity is 264.57 U/µg ,and the total purity is increased by 387.10 times.
In terms of biochemical properties, the experiment shows that the enzyme has the best catalytic activity at pH 9. The enzyme activity remains above 80% under the pH6 to 10 solution. In terms of enzyme kinetics analysis, the enzyme added 0.1% Tween 20 at pH10, which kcat/Km was 3.297 times that of the unadded Tween 20. The rate of the substrate hydrolyzed increases, and the Km is not changed by the addition of Tween 20, so the affinity of the enzyme for the substrate is not affected.Comparing the activity of the enzyme in different pH environments with 0.1% Tween 20, the Km (0.214 mM) of the enzyme at pH 6 is slightly greater than the Km (0.159 mM) at pH 10, so the affinity of the enzyme for the substrate at pH 6 is reduced. The kcat (184.36 sec-1) of the enzyme at pH10 is 4.32 times that of kcat (42.63 sec-1) at pH6, which shows that the rate of the enzyme hydrolyzing substrate at pH10 is 4.32 times that of pH6. The enzyme in pH10 environment with 0.1% Tween 20 is the best catalyzing condition, its Km is 0.159 mM, and kcat/Km is 1160.993 mM -1/sec.

目錄
摘要 i
Abstract iii
目錄 vi
圖目錄 x
表目錄 xiii
附錄 xiv
第一章 前言 1
1-1 高血脂治療藥物-statin 1
1-2 Simvastatin合成方式 2
1-3 酯解酶(esterases)與脂肪酶(lipases) 3
1-4 研究動機與目的 5
第二章 實驗材料 6
2-1 實驗步驟流程 6
2-2 實驗藥品 7
2-3 實驗儀器 8
2-4 電腦分析軟體 9
第三章 實驗方法 10
3-1 Clonostachys compactiuscula的保存、培養和發酵條件 10
3-2 Lovastatin酯解酶純化 11
3-2-1 提取菌體中酵素 11
3-2-2 管柱層析（column chromatography） 11
3-2-3 樣品電萃 ( whole gel eluter ) 12
3-3 聚丙烯醯胺凝膠電泳分析 13
3-3-1 Lovastatin酯解酶之電泳分析 13
3-3-2 蛋白水解酶之電泳及轉漬受質膠分析 14
3-4 蛋白質染色 15
3-4-1 硝酸銀染色步驟 15
3-4-2 鋅染色法 15
3-4-3 Coomassie blue 染色 16
3-5 Lovastatin酯解酶活性分析( lovastatin esterase activity assay ) 16
3-5-1 Lovastatin受質液配製 16
3-5-2 Lovastatin酯解酶活性測試 17
3-6 SDS-PAGE膠上 lovastatin酯解酶之活性回復 18
3-7 Lovastatin酯解酶之電泳膠體定位 18
3-8 Lovastatin酯解酶生化特性分析 19
3-8-1 含Tween20各pH環境下對酵素的活性影響 19
3-8-2 各pH環境對酵素的穩定性影響 19
3-8-3 酵素動力學 20
3-9 蛋白質定序 20
3-10蛋白質濃度測定 21
3-10-1 Bradford法總蛋白質定量 21
3-10-2 使用SDS-PAGE及Image J定量分析蛋白量 21
第四章 結果 23
4-1 優化菌種培養 23
4-2 Lovastatin酯解酶之電泳分析 24
4-2-1 改良二維電泳分析是否添加6-ACA 24
4-2-2 改良二維電泳蛋白處理方式 25
4-3 樣品中蛋白水解酶之電泳分析 26
4-3-1 SDS和pH對蛋白水解酶活性的呈現 26
4-3-2 樣品中蛋白酶 對IgY之水解能力測試 27
4-4 Lovastatin酯解酶純化 28
4-4-1 Mono-Q管柱層析及電泳純化分析 28
4-4-2 電萃及電泳純化分析 31
4-5 定量lovastatin酯解酶之純化過程損失率 32
4-6 SDS-PAGE上 lovastatin酯解酶之復性條件 33
4-7 Lovastatin酯解酶之電泳膠體定位 34
4-7-1 Bis-Tris native-PAGE和SDS-PAGE電泳 34
4-7-2 Lovastatin酯解酶之分子量 35
4-8 Lovastatin酯解酶 之生化特性分析 36
4-8-1 pH對酵素的活性和穩定性的影響 36
4-8-2 酵素動力學 37
4-9 蛋白質定序 40
第五章 討論與結論 42
5-1 優化菌種培養 42
5-2 Lovastatin酯解酶之電泳分析及膠體定位 43
5-2-1 Lovastatin酯解酶之二維電泳 43
5-2-2 Lovastatin酯解酶之分子量 45
5-3 Lovastatin酯解酶純化 46
5-4 定量lovastatin酯解酶之純化過程損失率 47
5-5 SDS-PAGE上 lovastatin酯解酶之活性復性 48
5-6 pH對酵素的活性和穩定影響 49
5-7 酵素動力學 50
5-8 蛋白質定序 52
第六章 參考文獻 100

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