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系統識別號 U0026-0812200910170442
論文名稱(中文) 氮化鋁粉體水解性質探討與抗濕技術開發
論文名稱(英文) Discussion of hydrolyzed properties of aluminum mitride powder and development of water-resistance technology
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 90
學期 2
出版年 91
研究生(中文) 謝承佑
學號 n3689133
學位類別 碩士
語文別 中文
口試日期 2002-06-07
論文頁數 107頁
口試委員 指導教授-鍾賢龍
口試委員-李威昌
口試委員-黃其清
關鍵字(中) 氮化鋁
水解
抗濕
關鍵字(英) aluminum nitride
water-resistance
hydrolysis
學科別分類
中文摘要 本論文研究分為三大部分,第一部分首先針對氮化鋁粉體在各種不同環境條件下的水解氧化速率進行分析探討,從中找出使氮化鋁水解的關鍵因素與各種水解氧化所可能產生的結果,便於針對水解的因素開發出適當的抗濕技術;第二部分即針對前一部分所得的結果與文獻中所記載類似的方法進行表面處理,主要採用有機物與環氧樹脂為主,經表面處理之氮化鋁粉體已可具備初步的抗濕性質,但在較為嚴苛的環境中依然會產生水解氧化反應;最後一部分則是針對第二部分的缺失再進行改進,改進的方式分為氮化鋁粉體在進行表面處理前先經過一道前處理的手續,接著再以有機物與環氧樹脂進行表面處理,經由此方式處理之氮化鋁粉體可比前一部分的抗濕效果更好;同時,此部份亦嘗試使用氮化鋁粉體與無機酸在水中進行表面處理,實驗結果證實此方式會具有最佳的抗濕效果。
英文摘要 This thesis includes three parts. The first part is focused on the hydrolysis properties of aluminum nitride powders in different kinds of environment. The main purpose is to find out the key factor that accelerates the hydrolysis rate of the aluminum nitride powders and the results after hydrolysis reaction. Based on these results, it is possible to develop the appropriate methods to prevent aluminum nitride powders from hydrolysis. The second part describes the method of surface treatment based on the results from part one and scientific literature. The main surface treatment chemicals are organic matters and epoxy resins. The aluminum nitride powders with surface treatment by this way are able to resist humidity. But they are still hydrolyzed in sterner environment. The final part describes the method to improve the defect in part two. There is one step added before surface treatment and then processed with the same way described in part two. Aluminum nitride powders with this surface treatment have better effect on water-resistance. Moreover, this part also tries to use inorganic acid as surface treatment chemicals and water as solvent. The experimental data show that aluminum nitride powders treated in this way will present excellent water-resistant properties.
論文目次 中文摘要…………………………………………………………………I
英文摘要…………………………………………………………………II
總目錄……………………………………………………………………III
表目錄……………………………………………………………………VII
圖目錄…………………………………………………………………X
第一章 緒 論…………………………………………………………1
1-1陶瓷簡介………………………………………………………………1
1-2 氮化鋁的特性與應用…………………………………………………2
1-3 氮化鋁的合成技術…………………………………………………3
1-4 研究動機……………………………………………………………4
1-5 文獻回顧……………………………………………………………6
1-5.1 氮化鋁表面披覆磷酸根離……………………………………6
1-5.2溶液法披覆SiO2………………………………………………6
1-5.3美國耐火陶瓷科技公司專利…………………………………8
1-5.4 射頻電漿化學氣相沉積披覆二氧化矽……………………8
1-5.5 化學表面處理…………………………………………………9
1-5.6 表面氧化處理………………………………………………13
第二章 本論文研發之方法…………………………………………15
2-1簡介………………………………………………………………15
2-2藥品………………………………………………………………15
2-3 實驗及產品測試設備…………………………………………………16
2-4 儀器分析………………………………………………………………18
第三章 氮化鋁水解氧化性質研究………………………………23
3-1 簡介………………………………………………………………23
3-2 原理……………………………………………………………23
3-3 實驗方法………………………………………………………24
3-4 實驗結果與討論……………………………………………25
3-4.1 氮化鋁粉體檢測…………………………………………25
3-4.2 氮化鋁粉體水解氧化性質………………………………35
(I) 水解對溶液pH值的影響……………………………35
(II) 水解對氮化鋁的影響…………………………………36
(III) 氮化鋁粉體粒徑對水解速度的影響…………………44
(IV) 氨氣對氮化鋁水解的影響……………………………46
(V) 氮化鋁粉體轉化率對水解的影響……………………48
3-5 結果討論…………………………………………………………72

第四章 氮化鋁抗濕處理………………………………………………74
4-1 簡介……………………………………………………………74
4-2 原理……………………………………………………………74
4-2.1 有機酸衍生物披覆……………………………………75
4-2.2 矽氧烷偶合劑披覆……………………………………76
4-2.3 熱固性塑膠披覆………………………………………82
4-3 實驗方法………………………………………………………84
4-3.1 氮化鋁粉體表面處理…………………………………84
4-3.2 表面處理粉體測試……………………………………86
4-4 實驗結果與討論………………………………………………88
4-4.1 披覆矽氧烷之抗濕效果………………………………88
4-4.2 披覆有機酸之抗濕效果………………………………99
(i) 單一官能基苯環有機酸衍生物…………………………99
(ii) 雙官能基苯環有機酸衍生物……………………………104
4-4.3 披覆偶合劑及環氧樹脂之抗濕效果……………………109
4-4.4 加速壽命測試……………………………………………113
4-4.5 水解測試…………………………………………………120
4-5 結論………………………………………………………………123

參考文獻…………………………………………………………164
參考文獻 參 考 文 獻
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系統識別號 U0026-0812200910201604
論文名稱(中文) 微水有機溶劑中利用脂肪分解酵素進行外消旋naproxen三氟乙酯之水解動態動力分割
論文名稱(英文) Lipase-catalyzed dynamic kinetic resolution of (R,S)-naproxen ester in isooctane
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 90
學期 2
出版年 91
研究生(中文) 林瀚淵
學號 n3689426
學位類別 碩士
語文別 中文
口試日期 2002-06-20
論文頁數 67頁
口試委員 口試委員-林松池
口試委員-陳特良
口試委員-陳志平
指導教授-蔡少偉
口試委員-吳昭燕
關鍵字(中) 脂肪分解酵素
水解
動態動力分割
關鍵字(英) lipase
hydrolysis
dynamic kinetic resolution
naproxen
學科別分類
中文摘要 過去研究指出利用Lipase MY進行naproxen三氟乙酯之水解動力分割可獲得相當優異的選擇性及反應性;此外,利用Lipase MY配合三辛基胺為鹼消旋觸媒,進行naproxen三氟乙硫酯之動態動力分割亦有相當好的成效。本論文將探討以外消旋naproxen三氟乙酯為反應物之動態動力分割製程,首先尋找可使之消旋之強鹼觸媒,以便配合Lipase MY進行水解動態動力分割。
本論文中尋得數種可將naproxen三氟乙酯消旋的有機鹼觸媒,且獲得消旋常數與鹼觸媒濃度具有線性關係。此外並發現當鹼觸媒消旋能力愈強,伴隨的無選擇性鹼催化水解亦愈嚴重。由於發現可溶性鹼觸媒會使酵素失活,因此改用固定化鹼觸媒配合Lipase MY進行水解動態動力分割,結果可成功克服傳統動力分割目標產物最高50 %轉化率之限制。最後,以Michaelis-Menten酵素反應機構配合一階消旋反應及酵素失活、酵素抑制作用之動態動力分割模式,可成功獲得與實驗值相當一致的理論結果。
英文摘要 Lipase MY has been successfully employed as the biocatalyst with excellent enantioselectivity and activity in the kinetic resolution of racemic naproxen trifluoroethyl ester via hydrolysis. Besides, a dynamic kinetic resolution process for the lipase-catalyzed hydrolysis of racemic naproxen trifluoroethyl thioester has also been developed by using Lipase MY and trioctylamine as the biocatalyst and racemization catalyst, respectively. Therefore, the purpose of this research is aimed to develop a dynamic kinetic resolution process with (R,S)-naproxen trifluoroethyl ester as the substrate. Stronger organic bases were firstly screened to effectively racemize the substrate. A linear relationship between the interconversion constant and the base concentration was found. However, the stronger was the base in racemization, the higher of hydrolysis of the base was observed. Since the base might deactivate the enzyme, a polymer-supported base was replaced as the racemization catalyst in the dynamic kinetic resolution process. The maximum 50% yield of the desired optical product in the standard kinetic resolution process was overcome. Agreements between the experimental data and theoretical results were found in which an enzymatic Michaelis-Menten kinetics, lipase deactivation and inhibition and a first-order reversible racemization kinetics were employed in the theoretical modelling.
論文目次 第一章 緒論
1-1 酵素 1
1-2 脂肪酵素 2
1-3 酵素於有機溶劑之行為 5
1-4 對掌性藥物 9
1-5 Naproxen 11
1-6 動態動力分割 14
1-7 研究動機 16

第二章 原理
2-1 鹼觸媒消旋 17
2-2 酵素催化水解動力分割 20
2-3 酵素催化水解動態動力分割 22

第三章 實驗方法
3-1 藥品與材料 24
3-2 實驗設備 25
3-3 分析方法 26
3-4 實驗步驟 28
3-4-1 合成naproxen三氟乙酯 28
3-4-2 酵素固定化 29
3-4-3 鹼觸媒消旋反應 29
3-4-4 不同基質濃度之水解反應初速率測定 29
3-4-5 不同抑制物濃度之抑制作用 30
3-4-6 不同鹼觸媒對動態動力分割外消旋naproxen三氟乙酯之影響 30

第四章 結果與討論
4-1 鹼觸媒消旋能力 31
4-2 酵素催化水解動力分割參數偶合 39
4-3 酵素的抑制作用與失活現象 43
4-4 以DBU為鹼觸媒之酵素催化水解動態動力分割 49
4-5 p-TBD之消旋能力 49
4-6 以p-TBD為鹼觸媒之酵素催化水解動態動力分割 52

第五章 結論與後續研究 61

參考文獻 63
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Crossley, R., Chirality and the biological activity of drugs, CRC Press, Florida, 1995.
Ebbers, E. J., Ariaans, G. J. A., Houbiers, J. P. M., Bruggink, A., Zwanenburg, B., Controlled racemization of optically active organic compounds: prospects for asymmetric transformation, Tetrahedron, 53, 9417-9476, 1997.
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系統識別號 U0026-0812200910240834
論文名稱(中文) 神經鞘磷脂水解的表現、純化及定性
論文名稱(英文) Expression, Purification and Characterization of Neutral Sphingomyelinase
校院名稱 成功大學
系所名稱(中) 生物科技研究所碩博士班
系所名稱(英) Institute of Biotechnology
學年度 90
學期 2
出版年 91
研究生(中文) 曾詩喬
學號 l6689402
學位類別 碩士
語文別 中文
口試日期 2002-07-20
論文頁數 70頁
口試委員 指導教授-蕭世裕
口試委員-楊俊佑
口試委員-鄭梅芬
口試委員-林聖哲
口試委員-林鴻儒
關鍵字(中) 枯草桿菌
中性神經鞘磷脂水解脢
關鍵字(英) Neutral sphingomyelinase
Bacillus subtilis
學科別分類
中文摘要 神經鞘磷脂代謝過程中扮演重要角色的磷脂水解酵素被稱之為神經鞘磷脂水解(Sphingomyelinase),此酵素可誘發細胞凋亡、細胞分化、細胞增生與體內固醇平衡等不同的細胞調控。本實驗室已由枯草桿菌中鑑定出中性神經鞘磷脂水解(Neutral Sphingomyelinase),此酵素分子量為34.3kDa,與已知之Bacillus cereus IAM1208 中之中性神經鞘磷脂水解在胺基酸序列上有98.6%相同,以N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM)為受質測定神經鞘磷脂水解之酵素動力學及pH、離子對酵素活性的影響,得到酵素動力學常數Km 11.06 μM 及Vmax 10.23 nmol /min(10μg sphingomyelinase),最佳的反應環境以pH 7.4尤佳;酵素的活性被鈣離子所抑制並被鎂離子活化,為瞭解不同離子與中性神經鞘磷脂水解的作用,以色胺酸螢光光譜分析不同濃度的鈣、鎂離子下的螢光強度及相對離子濃度活性,發現鈣離子色胺酸螢光光譜有一處明顯轉變,鎂離子色胺酸螢光光譜有二處明顯轉變,表示鈣離子在濃度10-6~10-8M有一處鍵結點、鎂離子在濃度10-7~10-8M與10-3~10-4M各有一處鍵結點,而在相對濃度的活性偵測可以發現鎂離子濃度低於10-4M時酵素活性消失,神經鞘磷脂代謝過程中扮演重要角色的磷脂水解酵素被稱之為神經鞘磷脂水解(Sphingomyelinase),此酵素可誘發細胞凋亡、細胞分化、細胞增生與體內固醇平衡等不同的細胞調控。本實驗室已由枯草桿菌中鑑定出中性神經鞘磷脂水解(Neutral Sphingomyelinase),此酵素分子量為34.3kDa,與已知之Bacillus cereus IAM1208 中之中性神經鞘磷脂水解在胺基酸序列上有98.6%相同,以N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM)為受質測定神經鞘磷脂水解之酵素動力學及pH、離子對酵素活性的影響,得到酵素動力學常數Km 11.06 μM 及Vmax 10.23 nmol /min(10μg sphingomyelinase),最佳的反應環境以pH 7.4尤佳;酵素的活性被鈣離子所抑制並被鎂離子活化,為瞭解不同離子與中性神經鞘磷脂水解的作用,以色胺酸螢光光譜分析不同濃度的鈣、鎂離子下的螢光強度及相對離子濃度活性,發現鈣離子色胺酸螢光光譜有一處明顯轉變,鎂離子色胺酸螢光光譜有二處明顯轉變,表示鈣離子在濃度10-6~10-8M有一處鍵結點、鎂離子在濃度10-7~10-8M與10-3~10-4M各有一處鍵結點,而在相對濃度的活性偵測可以發現鎂離子濃度低於10-4M時酵素活性消失,顯示低親和力鎂離子鍵結點與催化活性相關。
以往的研究顯示中性神經鞘磷脂水解在大腸桿菌中表現之產率不佳,為了改善其在大腸桿菌中表現之產率,本研究利用培養皿誘導、低溫培養及低濃度誘導物IPTG等方式改良表現系統,研究結果發現帶有枯草桿菌訊息胜之中性神經鞘磷脂水解在此表現系統中可以在大腸桿菌BL21(DE3)中被大量表現並減少包涵體產生的現象,其產率高達10mg/L。
顯示低親和力鎂離子鍵結點與催化活性相關。
以往的研究顯示中性神經鞘磷脂水解在大腸桿菌中表現之產率不佳,為了改善其在大腸桿菌中表現之產率,本研究利用培養皿誘導、低溫培養及低濃度誘導物IPTG等方式改良表現系統,研究結果發現帶有枯草桿菌訊息胜之中性神經鞘磷脂水解在此表現系統中可以在大腸桿菌BL21(DE3)中被大量表現並減少包涵體產生的現象,其產率高達10mg/L。
英文摘要 At the epicenter of the sphingomyelin-cell signaling pathway is a family of phospholipase called sphingomyelinase. These enzymes induce a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Sphingomyelinase may be useful for additional studies of apoptosis in experimental animals.
We have identified a neutral sphingomyelinase from Bacillus subtilis IAM1208. The enzyme, with a molecular mass of 34.3 kDa, has 98.6% amino acid sequence identity with sphingomyelinase from Bacillus cereus IAM 1208. When N-ω-trinitrophenylaminolauryl sphingomyelin (TNPAL-SM) was used as a substrate, the purified neutral-sphingomyelinase exhibited a Km of 11.34μM and a Vmax of 10.225 nmol /min/10μg of protein at 37℃, pH 7.4. The pH and ion effect on N-SMase activity was examined. The optimal pH of N-SMase was at pH 7.4 .The enzyme activity was inhibited by Ca2+ but was activated by Mg2+. The effect of bivalent cations on the activity and Tryptophan fluorescence spectra of N-SMase were also examined. In the Ca2+-dependence curve of tryptophan fluorescence intensity, one distinct transition was observed between 10-6 to 10-8M, In the Mg2+-dependence curve of fluorescence intensity, two distinct transition was observed between10-7 to 10-8 and 10-3 to 10-4M and the enzyme activity was disappeared in the presence of Mg2+ below 10-4 M, suggesting that the binding of to the low-affinity site is essential for the catalysis.
In literature, the expression of sphingomyelinase of Bacillus cereus was not efficient in E. coli. To improve the yield sphingomyelinase of Bacillus cereus, we have modified the expression and induction system by changing the plating method, lowered temperature, and lowered concentration of IPTG. In this report, we have expressed sphingomyelinase from Bacillus subtilis in E. coli and decreased the amount of sphingomyelinase in inclusion bodies with the yield of 10mg protein in 1L of Bacteria culture.
論文目次 中文摘要 I
英文摘要 II
目錄 IIV
圖目錄 VII
表目錄 X

壹、前言 1
貳、文獻探討 3
一、神經鞘磷脂生物功能與疾病 3
1. 神經鞘磷脂 3
2. 尼曼-匹克氏病 4
二、中性神經鞘磷脂水解 5
1. 動物中性神經鞘磷脂水解生理功能 5
1.1神經鞘磷脂循環與細胞凋亡 5
1.2膽固醇調節與心血管疾病 6
2. 微生物之神經鞘磷脂水解 7
2.1溶血現象 7
2.2致病能力 9
三、Bacillus cereus之中性神經鞘磷脂水解 9
1. B. cereus神經鞘磷脂水解性質 9
2. B. cereus神經鞘磷脂水解純化與表現 11
四、Bacillus subtilis之中性神經鞘磷脂水解 12
參、材料與方法 13
一、材料 13
二、儀器 13
三、枯草桿菌神經鞘磷脂水解之純化 14
1. 枯草桿菌培養 15
2. 快速蛋白質液相層析儀(FPLC)純化 15
3. 蛋白質電泳分析 16
四、大腸桿菌之轉殖、表現與蛋白質純化 17
1. 勝任細胞的製備 17
2. Transformation轉型作用 17
3. 轉殖大腸桿菌之誘導作用 18
4. 轉殖大腸桿菌之神經鞘磷脂水解純化 18
5. 蛋白質定量 19
6. TCA蛋白質沈澱法 19
7. 質譜儀分析與N端胺基酸定序 20
五、神經鞘磷脂水解之性質研究 20
1. 活性測試 20
2. 動力學測定 20
3. 最適反應環境測定 21
4. 色胺酸螢光光譜分析 23
六、無訊息胜中性神經鞘磷脂水解選殖 24
肆、結果 27
一、枯草桿菌神經鞘磷脂水解之純化 27
二、大腸桿菌的表現與純化 27
三、酵素活性測試 28
伍、討論 31
一、枯草桿菌中性神經鞘磷脂水解純化 31
二、大腸桿菌之培養皿誘導法 31
三、中性神經鞘磷脂水解性質探討 34
陸、結論 37
柒、參考文獻 38
圖 42
表 69
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系統識別號 U0026-0812200910242588
論文名稱(中文) 以環狀糊精為基材製備親和性吸附劑以及對澱粉水解酵素吸附脫附之探討
論文名稱(英文) Investigation of beta-cyclodextrin based affinity adsorbents for the adsorption and desorption of alpha-amylase
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 90
學期 2
出版年 91
研究生(中文) 周泰成
學號 n3689120
學位類別 碩士
語文別 中文
口試日期 2002-07-22
論文頁數 65頁
口試委員 指導教授-許梅娟
口試委員-周明顯
口試委員-王鴻博
口試委員-陳阿煌
口試委員-李彩雲
關鍵字(中) 固定化金屬離子親和性層析法
Beta 型環狀糊精
環氧氯丙烷
DADPA
Alpha 型澱粉水解酵素
EDTA
關鍵字(英) EDTA
Alpha-amylase
DADPA
EPI
immobilized metal affinity chromatography
Beta-CD
學科別分類
中文摘要 純化在生化產業中是非常重要的步驟,其中親和性層析法 (affinity chromatography) 頗受重視是因其專一性的純化特性,可以有效且快速的將產物分離,藉此達到降低成本的目的,並且提高產業的競爭力。
本論文主要是以固定化金屬離子親和性層析法 (immobilized metal affinity chromatography) 對alpha 型澱粉水解酵素(alpha-amylase) 進行吸脫附的研究與探討。研究是以 beta 型環狀糊精 (beta-cyclodextrin,beta-CD) 與環氧氯丙烷 (epichlorohydrin, EPI) 形成的交聯物為擔體,再分別鍵結IDA或cibacron blue F3G-A或DADPA為配位基並螯合銅離子製備固定化金屬離子親和性吸附劑;其中以 beta-CD CL-DADPA-Cu2+ 的吸附效果最高可達99%,而以EDTA脫附時,其脫附效果也高達98%。
綜合上述之結果,本研究是以擔體鍵結DADPA並螯合銅離子所製備而成的吸附劑,具有最好的吸附與脫附效果。
英文摘要 Purification is essential in the biochemical industry. The approach of affinity chromatography is widely applied because of its specificity as well as its separate efficiency.
The aim of this dissertation is to discuss the absorption and desorption the immobilized metal affinity adsorbent for alpha-amylase. In this work, the matrix beta-CD was cross-linked with EPI, then further immobilized with the ligand such as IDA, cibacron blue F3G-A, and DADPA respectively. In addition Cu2+ was chelated on the matrix to form immobilized metal affinity adsorbent. Among these adsorbents being studied, beta-CD CL-DADPA-Cu2+ shows best adsorption result of 99%. Meanwhile, of EDTA shows best result of desorption which can reach up to 98%.
In conclusion, the affinity adsorbent beta-CD CL-DADPA-Cu2+ can reach best performance on adsorption and desorption.
論文目次 表目錄 I
圖目錄 II
第一章 緒論 1
1-1 Alpha-Amylase之簡介 1
1-2 生化產物之分離與純化 1
1-3 層析法之定義與分類 2
1-3-1 親和性層析 (Affinity chromatography) 之原理 3
1-4 擔體 (Matrix) 4
1-5 配位基 (Ligands) 5
1-6 固定化金屬離子親和性層析
(Immobilized metal-ion affinity chromatography ,IMAC) 6
1-7 環狀糊精 (Cyclodextrin) 之簡介 7
1-8 環狀糊精在親和性層析之應用 8
第二章 實驗材料與方法 10
2-1 酵素之活性測定方法 10
2-2 Beta-Cyclodextrin CL (-CD CL) 擔體之製備 10
2-2-1 以環氧氯丙烷 (epichlorohydrin, EPI) 為交聯劑製備 beta-CD CL擔體 10
2-2-2 以不同交聯劑製備beta-CD CL擔體 10
2-3 不同吸附劑之製備 11
2-3-1 A1吸附劑之製備 11
2-3-2 B1吸附劑之製備 11
2-3-3 C1吸附劑之製備 12
2-3-4 A2、B2、C2吸附劑之製備 12
2-3-5 D1、D2、D3吸附劑之製備 12
2-4 Alpha-amylase之吸附行為 12
2-4-1 以不同吸附劑吸附 alpha-amylase溶液 12
2-4-2 以 D2吸附劑吸附不同濃度之 alpha-amylase溶液 12
2-4-3 以不同粒徑之D2吸附劑吸附 alpha-amylase溶液 13
2-4-4 以不同交聯程度擔體製備之D2吸附劑吸附 alpha-amylase溶液 13
2-4-5 以不同濃度配位基製備之D2吸附劑吸附 alpha-amylase溶液 13
2-4-6 以D2 吸附劑吸附發酵液中之 alpha-amylase 13
2-5 脫附吸附劑上之 alpha-amylase 13
2-5-1 以咪唑溶液脫附不同質量的吸附劑上之 alpha-amylase 13
2-5-2 以不同濃度之咪唑溶液脫附於吸附劑上之 alpha-amylase 13
2-5-3 以不同濃度之EDTA溶液脫附於吸附劑上之 alpha-amylase 14
2-5-4 以EDTA溶液脫附不同量的吸附劑上之 alpha-amylase 14
2-5-5 以EDTA溶液脫附與發酵液反應之A2 吸附劑 14
2-6 銅離子之濃度校正 14
2-7 實驗藥品 17
2-8 實驗儀器 19
第三章 結果與討論 20
3-1 Beta型環狀糊精之交聯反應 20
3-2 以 beta-CD CL為擔體製成之親和性吸附劑對 alpha-amylase吸附之探討 22
3-2-1 以IDA為配位基製備親和性吸附劑進行 alpha-amylase之吸附探討 24
3-2-2 以cibacron blue F3G-A為配位基所製成之親和性吸附劑
之探討 24
3-2-3 以DADPA為配位基所製成之親和性吸附劑之探討 25
3-3 以 beta-CD CL-DADPA-Cu2+ 為吸附劑之探討 30
3-3-1 以吸附劑吸附不同濃度 alpha-amylase之探討 30
3-3-2 以不同量之吸附劑吸附 alpha-amylase之探討 30
3-4 以咪唑溶液脫附已吸附 alpha-amylase之
beta-CD CL-DADPA-Cu2+ 吸附劑之探討 35
3-4-1 以咪唑溶液脫附不同量已吸附 alpha-amylase之吸附劑之探討 35
3-4-2 以不同濃度之咪唑溶液脫附已吸附 alpha-amylase之吸附劑之探討 41
3-5 EDTA 溶液脫附已吸附 alpha-amylase之
-CD CL-DADPA-Cu2+ 吸附劑之探討 44
3-5-1 以不同濃度EDTA溶液對已吸附 alpha-amylase之吸附劑之探討 44
3-5-2 以EDTA溶液對不同量已吸附 alpha-amylase之吸附劑之探討 44
3-6 不同交聯比例 (beta-CD:EPI) 之擔體製備 beta-CD CL-DADPA-Cu2+ 48
3-6-1 以不同交聯比例 (beta-CD:EPI) 之 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase吸附之探討 48
3-7 以不同濃度之配位基製備 beta-CD CL-DADPA-Cu2+ 50
3-7-1 以不同濃度之配位基製備 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase吸附之探討 50
3-8 不同粒徑之親和性吸附劑 53
3-8-1 以不同粒徑之親和性吸附劑對 alpha-amylase吸附之探討 53
3-9 不同交聯程度幾丁聚醣 (chitosan) 為擔體製備 chitosan-EDTA-Cu2+ 對 alpha-amylase進行吸附之探討 58
3-10 以 beta-CD CL-DADPA-Cu2+ 為吸附劑吸附與脫附發酵液中之 alpha-amylase 60
第四章 結論 61
參考文獻 63

表目錄
表1-1 各種層析法吸附劑之專一性 6
表2-1 不同吸附劑之組成與代號 11
表3-1-1 不同體積EPI對 beta-CD交聯程度之影響 21
表3-1-3 以不同交聯劑交聯 beta-CD之探討 20
表3-2-1 以 beta-CD CL為擔體製成之不同親和性吸附劑對 alpha-amylase之吸附 22
表3-2-2 以IDA為配位基製成不同親和性吸附劑對 alpha-amylase之吸附 24
表3-2-3 銅離子於 beta-CD CL-DADPA上鍵結情形 24
表3-2-4 以cibacron blue F3G-A為配位基製成之不同親和性吸附劑對 alpha-amylase之吸附 26
表3-2-5 以DADPA為配位基製成之不同親和性吸附劑對 alpha-amylase之吸附 28
表3-2-6 銅離子於 beta-CD CL-DADPA上鍵結情形 28
表3-3-1 以 beta-CD CL-DADPA-Cu2+ 為吸附劑對不同 alpha-amylase濃度之吸附 31
表3-3-2 以不同量 beta-CD CL-DADPA-Cu2+ 為吸附劑對10 mL alpha-amylase之吸附 31
表3-4-1 以10 mL咪唑溶液對已吸附 alpha-amylase之不同量吸附劑之脫附 41
表3-4-2 以不同濃度之咪唑溶液對已吸附 alpha-amylase吸附劑之脫附 41
表3-5-1 以不同濃度之EDTA對已吸附 alpha-amylase吸附劑之脫附 45
表3-5-2 以EDTA溶液對不同量已吸附 alpha-amylase吸附劑之脫附 45
表 3-6-1 不同交聯比例 (beta-CD:EPI) 之 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase之吸附 48
表3-7-1 以不同濃度之配位基製備螯合金屬親和性吸附劑對 alpha-amylase之吸附與脫附 50
表3-7-2 銅離子於 beta-CD CL-DADPA上鍵結情形 52
表3-7-3 DADPA鍵結於 beta-CD CL殘餘量之比較 52
表3-8-1 不同粒徑之平均粒徑與比表面積 53
表3-8-2 以不同粒徑之親和性吸附劑對 alpha-amylase之吸附 53
表3-9 以不同交聯程度幾丁聚醣為擔體製備親和性吸附劑對 alpha-amylase 之吸附 58
表3-10-1 以吸附劑吸附與脫附發酵液中之 alpha-amylase 60

圖目錄
圖1-1 Alpha-Amylase之胺基酸序列 1
圖1-2 蛋白質純化分離之流程圖 2
圖1-3 親和性層析之過程示意圖 4
圖1-4 固定化金屬離子吸附劑之結構 8
圖1-5 (a) Alpha 型環狀糊精、(b) Beta 型環狀糊精、(c) Gama 型環狀糊精之化學結構式 9
圖2-1 Alpha-Amylase溶液之濃度檢量線 15
圖2-2 硫酸銅溶液於波長600 nm下濃度對吸光值之檢量線 16
圖3-2-1 Beta-CD CL-CBF3GA、beta-CD CL-CBF3GA-Cu2+、beta-CD CL-CBF3GA-Cu2+ (含水)、beta-CD CL-DADPA-Cu2+ 與beta-CD CL-DADPA-Cu2+ (含水) 之吸附曲線 23
圖3-2-2 Beta-CD CL-CBF3GA與 beta-CD CL-CBF3GA-Cu2+ 之吸附曲線 27
圖3-2-3 Beta-CD CL-DADPA-Cu2+ 與含水 beta-CD CL-DADPA-Cu2+ 之吸附曲線 29
圖3-3-1 以 beta-CD CL-DADPA-Cu2+ 吸附不同濃度 alpha-amylase之吸附曲線 32
圖3-3-2 以不同量 beta-CD CL-DADPA-Cu2+ 吸附 alpha-amylase之吸附曲線 33
圖3-3-3 吸附平衡曲線圖 34
圖3-4-1 咪唑溶液脫附之示意圖 35
圖3-4-2 Beta-CD之XPS圖 36
圖3-4-3 Beta-CD CL之XPS圖 37
圖3-4-4 Beta-CD CL-DADPA-Cu2+ 之XPS圖 38
圖3-4-5 Beta-CD CL-DADPA-Cu2+ 吸附 alpha-amylase之XPS圖 39
圖3-4-6 以咪唑脫附後吸附劑之XPS圖 40
圖3-4-7 以咪唑溶液對已吸附 alpha-amylase之不同量吸附劑之脫附曲線 42
圖3-4-8 以不同濃度之咪唑溶液對已吸附 alpha-amylase吸附劑之脫附曲線 43
圖3-5-1 以EDTA脫附 alpha-amylase之示意圖 44
圖3-5-2 以不同濃度之EDTA對已吸附 alpha-amylase吸附劑之脫附曲線 46
圖3-5-3 以EDTA溶液對不同量已吸附 alpha-amylase吸附劑之脫附曲線 47
圖3-6-1 不同交聯比例 (beta-CD:EPI) 之 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase之吸附曲線 49
圖3-7-1 以不同濃度之配位基製備 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase之吸附曲線 51
圖3-8-1 以不同粒徑之 beta-CD CL-DADPA-Cu2+ 對 alpha-amylase之吸附曲線 54
圖3-8-2 以顯微鏡拍攝不同粒徑之 beta-CD CL-DADPA-Cu2+ 55
圖3-8-3 各種粒徑 beta-CD CL-DADPA-Cu2+ 之SEM圖 56
圖3-8-4 Beta-CD CL-DADPA-Cu2+ 攪拌前後之SEM圖 57
圖3-9 以不同交聯程度幾丁聚醣為擔體製備親和性吸附劑吸附 alpha-amylase之吸附曲線 59
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22. R. J. Todd, R. D. Johnson and F. H. Arnold, “Multiple-site binding interactions in metal-affinity chromatography I. equilibrium binding of engineered histidine containing cytochromes c”, Journal of Chromatography A, 622, PP. 13-26, 1994.
23. 劉國詮,生物工程下游技術-細胞培養、分離純化、分析檢測,曉園出版社,民國 86 年九月,1997.
24. S. Bernhardt, P. Glöckner and H. Ritter, “Cyclodextrins in polymer synthesis: Influence of methylated beta-cyclodextrin as host on the free radical copolymerization reactivity ratios of hydrophobic acrylates as guest monomers in aqueous medium”, Polymer Bulletin, 46, pp. 153-157, 2001.
25. L. M. Hamilaton, C. T. Kelly and W. M. Fogarty, “Review: cyclodextrins and their interaction with amylolytic enzymes”, Enzyme and Microbial Technology, 26, pp. 561-567, 2000.
26. Szejtli, Jozsef, Fenyvesi, Eva, Zoltan, Sandor, Zsadon, Bela, Tudos and Ferenc, “Cyclodextrin-polyvinyl alcohol polymers and a process for the preparation thereof in a pearl, foil, fiber or block form”, United States Patent, 4,274,985, 1981.
27. W. L. Hinze and D. W. Armstrong, Ordered Media in Chemical Separations, American Chemical Society, New York, 1987.

------------------------------------------------------------------------ 第 5 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200910245684
論文名稱(中文) 酯水解酵素固定於矽藻土之研究
論文名稱(英文) Immobilization of Esterase on Celite
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 90
學期 2
出版年 91
研究生(中文) 孫鍾仁
學號 l3689101
學位類別 碩士
語文別 中文
口試日期 2002-07-29
論文頁數 34頁
口試委員 口試委員-葉茂榮
口試委員-黃福永
口試委員-黃得時
指導教授-蕭世裕
關鍵字(中) 高血壓
酯水解酵素
酵素
固定化
矽藻土
關鍵字(英) Celite
Captopril
Immobilization
Enzyme
Esterase
Packed-bed reactor
學科別分類
中文摘要 本篇論文是將Pseudomonas putida之酯水解酵素(esterase)在大腸桿菌(E. coli)大量表現。酯水解酵素可對乙醯硫代異丁胺(DL-β-acetylthioisobutyamide 簡稱ATIA)進行酵素光學分割而得到乙醯硫代異丁酸(D-β-acetylthioisobutyric acid簡稱DAT)。DAT為合成降血壓藥物Captopril的前驅物。
在以酯水解酵素進行乙醯硫代異丁胺水解時,溶液的pH值並不會有大的改變,所以酯水解酵素不會因pH值的改變而失去活性,故可以重覆再利用。固定菌體細胞在反應器的最大優點是可以省去製備純化酵素的繁雜步驟,在分離純化DAT也很容易,是一非常好的設計。
英文摘要 In this paper, a stereoselective esterase cloned from Pseudomonas putida was expressed in E.coli and used for optical resolution of DL-β-acetylthioisobutyamide (ATIA) to get D-β-acetylthioisobutyric acid (DAT). DAT is a precursor of an antihypertensive drug ---Captopril. During hydrolysis of DL-β-acetylthioisobutyamide by the esterase, the pH of the bulk solution was not changed. The activity of the enzyme can be maintained and reused. The advantages of using immobilized cell in the packed-bed reactor is to avoid the multiple steps of enzyme purification and immobilization. In addition, the purification of D-β-acetylthioisobutyric acid from the reaction mixture is also facilitated in the packed-bed reactor in.
論文目次 中文摘要 Ⅰ
英文摘要 Ⅱ
目錄 Ⅲ
圖目錄 V

第一章 緒論 1
1-1 高血壓簡介 1
1-2 Captopril的合成 5
1-3 矽藻土的簡介 11
第二章 實驗材料與方法 14
2-1 藥品 14
2-2 儀器 15
2-3 酯水解酵素之受質的合成 16
2-4 製備勝任細胞(Competent Cell) 16
2-5 Transformation轉型作用 19
2-6 誘導 20
2-7 包埋固定化及取樣偵測 21
第三章 結果與討論 22
3-1乙醯硫代異丁胺(DL-β-acetylthioisobutyamide)之合成 22
3-2菌體的活性測試 23
第四章 結論 25
參考文獻 33


圖目錄
圖一、繼發性高血壓示意圖 1
圖二、Captopril的結構及其抑制的機制 4
圖三、結晶分割法生成DAT 6
圖四、酵素分割法生成DAT 8
圖五、酯水解酵素分割DL-MATI 9
圖六、DAT合成Captopril 10
圖七、矽藻土的微粒結構 11
圖八、光學切割反應器示意圖 13
圖九、乙醯硫代異丁胺(ATIA)之HPLC層析圖 26
圖十、乙醯硫代異丁胺(ATIA)之1H-NMR圖 27
圖十一、乙醯硫代異丁酸甲酯(MATI)之1H-NMR圖 28
圖十二、乙醯硫代異丁胺(ATIA)之13C-NMR圖 29
圖十三、乙醯硫代異丁酸甲酯(MATI)之13C-NMR圖 30
圖十四、乙醯硫代異丁酸甲酯(MATI)之13C-DEPT圖 31
圖十五、乙醯硫代異丁胺水解情形 32
參考文獻 1. Peter Semple博士著, 高怡博士譯, 陳綺君編輯(1991)高血壓預
防和護理, 香港珠海出版有限公司
2. 新城之介著, 劉秀香譯(1990)高血壓醫療與食療, 王家出版社有限公司
3. 菊池由夫著, 劉晶, 劉淼譯(1995)高血壓和動脈硬化的防治, 書泉出版社
4. 陳裕仁著 (1999) 乙醯硫代異丁酸甲酯的酵素光學分割研究,國立成功大學化學系研究所, 碩士論文
5. J. W. Skiles, J. T. Suh, B. E. Williams, P. R. Menard, J. N. Barton, B. Loev, H. Jones, E. S. Neiss, A. Schwab, W. S. Mann, A. Khandwala, P. S. Wolf and I. Weinryb(1986)J. Med. Chem., 29, 784-796
6. J. W. Skiles, J. T. Suh, B. E. Williams, R. D. Youssefyeh, H. Jones, B. Loev, E. S. Neiss, A. Schwab, W. S. Mann, A. Khandwala, P. S. Wolf and I. Weinryb(1985)J. Med. Chem., 28, 57-66
7. Qu-Ming Gu, D. R. Reddy and C. J. Sih(1986)Tet. Lett., 27(43), 5203-5206
8. A. Sakimae, E. Ozaki, H. Toyama, N. Ohsuga, R. Numazawa, I. Muraoka, E. Hamada and H. Ohnishi (1993)Biosci. Biotech. Biochem., 57 ( 5 ), 782-786
9. A. Sakimae, R. Numazawa and H. Ohnishi(1992)Biosci. Biotech. Biochem., 56 ( 8 ), 1241
10. T. Sawayama, M. Tsukamoto, T. Sasagawa, S. Naruto, J. Matsumoto and H. Uno(1989)Chem. Pharm. Bull. 37 ( 5 ), 1382-1383
11. A. Sakimae, A. Hosoi, E. Kobayashi, N. Ohsuga, R. Numazawa, I.
Watanabe and H. Ohnishi ( 1992) Biosci. Biotech. Biochem., 56 (8), 1252-1256
12. E. Ozaki, A. Sakimae and R. Numazawa(1995)Biosci. Biotech.
Biochem., 59(7), 1204-1207
13. D. Bianchi and P. Cesti(1990)J. Org. Chem., 55, 5657-5659
14. 潤麟實業有限公司, 矽藻土規格及說明。
15. 林敬二, 楊美惠, 楊寶旺, 廖德章, 薛敬和(1993)英中日化學大
辭典, 高立圖書有限公司
16. 黃榮茂, 王禹文, 林聖富, 楊得仁(1992)化學化工百科辭典, 曉
園出版社
17. Y.-J. Yuan, S.-H. Wang, Z.-X. Song, R.-C. Gao(2002)J. Chem. Technol. Biotechnol., 77, 602-606
18. J. S. Shin, B. G. Kim, D. H. Shin(2001)Enzyme Microbial Technol., 29, 232-239

------------------------------------------------------------------------ 第 6 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200910344446
論文名稱(中文) 酯水解酵素固定於幾丁聚醣之研究
論文名稱(英文) Immobilization of Esterase on Chitosan
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 91
學期 1
出版年 92
研究生(中文) 何世湧
學號 l3689118
學位類別 碩士
語文別 中文
口試日期 2002-07-29
論文頁數 109頁
口試委員 指導教授-蕭世裕
口試委員-黃福永
口試委員-黃得時
口試委員-葉茂榮
關鍵字(中) 幾丁聚醣
固定化
酯水解酵素
關鍵字(英) Chitosan
Esterase
Immobilized
學科別分類
中文摘要   我們將Pseudomonas putida 之酯水解酵素(esterase)在大腸桿菌(E.coli)大量表現。酯水解酵素可對乙醯硫代異丁酸甲酯(methyl DL-β-acetylthioisobutyrate 簡稱MATI)進行酵素光學分割而得到乙醯硫代異丁酸(D-β-acetylthioisobutyric acid 簡稱DAT)。DAT 為合成降血壓藥物Captopril 的前驅物。

  為了能使酵素重複使用及增加其穩定性,我們利用大腸桿菌(E.coli)表現的Pseudomonas putida 酯水解酵素(esterase),並將純化的酯水解酵素用戊二醛1%(w/v)固定在天然的幾丁聚醣薄膜上,本實驗研究將對游離酵素與固定化酵素的動力學參數、最佳反應溫度及pH 值、熱穩定、儲存的穩定性與操作的穩定性做偵測。

  實驗結果發現游離酵素與固定化酵素的最佳反應溫度都在67℃,最佳反應pH 值分別在pH7.5 與pH8.5。而游離酵素的KM =26.1 mM,Vmax=833.0 μmol/min‧mg protein,固定化酵素的KM =197.8 mM,Vmax=3.6 μmol/min‧mg protein。雖然從以上可以觀察到游離酵素與固定化酵素的動力學參數有明顯的不同,但是固定化酵素能有效的改善對於溫度與酸鹼值的穩定性。
英文摘要   In this paper, a stereoselective esterase cloned from Pseudomonas putida was expressed in E.coli and used for optical resolution of methyl DL-β-acetylthioisobutyrate ( MATI ) to get D-β-acetylthioisobutyric acid (DAT). DAT is a precursor of an antihypertensive drug ---Captopril.

  In order to reuse and increase the stability of esterase. In this study,esterase from pseudomonas putida was immobilized onto chitosan film using glutaraldehyde (1%,w/v) as cross-linking reagent. Esterase was immobilized on the chitosan film that is a natural polymer. The studies were done on free esterase and immobilized esterase on chitosan film to determine the kinetic parameters, optimum temperature, optimum pH, thermal stability,storage stability, and operational stability.

  The result showed that optimum temperature for free esterase and immobilized esterase on chitosan film is 67°C.Optimum pH is 7.5 , 8.5 for free esterase and immobilized esterase respectively.It was found that KM =26.1 mM,Vmax=833.0 μmol/min‧mg protein for free esterase and KM =197.8 mM,Vmax=3.6 μmol/min‧mg protein for immobilized esterase on chitosan. The kinetic parameters of the immobilized esterase were significantly changed but the thermal and pH stabilities of the immobilized esterase were improved.
論文目次 中文摘要  Ⅰ
英文摘要  Ⅱ
目錄  Ⅲ
圖目錄  Ⅴ
表目錄  Ⅶ

(一) 緒論  1
  1. 高血壓簡介  1
  2. 高血壓的預防與治療  2
  3. Captopril 的合成  7
  4. 固定化的原理  9
  5. 幾丁質(chitin)與幾丁聚醣(chitosan)的應用  11
  6. 研究目的  20

(二) 藥品及儀器  21
  1. 藥品  21
  2. 儀器  23

(三)實驗方法  24
  1. 合成MATI(methyl DL-β-acetylthioisobutyrate)  24
  2. 大腸桿菌(E-coli)之轉殖與表現  24
  3. 酯水解酵素(esterase)的純化  29
  4. 酯水解酵素(esterase)的活性測試及定量  32
  5. 製備幾丁聚醣(chitosan)薄膜  34
  6. 酯水解酵素(esterase)的固定化  34
  7. 固定後之蛋白質定量  36
  8. 固定化酵素與游離酵素之活性測試及定量  36
  9. 逆向管柱層析  36
  10. 以掃瞄式電子顯微鏡觀測酵素固定化結果  37
  11. 固定化酵素與游離酵素的物理、化學性質之測試  37

(四) 結果與討論  45
  1. 合成MATI(methyl DL-β-acetylthioisobutyrate)  45
  2. 大腸桿菌(E-coli)之轉殖與表現  45
  3. 轉殖之大腸桿菌的大量培養與誘導作用  46
  4. 酯水解酵素(esterase)的純化  46
  5. 製備幾丁聚醣(chitosan)薄膜  51
  6. 酯水解酵素(esterase)的固定化  52
  7. 固定化酵素之活性測試及定量  53
  8. 以掃瞄式電子顯微鏡觀測酵素固定化結果  55
  9. 固定化酵素與游離酵素的物理、化學性質之比較  55

(五) 結論  59

(六) 參考文獻  60

附錄一  90
附錄二  102
附錄三  105
參考文獻 [1] A.Sakimase, R. Numazawa and H. Ohnishi Biosci. Biotech. Biochem.,56(8),1341 (1993)

[2] A. Sakimase, A. Hosoi, E. Kobayashi, N.Ohsuga, R.Numazawa, I. Watanabe and H. Ohnishi Biosci. Biotech. Biochem. 56(8),

[3] Akio Sakuragawa, Tetsuyuki Taniai, Tadao Okutani Analysis Chimica Acta 374,191-200 (1998)

[4] Barbara Krajewska,Maciej Leszko and Wieslawa Zaborska Urease immobilized on chitosan membrane: Preparation and properties J. Chem. Tech. Biotechnol. 48,351-360 (1990)

[5] Baileey JE, Ollis DF. Applied enzyme catalysis. In: Biochemical engineering fundamentals, 2nd edition, Singapore: McGraw-Hill Int, 180¡V189 (1986)

[6] Bohle, M., Bahadur, A., Immobilization of urease on various cellulosic supports, Cellulose Chem. Technol. 24 563-570

[7] Cavaille, D., & Combes, D. Characterization of beta-galactosidase from Kluyveromyces lactis. Biotechnology and Applied Biochemistry, 22(1), 55-64 (1995)

[8] D. Bianchi and P. Cesti J. Org. Chem., 55, 5657-5659¡]1990¡^

[9] E. Ozaki, A.Sakimase and R. Numazawa Biosci. Biotech. Biochem. 59(7), 1204-1207 (1995)

[10] Fahmy, A.; Bagos, V.B.; Mohammed, T.M.: Immobilization of citrus vulgaris urease on cyanuric chloride DEAE-cellulose ether: preparation and properties. Bioresource Tech. 64: 121-129 (1998)

[11] Iyengar L, Prabhakara, R. Urease bound to chitin with glutaraldehyde. Biotechnol. Bioeng.; 21:1333¡V1343 (1979)

[12] J. W. Skiles, J. T. Suh, B. E. Williams, P. R. Menard, J. N. Barton, B. Loev, H. Jones, E. S. Neiss, A. Schwab, W. S. Mann, A. Khandwala, P. S. Wolf and I. Weinryb J. Med. Chem., 29,784-796 (1986)

[13] J. W. Skiles, J. T. Suh, B. E. Williams, R. D. Youssefyeh, H. Jones, B. Loev, E. S. Neiss, A. Schwab, W. S. Mann, A. Khandwala, P. S. Wolf and I. Weinryb J. Med. Chem., 28, 57-66(1985)

[14] J. P. Chen and S. H. Chiu Bioprocess Engineering ,21,323-330(1999)

[15] Kennedy JF, Melo EHM, Immobilized enzymes and cells. Chem Eng Prog: 81¡V89 (1990)

[16] Krajewska B.; Leszko, M.; Zaborska, W.: Urease immobilized on chitosan membrane: preparation and properties. J. Chem. Tech. Biotechnol. 48 337-350 (1990)

[17] Madeira,V.M.C., Incorporation of urease into liposomes. Biochim. Biophys. Acta, 499 202-211 (1977)

[18] Martins, M.B.F.; Cruz, M.E.M.; Cabral, J.M.S.; Kennedy, J.F.: Urease immobilization on an alkylamine derivative of titanium (IV)-porous silica, kinetic and operational stability. J. Chem. Tech. Biotechnol. 39 201-213 (1986)

[19] Pozniak, G.; Krajewska, B.; Trochimczuk, W.: Urease immobilized on modiAed polysulphone membrane: preparation and properties. Biomaterials 16 129-134 (1995)

[20] Quinn Z.K. Zhou, Xiao Dong Chen Journal of Food Engineering 48,69-74 (2001)

[21] Qu-Ming Gu, D. R. Reddy and C. J. Sih Tet. Lett., 27(43), 5203-5206(1986)

[22] Ramachandran,K.B. and Perlmutter,D.D.,Effects of immobilization on the kinetics of enzyme-catalyzed reaction. ¢º.Urease in a packed-column differential reactor system. Biotechnol. Bioeng.,18 685-699 (1976)

[23] Safro K.; Petach, H.; Hederson, W.: Polymeric phosphine oxide polyether-derived copolymer as a support for urease immobilization. Enzyme Microb. Technol. 17 804-808 (1995)

[24] T. Sawayama, M. Tsukamoto, T. Sasagawa, S. Naruto, J. Matsumoto and H. Uno Chem. Pharm. Bull. 37(5), 1382-1383(1989)

[25] 陳裕仁乙醯硫代異丁酸甲酯的酵素光學分割研究,國立成功大學化學系研究所,碩士論文(1999)

------------------------------------------------------------------------ 第 7 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200910383690
論文名稱(中文) 利用 NBO 理論探討有機酸衍生物的水解反應
論文名稱(英文) Investigation of Hydrolysis of Carboxylic Acid Derivatives by NBO Theory
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 91
學期 2
出版年 92
研究生(中文) 梁瑾瑩
學號 l3690101
學位類別 碩士
語文別 中文
口試日期 2003-06-27
論文頁數 152頁
口試委員 指導教授-蘇世剛
口試委員-王小萍
口試委員-黃守仁
關鍵字(中) 水解
孤電子對
天然鍵性軌域
有機酸衍生物
關鍵字(英) hydrolysis
carboxylic acid derivatives
natural bond orbital
lone pair
學科別分類
中文摘要 本篇論文主要是以天然鍵性軌域理論(NBO)的『donor-acceptor interaction』概念為基礎,探討有機酸衍生物的水解反應。首先我們討論無攻擊劑狀態時,有機酸衍生物分子內的電荷轉移情形,再衍生到這些分子發生水解反應時,分子內及分子間的電荷移轉。

計算有機酸衍生物,我們發現碳醯基氧上孤對電子對具有削弱鄰近σC-X*鍵的能力,且愈易離去的離去基,其『lpC=O→σC-X*』能量越大。主要是因為愈易離去的離去基,本身分子中的σC-X* 軌域能量較穩定,所以『lpC=O→σC-X*』能量較大。

在模擬水解反應部分,我們分成有機酸衍生物(CH3COX,X=F、Cl、Br、OCH3)的中性水解反應及乙酸甲酯(CH3COOCH3)的鹼性水解反應。我們得到無論中性與鹼性的狀況,攻擊劑上的孤電子對主要都是扮演削弱πC=O的角色,且削弱πC=O之後,間接提升碳醯基氧上孤電子對的軌域能量,縮短孤電子對與σC-X*的能差。攻擊劑以這樣的方式幫助反應進行,但是最後離去基的離去仍取決於σC-X*本身的軌域能階高低。模擬乙醯氟與乙酸甲酯的中性水解及乙酸甲酯的鹼性水解,我們發現它們本身σC-X*軌域能量較乙醯溴及乙醯氯來的高,因此就算攻擊劑有幫助反應進行,最後離去基仍未離去。






英文摘要 Donor-acceptor interaction concept of the natural bond orbital theory was applied to evaluate charge transfer in carboxylic acid derivatives and in hydrolysis of carboxylic acid derivatives in our study respectively.

In our calculations of carboxylic acid derivatives, lone pair on the oxygen atom of carbonyl group was observed to donate to neighbor σC-X* orbital. And the better leaving group has more 『lpC=O→σC-X*』interaction energies by it’s lowerσC-X* orbital energy.

For hydrolysis of carboxylic acid derivatives, we calculate CH3COX(X= F、Cl、Br、OCH3)in water and CH3COX(X= OCH3)in base-catalyzed surroundings respectively. The results are similar in different cases, that lone pair on nucleophilic reagent mainly donates to πC=O* orbital, result in energy increasing of lone pair on the oxygen atom of carbonyl group. Furthermore, the energy gap between the lone pair on the oxygen atom of carbonyl group and σC-X* orbital will get smaller. The nucleophilic reagent helps hydrolysis reaction processing with this way. However the leaving group leaves or not depending on σC-X* orbital energy. To simulating hydrolysis of acetyl fluoride and methyl acetate in water and hydrolysis of methyl acetate in base-catalyzed surroundings, we found that their σC-X* orbital energy is higher than acetyl chloride and acetyl bromide. Although the nucleophile reagent helps reaction processing, the leaving group still did not leave.






論文目次 目錄
中文摘要… … … … … … … … … … … … … … … … … … … … … … … … … … .. I
英文摘要… … … … … … … … … … … … … … … … … … … … … … … … … … .. II
目錄… … … … … … … … … … … … … … … … … … … … … … … … … … … … .. III
表目錄… … … … … … … … … … … … … … … … … … … .… … … … … … … … . VII
圖目錄… … … … … … … … … … … … … … … … … … … … … … … … … … … .. VIII
第一章序論… … … … … … … … … … … … … … … … … … … … … … … … … 1
1-1 研究背景… … … … … … … … … … … … … … … … … … … … .… … … . 1
1-2 研究動機… … … … … … … … … … … … … … … … … … … … … … … .. 6
1-3 研究方向… … … … … … … … … … … … … … … … … … … … … … … .. 7
第二章理論背景… … … … … … … … … … … … … … … … … … … ..… .… … . 10
2-1 天然鍵性軌域( natural bond orbital,NBO )… … … … … ..… .… … … 10
2-2 NBO Fock matrix 值… … … … … … … … … … … … … … … … … … .. 12
2-3 軌域能差… … … … … … … … … … … … … … … … … … … … … … … .. 15
第三章計算方法與模擬水解反應的做法… … … … … … … … … … … … … 16
3-1 有機酸衍生物的最佳化計算..… … … … … .… ...… … … … … … … … 16
3-1-1 前言… … … … … … … … … ...… … … … ..… .… … … … … … … . 16
3-1-2 輸入格式及輸入指令的函數說明… … … ...… … ..… … … … .. 16
3-2 模擬水解反應之計算… ..… … … … … … … … … .… .… … … … … … .. 19
3-2-1 方法一..… … … … … … … … … … … … … … … … … … … … … . 19
3-2-1-1 起始距離的獲得… … … … … … … … … .… … … … ... 20
3-2-1-2 最末距離的獲得… … … … … … … .… … … … … … ... 21
3-2-2 方法二… … … … … … … … … … … … … … … … … … … … … ... 24
第四章結果與討論… … … … … … … … … … … … … … … … … … … … … … 31
4-1 前言… … … … … … … … … … … … … … … … … … .… … … … … … … . 31
4-2 實驗結果… … … … ...… … … … … … … … .… … … … … … … … … … .. 34
4-2-1 有機酸衍生物的計算… … … … … … … … … … … … … … … ... 35
4-2-1-1 孤電子對的比較… … … … … … … … .… … … … … ... 35
4-2-1-2 不同離去基之『donor-acceptor interaction』分析.. 36
4-2-1-3 結果總結推論… … … … … … … … … … … … … … … 40
4-2-2 模擬水解反應能障與實驗活化能比較… … … … … … … … . 40
4-2-2-1 中性下模擬乙醯氯水解反應… … … … … … … … … 40
4-2-2-2 鹼性下模擬乙酸甲酯水解反應… … … … … … … … 42
4-2-2-3 法蘭克-康登因子的計算… … … … … … … … .… … . 87
4-2-3 模擬水解反應過程,分子的構形變化… … … … … … … … . 43
4-2-4 中性下模擬乙醯氯水解反應… … … … … … … … … … … … . 46
4-2-4-1『donor-acceptor interaction』分析… … … … … … … 46
4-2-4-2 其他結果… … … … … … … … … … … … … … … … … 60
4-2-4-3 總結推論… … … … … … … … … … … … … … … … … 62
4-2-5 中性下模擬乙醯溴水解反應結果… … … … … … … … … … ... 63
4-2-5-1 相對分子總能… … … … … … … … … … … … … … … 63
4-2-5-2『donor-acceptor interaction』分析… … … … … … … 64
4-2-5-3 其他結果… … … … … … … … … … … … … … … … … 74
4-2-5-4 總結推論… … … … … … … … … … … … … … … … … 76
4-2-6 中性下模擬乙醯氟水解反應結果… … … … … … … … … … ... 77
4-2-6-1 中性下模擬乙醯氟水解反應的分子總能… … … … 77
4-2-6-2『donor-acceptor interaction』分析… … … … … … … 78
4-2-6-3 其他結果… … … … … … … … … … … … … … … … … 88
4-2-6-4 總結推論… … … … … … … … … … … … … … … … … 90
4-2-7 中性下模擬乙酸甲酯水解反應結果… … … … … … … … … ... 90
4-2-7-1 中性下模擬乙酸甲酯水解反應的分子總能… … … 90
4-2-7-2『donor-acceptor interaction』分析… … … … … … … 91
4-2-7-3 其他結果… … … … … … … … … … … … … … … … … 101
4-2-7-4 總結推論… … … … … … … … … … … … … … … … … 103
4-2-8 鹼性下模擬乙酸甲酯水解反應結果… … … … … … … … … ... 103
4-2-8-1『donor-acceptor interaction』分析… … … … … … … 105
4-2-8-2 其他結果… … … … … … … … … … … … … … … … … 116
4-2-8-3 總結推論… … … … … … … … … … … … … … … … … 118
4-3 實驗討論… … … … … … … … … … … … … … … .… … … … … … … … . 118
4-3-1 中性下模擬有機酸衍生物水解反應的比較… … … … … … ... 118
4-3-2 中性及鹼性下模擬乙酸甲酯水解反應的比較… … … … … ... 122
第五章結論.… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… … … … ... 125
第六章未來展望… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… … … … . 127
參考文獻… ..… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… .… … … ... 129
附錄… … … … … … … … … … … … … … … … … … … … … … … … … … … … .. 131
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------------------------------------------------------------------------ 第 8 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200910402203
論文名稱(中文) 利用PEI修飾脂肪分解酵素進行外消旋Naproxen三氟乙硫酯之水解動態動力分割
論文名稱(英文) none
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 91
學期 2
出版年 92
研究生(中文) 侯輝昌
學號 n3690115
學位類別 碩士
語文別 中文
口試日期 2003-06-24
論文頁數 56頁
口試委員 口試委員-李文乾
口試委員-陳特良
口試委員-張嘉修
指導教授-蔡少偉
關鍵字(中) 動態動力分割
固定化酵素
動力參數
鹼水解
Naproxen三氟乙硫酯
聚乙烯亞胺
關鍵字(英) dynamic kinetic resolution
trifluoroethyl thioester
Lipase MY
PEI
Naproxen
poltethyleneimine
DBU
學科別分類
中文摘要 本實驗室過去以外消旋Naproxen三氟乙硫酯為基質,利用Lipase MY配合三辛基胺為鹼觸媒之動態動力分割反應已有相當好的成果,但是在長時間反應時,酵素會漸漸失活而降低反應速率。許多文獻報導以聚乙烯亞胺(PEI, polyethyleneimine)修飾酵素,可提升酵素的反應性及穩定性。因此,本論文之研究目的在於使用PEI修飾後的固定化酵素,配合有機鹼1,8-Diazabicyclo[5.4.0]undec-7-ene(1,5-5) (DBU)進行外消旋Naproxen三氟乙硫酯之動態動力分割。

論文中嘗試改變PEI修飾固定化酵素條件,並配合動力分割進行條件篩選,發現經由5 % PEI水溶液、攪拌60分鐘處理的固定化酵素,在動力分割方面約提高了2倍酵素活性,故進一步選擇此條件下處理的固定化酵素以進行外消旋Naproxen三氟乙硫酯之動態動力分割反應。利用實驗值擬合出動力參數,進而比較理論值與實驗值,推測反應進行時應考慮產物抑制、鹼水解之影響。

在添加10 mM DBU進行分割時,發現酵素反應性提高約10倍,顯示有機鹼會對經由PEI修飾後的固定化酵素有活化效果。
英文摘要 A dynamic kinetic resolution process of using racemic Naproxen trifluoroethyl thioester as the substrate as well as Lipase MY and trioctylamine as the biocatalyst and recemization catalyst, respectively, has been developed. However, enzyme deactivation was found for a long period time of reaction. Literatures have reported that PEI (polyethyleneimine) could be used to modify enzymes in order to increase enzyme activity and stability. Therefore, the purpose of this research is aimed to develop a dynamic kinetic resolution process of using (R, S)-Naproxen trifluoroethyl thioester as the substrate. Immobilized lipase modified with 5 % PEI solution with agitation for 60 minutes can increase two folds of enzyme activity. By using this condition for the dynamic kinetic resolution process, the kinetic parameters were estimated and experimented data were compared with theoretical results by further considering the effect of product inhibition and hydrolysis by the organic base. Besides, the activity of the modified enzyme increased ten times when adding 10 mM of DBU during the dynamic kinetic resolution.
論文目次 第一章 緒論
1-1 酵素 1
1-2 脂肪酵素 2
1-3 有機溶劑中之酵素反應 3
1-4 對掌性藥物 6
1-5 Naproxen 8
1-6 動態動力分割 8
1-7 聚乙烯亞胺 11
1-8 研究動機 11

第二章 原理
2-1 鹼觸媒的消旋反應 14
2-2 酵素催化動力分割反應 16
2-3 酵素催化水解之動態動力分割 19

第三章 實驗方法
3-1 藥品與材料 21
3-2 實驗儀器設備 22
3-3 分析方法 23
3-4 實驗方法 23
3-4-1 合成Naproxen三氟乙硫酯 23
3-4-2 酵素固定化程序 25
1. 未含有PEI的酵素固定化 25
2. 利用PEI修飾的酵素固定化 26
3-4-3 篩選PEI修飾固定化酵素之條件 26
3-4-4 測定不同基質濃度下的酵素水解初速率 27
3-4-5 測定產物抑制動力學參數 27
3-4-6 測定不同基質濃度下鹼的水解速率常數 27
3-4-7 測定不同基質濃度下動態動力分割之動力學常數 28
3-4-8 不同濃度鹼觸媒對外消旋Naproxen三氟乙硫酯之水解反應 28

第四章 結果與討論
4-1 利用PEI修飾固定化酵素之條件篩選 29
4-2 酵素催化水解動力分割之參數擬合 30
4-3 動力分割考慮及不考慮產物抑制現象 33
4-4 以DBU進行為鹼觸媒進行水解動態動力分割之參數擬合 37
4-5 以DBU為鹼觸媒進行動態動力分割 41
4-6 不同濃度DBU鹼觸媒進行動態動力分割 47

第五章 結論與後續研究 50

參考文獻 52
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------------------------------------------------------------------------ 第 9 筆 ---------------------------------------------------------------------
系統識別號 U0026-0812200910414629
論文名稱(中文) 酵素水解牛第一型膠原蛋白之研究
論文名稱(英文) Research on Proteolytic Cleavage of Bovine Type I Collagen
校院名稱 成功大學
系所名稱(中) 化學系碩博士班
系所名稱(英) Department of Chemistry
學年度 91
學期 2
出版年 92
研究生(中文) 石凱元
學號 l3690121
學位類別 碩士
語文別 中文
口試日期 2003-07-21
論文頁數 52頁
口試委員 口試委員-葉茂榮
口試委員-黃得時
口試委員-黃福永
指導教授-蕭世裕
關鍵字(中) 胃蛋白酵素
水解
牛第一型膠原蛋白
關鍵字(英) Bovine collagen type I
proteolytic
pepsin
學科別分類
中文摘要 膠原蛋白是構成身體結締組織的主要成分,由於其特殊的三股螺旋結構,以及單體與單體間聚合形成生物高分子的特性,使其在醫藥生技、組織工程、化妝品以及食品添加等生物科技上的用途,快速而蓬勃地發展。
本研究以牛膠原蛋白第一型(bovine collagen type I)為材料,利用胃蛋白酵素(pepsin)在弱酸中進行水解,以製造分子量不同之膠原蛋白片段。研究結果顯示在4℃下,胃蛋白酵素水解反應之主要產物以MALDI-TOF MS測量產物為93 kDa之片段,反之在高溫下(40℃),胃蛋白酵素會將膠原膠原蛋白切成許多分子量較小之片段。
經胃蛋白酵素處理之膠原蛋白不但在水溶液中之溶解度增加,而且對骨細胞增生之活性亦增加兩倍。
英文摘要 Collagen is the most abundance biopolymer in connective tissue. Its special triple helical structure and biopolymer property has made it widely used in biopharmaceutical, tissue engineering, cosmetics and food industries.

In this research, we used pepsin to cleave bovine type I collagen and studied pepsin-cleaved collagen fragments. Under low temperature condition (4℃), pepsin only cleaved off only a small fragment from collagen and yield a 93 kDa collagen fragment as determined by MALDI mass spectrometry. Under higher temperature condition (40℃), the collagen was cleaved into many small fragment.

The cleavage of collagen by pepsin not only improves its water solubility but also improves its biological activity. The result of mouse osteoblast cell proliferation assay showed that pepsin-cleavage collagen is approximately two fold better than the uncleavaed collagen.
論文目次 中文摘要…………………………………………………………………… i
英文摘要…………………………………………………………………… ii
目錄………………………………………………………………………… iii
表目錄……………………………………………………………………… v
圖目錄……………………………………………………………………… vi

緒論
一、膠原蛋白簡介………………………………………………………… 1
二、膠原蛋白在生物科技上之應用與潛力……………………………… 4
三、台灣地區市場概況…………………………………………………… 8
文獻回顧…………………………………………………………………… 10
實驗材料、儀器與方法
一、材料…………………………………………………………………… 12
二、儀器…………………………………………………………………… 14
三、方法
1.膠原蛋白溶液配製處理………………………………………………… 15
2.羥脯胺酸定量…………………………………………………………… 15
3.膠原蛋白之溶解度測試………………………………………………… 16
4.不同溫度對胃蛋白酵素水解膠原蛋白的影響………………………… 16
5.等比例胃蛋白酵素水解不同濃度之膠原蛋白醋酸溶液……………… 16
6.以不同比例胃蛋白酵素水解膠原蛋白醋酸溶液……………………… 17
7.以固定化胃蛋白酵素水解膠原蛋白醋酸溶液………………………… 17
8.經胃蛋白酵素水解之膠原蛋白結構分析……………………………… 17
9.經胃蛋白酵素水解之膠原蛋白活性分析……………………………… 17
結果與討論………………………………………………………………… 19
結論………………………………………………………………………… 24
參考文獻…………………………………………………………………… 25
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