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研究生: 陳秋樺
論文名稱: 碳六十固定化脂肪酶酯類光學異構物壓電晶體感測器研製與應用
指導教授: 施正雄
Shih, Jeng-Shong
學位類別: 碩士
Master
系所名稱: 化學系
Department of Chemistry
論文出版年: 2003
畢業學年度: 91
語文別: 中文
論文頁數: 132
中文關鍵詞: 碳六十脂肪分解酵素壓電感測器光學異構物
英文關鍵詞: fullerene, lipase, piezoelectric crystal sensor, optical isomer
論文種類: 學術論文
相關次數: 點閱:182下載:0
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  • 本研究合成碳六十(C60)-固定化酵素:碳六十/脂肪分解酵素,並研製一酯類光學異構物壓電晶體感測器。將塗佈碳六十的石英壓電晶體感測器做為研究碳六十與脂肪分解酵素之間作用力的工具,結果顯示酵素能被碳六十所吸附並呈現不可逆的化學吸附現象。
    探討塗佈碳六十的石英壓電感測器與脂肪分解酵素之間的pH及溶劑效應。碳六十與脂肪分解酵素作用的最適pH為7,最適溫度為31oC,在有機溶劑(甲醇、乙醇)存在時,碳六十與脂肪分解酵素的結合能力會降低。
    將碳六十與酵素反應生成不溶於水的碳六十/脂肪分解酵素,並以IR鑑定產物,將產物譜圖與碳六十及酵素的譜圖比較後,能夠得知產物是由碳六十及酵素鍵結而成。以孔徑大小約5~10μm的多孔性二氧化矽材質的濾片浸泡於碳六十/甲苯溶液中,帶溶液揮發後得到碳六十濾片,將碳六十濾片浸泡於酵素(脂肪分解酵素)溶液中,製作碳六十/脂肪分解酵素,經測試後,發現所製作的碳六十/脂肪分解酵素濾片具有活性。當浸泡酵素溶液的濃度增加時,碳六十/脂肪分解酵素的催化能力也隨之增加。自行製作的碳六十/脂肪分解酵素濾片可以催化L-form胺基酸酯類水解產生L-form胺基酸,而對於D-form的胺基酸酯類則無水解反應發生。
    使用塗佈C60-cryptand[2,2]的石英壓電感測器以及固定化的碳六十/脂肪分解酵素來偵測水溶液中的L-form胺基酸酯類。利用L-form胺基酸(L-form胺基酸酯類經碳六十/脂肪分解酵素水解後的產物)可被塗佈C60-cryptand[2,2]的石英晶體吸附的原理,使用自製的碳六十/脂肪分解酵素濾片研製一酯類光學異構物壓電晶體感測器。所研製的酯類光學異構物壓電晶體測器能成功的分辨D-form與L-form的胺基酸酯類,且對於L-form的胺基酸酯類的偵測具有良好的靈敏度及再現性。

    Immobilized fullerene C60-lipase was synthesized and applied in a piezoelectric crystal sensor for optical isomer of esters. The C60 coated piezoelectric quartz crystal sensor was employed to study the interaction between C60 and lipase. The partially irreversible response for lipase was obserred by the desorption study, which implied that lipase could be adsorbed on C60 by chemisorption.
    Effects of pH and solvents on the response of the C60 coated piezoelectric crystal sensor for lipase was also studied. Optimum pH was found to be at 7 and optimum temperature was 31oC. The reactivity between C60 and lipase was decreased in organic solvents such as methanol and ethanol.
    The water insoluble C60-lipase was identified with IR spectrography. The C60-lipase coated SiO2 chip with pore size of about 5~10 μm was prepared. The activity of immobilized C60-lipase was investigated and was still activated. The increased concentration of lipase resulted the increase in the ability of C60-lipase catalyze the hydrolysis of L-amino acid esters. The immobilized C60-lipase could catalyze the hydrolysis of L-amino acid esters, but not D-amino acid esters.
    The C60-cryptand[2,2] coated piezoelectric (PZ) crystal sensor with immobilized C60-lipase was prepared to detect L-amino acid esters in aqueous solutions. The home-made optical isomer PZ sensor for esters was based on the adsorption of L-amino acid, the product of the hydrolysis of L-amino acid ester with C60-lipase, onto the C60-cryptand[2,2] coated quartz crystal. The optical isomer PZ sensor for esters based on C60-lipase was successfully to be applied to distinguish between D-form and L-form amino acid esters. The optical isomer PZ sensor for esters exhibited good sensitivity and reproducibility for L-amino acid esters in aqueous solutions.

    中文摘要...............................................Ⅰ 英文摘要...............................................Ⅱ 目錄...................................................IV 圖目錄................................................VII 表目錄.................................................XI 第一章、 緒論...........................................1 1-1碳六十化學...........................................1 1-1-1碳六十(C60)的發現..................................1 1-1-2碳六十基本性質.....................................3 1-1-3碳六十的化學反應...................................6 1-2冠狀醚及大環胺醚.....................................9 1-2-1冠狀醚及大環胺醚簡介...............................9 1-2-2大環胺醚的應用....................................11 1-3酵素化學............................................12 1-3-1酵素的發現........................................12 1-3-2酵素的特性........................................12 1-3-3固定化酵素(immobilized enzymes)...................13 1-3-4脂肪分解酵素(lipase)..............................18 1-4壓電晶體............................................21 1-4-1壓電晶體之壓電性..................................21 1-4-2石英振盪器........................................23 1-4-3石英振盪器的線路..................................25 1-4-4振盪頻率的量測....................................27 1-4-5石英微量天平(Quartz Crystal Microbalance,QCM)....29 1-4-6石英壓電晶體在分析化學領域上的運用................32 第二章、實驗部分.......................................41 2-1藥品及儀器..........................................41 2-2石英晶片的處理......................................41 2-2-1石英電極之表面塗佈液(coating solution)............42 2-2-2表面塗佈法........................................43 2-3實驗系統............................................44 2-4實驗裝置及步驟......................................45 2-4-1碳六十與酵素間作用力的研究........................45 2-4-2碳六十-酵素之合成................................47 2-4-3以pH meter檢驗碳六十-酵素之活性..................48 2-4-4光學異構物壓電晶體之研究..........................49 2-4-4.1C60-cryptand[2,2]之合成........................49 2-4-4.2碳六十-酵素濾片之製作..........................50 2-4-4.3光學異構物壓電晶體感測系統......................51 第三章、實驗結果與討論.................................52 3-1碳六十與酵素間的吸附................................52 3-1-1塗佈C60/PVC石英晶體對酵素的感應...................52 3-1-2 C60/PVC的塗佈量效應及酵素的濃度效應..............52 3-1-3酵素的脫附行為....................................52 3-2碳六十固定化酵素....................................57 3-2-1碳六十/脂肪分解酵素合成條件的探討.................57 3-2-1.1溫度效應........................................57 3-2-1.2 pH值效應.......................................57 3-2-1.3有機溶劑對C60-Lipase合成的影響..................57 3-2-2碳六十/脂肪分解酵素的鑑定.........................62 3-2-3碳六十/脂肪分解酵素的活性.........................62 3-2-4碳六十-大環胺醚的鑑定............................65 3-3研製光學異構物壓電感測器............................67 3-3-1胺基酸酯類光學異構物的偵測........................67 3-3-1.1 D-form與L-form胺基酸酯類光學異構物的比較.......67 3-3-1.2表面塗佈物種效應................................70 3-3-1.3碳六十濾片浸泡酵素濃度的影響....................70 3-3-1.4碳六十濾片浸泡酵素時間的影響....................70 3-3-1.5 不同的胺基酸酯類之感應訊號.....................74 3-3-2 L-Aspartic acid dimethyl ester 偵測..............77 3-3-2.1塗佈C60-Cryptand [2,2]/PVC石英晶體之感應情形....77 3-3-2.2濾片數目(C60-Lipase量)的影響....................77 3-3-2.3濃度效應........................................81 3-3-2.4回收率(Recovery)................................83 3-3-3 L-Glutamic acid γ-methyl ester 偵測..............85 3-3-3.1塗佈C60-Cryptand[2,2]/PVC石英晶體之感應情形.....85 3-3-3.2 C60-Cryptand[2,2]/PVC的塗佈量效應..............85 3-3-3.3濾片數目(C60-Lipase量)的影響....................88 3-3-3.4 L-Glutamic acid γ-methyl ester的濃度效應.......88 3-3-3.5再現性..........................................92 3-3-4 L-Glutamic acid γ- benzyl ester 偵測.............93 3-3-4.1塗佈C60-Cryptand [2,2]/PVC石英晶體之感測情形....93 3-3-4.2脫附訊號........................................93 3-3-4.3濾片數目(C60-Lipase量)的影響....................93 3-3-4.4 L-Glutamic acid γ-benzyl ester濃度效應.........93 3-3-5 L-Glutamic acid γ- ethyl ester 偵測..............99 3-3-5.1塗佈C60-Cryptand[2,2]/PVC 石英晶體之感測情形....99 3-3-5.2 L-Glutamic acid γ-benzyl ester的濃度效應.......99 3-3-5.3脫附訊號........................................99 3-3-5.4鹽類的影響.....................................103 3-3-5.5有機溶劑的影響.................................103 3-3-5.6光學異構物的影響...............................103 3-3-6濾片儲存活性.....................................114 第四章、結論..........................................115 參考文獻..............................................116

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