本論文可以分為兩部分，第一部份是以固定化金屬離子親和性吸附劑 (immobilized metal ion affinity adsorbent) 對 a 型澱粉水解酵素 (a-amylase) 進行分離的探討，第二部份則是利用液相層析與毛細電泳對化療促進劑—甲氧基胺 (methoxyamine)，建立分析方法。首先以 b 型環糊精 (b-cyclodextrin) 為基材、環氧氯丙烷 (epichlorohydrin, EPI) 為交聯劑進行交聯反應，EPI 不只能改善 b 型環糊精的機械強度同時也對交聯後的基材產生活化作用，以便可再耦合金屬離子，形成固定化金屬離子親和性吸附劑 b-CDcl-IDA-Cu2+。吸附劑只需 2 分鐘便能夠吸附完活性為 750 U/mL 的 Bacillus licheniformis a-amylase (BLA)，而對活性達 38,900 U/mL、對應濃度為 1.0 mg/mL 左右的 BLA 吸附則需要兩個小時達平衡；以 500 mg 吸附劑對 BLA 進行快速吸附、脫附，連續吸、脫循環可達 50 次以上，且具有平均 97% 的回收效果，應用至 50 次吸附再經一次脫附的濃縮實驗中，則可回收 93% 的 BLA 酵素，且可高達超過 46 倍的濃縮效果。

　　將吸附劑應用於 Bacillus amyloliquefaciens a-amylase (BAA) 醱酵液，發現添加 PEG 有利於吸附，且藉由調整 PEG 的分子量以及添加量可以達到 95% 以上的吸附效果。在脫附劑 imidazole 中加入 200 mM NaCl 能夠改善 BAA 的脫附效果，使其提升超過 35% 之效益，而達到 95% 以上的脫附。對澄清與未澄清的 BAA 醱酵液之分離則分別可達 87% 與 81% 的回收效果。經吸附劑吸附固定之 BLA 只能展現原有活性的 5%，顯示吸附位置是作用在與酵素活性相關的活性座上，但究竟吸附位置是與基質作用的鍵結座或是進行水解反應的催化座，仍不明確。對解酯酵素 (esterase) 進行吸附實驗，結果顯示在混合 a 型澱粉水解酵素與解酯酵素的系統中，吸附劑對 a 型澱粉水解酵素的吸附幾乎不受到解酯酵素存在之影響，然而，對解酯酵素的吸附卻反而受到 a 型澱粉水解酵素的抑制。

　　在第二部份中，首先利用紫外-可見光光度計、LC-MS 與化學還原反應釐清分析物甲氧基胺與衍生劑4-二乙胺基苯甲醛(4-(diethylamino)benzaldehyde, DEAB) 的衍生反應，針對液相層析與毛細電泳的特性對衍生產物進行探討，以發展各自的分析方法。在高鹽類緩衝與有機相存在的流動相條件中，液相層析方法可以在 310 nm 測得衍生產物 4-(diethylamino)benzaldehyde o-methyloxime (DBMO) 的訊號，其線性範圍落在 0.10-10.0 mM 之間。另一分析方法則是使用 pH 2.5 的緩衝液於毛細電泳中，可針對質子化的衍生產物 protonated 4-(diethylamino)benzaldehyde o-methyloxime (DBMOH+) 與內標準品 N,N-dimethyl-p-toluidine (DMPT) 的相對波峰面積比製作出校正曲線，此方法則是在波長 200 nm 下進行分析，可得到在 5.0-500 mM 的線性範圍。

　This thesis is divided into two parts. The first part is aimed at the investigation of immobilized metal ion affinity adsorbent for a-amylase separation and the second part is on the development and validation of analysis methods, liquid chromatography and capillary electrophoresis, for a novel chemotherapeutic enhancer, methoxyamine. b-Cyclodextrin (b-CD) and epichlorohydrin (EPI) were chosen as the matrix material and crosslinker, respectively. Thus the cross-linked b-cyclodextrin (b-CDcl) could react with iminodiacetic acid (IDA) to form b-CDcl-IDA. The immobilized metal ion adsorbent, b-CDcl-IDA-Cu2+, was prepared by chelating metal ions with the carboxylic groups from IDA. The adsorbent could adsorb 750 U/mL Bacillus licheniformis a-amylase (BLA) within 2 min. However, it took 2 hours to reach the adsorption equilibrium as the BLA activity was raised to 38,900 U/mL. A rapid and repeated BLA adsorption-desorption procedure was executed 50 times with 500 mg of adsorbent and the average recovery of 97% was achieved. In total, 93% of recovery and 46-fold concentration could thus be obtained.

　The presence of PEG could facilitate a-amylase adsorption from Bacillus amyloliquefaciens a-amylase (BAA) fermentation broth. 95% of adsorption could be obtained by changing the molecular weight and the amount of PEG. Adding 200 mM NaCl to the desorption agent, imidazole, the desorption of BAA raised by 35% to more than 95%. The BAA were separated from the fermentation broths by the prepared adsorbents and the BAA recovery were higher than 80%. Esterase was used to examine the specific affinity of adsorbents for a-amylase. In the mixture of a-amylase/esterase, the adsorption of a-amylase was nearly not influenced by esterase. On the contrary, the esterase adsorption was inhibited by a-amylase.

　In the second part, UV-Visible photometer, LC-MS and chemical reduction were used to discover the derivatization reaction of derivating agent, 4-(diethylamino)benzaldehyde (DEAB), with methoxyamine. The high performance liquid chromatography (HPLC) method quantitated the main derivate, 4-(diethylamino)benzaldehyde o-methoxyloxime (DBMO), at the wavelength of 310 nm. A linear calibration range of 0.10-10.0 mM was obtained. Another method is using pH 2.5 phosphate buffer as the separation buffer for capillary electrophoresis (CE). The relative peak area of protonated 4-(diethylamino)benzaldehyde o-methoxyloxime (DBMOH+) to the internal standard, N,N-dimethyl-p-toluidine (DMPT), was adopted to the calibration which had a linear range of 5.0-500 mM at the wavelength of 200 nm.

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