近年來利用酵素進行非對掌異構物之光學分割研究廣為受到重視，而酵素固定化技術的進步也使酵素的應用更具高穩定性和可重複使用性。
本實驗利用固定於幾丁聚糖之脂肪酵素（Pseudomonas fluorescens IFO 12055，EC 3.1.1.3）催化S,R-MAMP（R,S-Methyl-3-Acetylthio- 2-Methylpropionate）不對稱水解轉換以生產S-AMPA（S-Acethylthio-2-Methylpropionic Acid）。

在MAMP的合成純化方面，成功合成出高純度的反應基質，並可達到良好的製備再現性。

實驗發現，MAMP與AMPA在水溶液中皆可能有負反應的產生，例如因為硫酯鍵（thioester-bonding）水解而生成methyl β-mercaptoisobutyrate與β-mercaptoisobutyric acid。

由膠層過濾層析法與SDS-PAGE分析，Pseudomonas fluorescens IFO 12055脂肪酵素 (68.0kDa)含有兩個分子量相同或及接近之subunit，其分子量約為36.0kDa。固定化酵素與自由酵素最適操作條件均為50℃, pH=8.0，且固定化增加了酵素對酸的忍受度。兩者Km值相近，惟固定化酵素（41.18μmole AMPA /hr, mg enzyme）之Vmax小於自由酵素（71.06μmole AMPA /hr, mg enzyme），顯示固定化造成酵素部分失活。固定化酵素在操作30批次後仍能保有80%以上之活性，具有良好重複使用利用性。固定純化酵素之單位活性約是粗酵素的2.38倍。

在30℃ pH=7.6 條件下以固定化酵素進行光學分割，可得到光學純度88%（e.e.）的S-AMPA。

The present research was mainly concerned with the immobilization of lipase（EC 3.1.1.3）from Pseudomonas fluorescens IFO 12055 on chitosan bead, for preparing S-AMPA（S-Acethylthio-2-Methylpropionic Acid） by enantioselective hydrolysis of racemic MAMP（Methyl-3-Acetylthio- 2-Methylpropionate）. We found that MAMP and AMPA could hydrolyze not only the ester-bonding but also the thioester-bounding. The Molecular Weight of the lipase estimated by Gel Filtration and SDS-PAGE was 68kDa. This enzyme was constituted of two subunits （about 36kDa）.The optimum temperature for free lipase and immobilized lipase on chitosan bead was 50℃, and optimum pH was 8.0. The Km（Michaelis constant）value of the immobilized enzyme was close to that of free enzyme. The Vmax value of the immobilized enzyme was lower than that of the free enzyme. Immobilized enzyme retains more than 80% of its activity for 30 batchs. It was confirmed that S-AMPA with 88%（e.e.）of optical purity was produced when reaction was done at 30℃ pH=7.6 by immobilized enzyme.

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