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。

　　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.

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