本論文包括三個研究主題：（1）利用偽單孢菌IFO12996酯水解酵素對乙醯硫代異丁酸甲酯及乙醯硫代異丁醯胺的酵素光學分割研究；（2）偽單孢菌酯水解酵素固定在磁性奈米粒子上的研究：（3）利用基因工程重組大腸桿菌固定在填充床生物反應器內的矽藻土來催化水解乙醯硫代異丁醯胺。
第一部份的研究是利用基因工程的方法，將偽單孢菌(Pseudomonas putita) IFO12996酯水解酵素 (PpEST) 的基因轉殖到大腸桿菌，進行大量表現與酵素純化，其後再將酵素對乙醯硫代異丁酸甲酯（DL-β-acetylthioisobutyrate，簡稱DL-MATI）及乙醯硫代異丁醯胺（DL-β-acetylthioisobutyramide ，簡稱DL-ATIA）進行光學分割可得到乙醯硫代異丁酸 （D-β-acetylthioisobutyric acid又名S-(-)-3-acetylthio-2-methylpropionic acid，簡稱DAT）， DAT為合成血管收縮素轉化酶抑制劑的重要前驅物。純化出的酯水解酵素分子量約為33 kDa，其酵素反應最適當的酸鹼值約在8～10之間，最適當的反應溫度約在57~67℃之間。酵素水解DL-MATI和DL-ATIA得到DAT的光學純度（e.e.值）均為97%，光學選擇性(enantioselectivity value， E-value) 也大於150。由動力學參數指出，DL-ATIA是酯水解酵素比較差的受質，但是由於DL-ATIA的水溶性是DL-MATI的20倍且當濃度達到780 mM時對酯水解酵素也沒有受質抑制的情形發生，所以當運用在工業上大量生產DAT時，相較於DL-MATI而言 ，DL-ATIA是一個較好的選擇。
第二部分則是探討酯水解酵素利用共價鍵結固定在戊二醛衍生化後的磁性奈米粒子上的特性。四氧化三鐵磁性奈米粒子(Fe3O4 nanoparticles)是利用Fe3+和Fe2+在氨水裡面共沈澱而得，並且以3-丙胺三乙氧基矽烷和戊二醛進行表面修飾，利用奈米粒子及酵素上的胺基藉著戊二醛進行固定化。利用XRD及FT-IR的光譜數據可以得知所有的修飾反應及固定化反應皆發生在磁性奈米粒子的表面，並不會影響到內部的結晶構型。而TEM的結果顯示出奈米粒子在固定化前後的粒徑並無明顯的改變，平均粒徑約在11.8 nm左右。經由計算最大固定化效率可知當酵素與磁性奈米粒子的重量比為0.058時，平均一顆Fe3O4可吸附4個酯水解酵素。在動力學參數分析指出，當酵素固定後尚保持63％的活性，在酸鹼值及溫度方面的特性皆與固定化前的酵素相似。而固定化酵素水解DL-MATI得到DAT的光學活性(e. e. value)也高達97.2%，且在重複使用10次後的活性也有84％。
最後一部分則是將此具Pseudomonas putita IFO12996酯水解酵素 (PpEST) 的基因轉殖大腸桿菌固定於填充床生物反應器之矽藻土(Celite 580)上，而此生物反應器用來水解DL-ATIA可以生產產率達到34.5％及光學活性97％的DAT。此方法提供了一種新穎、簡單、實際以及具有經濟價值的方法來生產DAT。而此方法在重複使用五次後，大腸桿菌還保有60%的活性。

There are three major themes in this thesis, including (1) Enzymatic resolution of methyl DL-β-acetylthioisobutyrate and DL-β-acetylthioisobutyramide using a stereoselective esterase from Pseudomonas putida IFO12996, (2) Preparation and characterization of Pseudomonas putida esterase immobilized on magnetic nanoparticles and (3) Steroselective hydrolysis of DL-β-acetylthioisobutyramide catalyzed by genetically engineered E. coli immobilized on celite 580 in a packed bed bioreactor
In the first subject, esterase (PpEST) from Pseudomonas putida IFO12996 catalyzes the stereoselective hydrolysis of methyl DL-β-acetylthioisobutyrate (DL-MATI) and DL--acetylthioisobutyramide (DL-ATIA) to give D-β-Acetylthioisobutyric acid (also known as S-(-)-3-acetylthio-2-methylpropionic acid, DAT). DAT is a key intermediate for the synthesis of a series of angiotensin converting enzyme inhibitors. To use enzyme for the DAT production, the PpEST gene of Pseudomonas putida IFO12996 was cloned and expressed in E. coli. PpEST with a molecular weight of 33 kDa could hydrolyze DL-MATI and DL-ATIA to give DAT with enantiometric excess value (e.e. value) about 97% and enantioselectivity value (E-value) ＞150, respectively. The kinetic constants of PpEST for DL-MATI and DL-ATIA were examined and they showed that DL-ATIA was a poorer substrate than DL-MATI for PpEST. However, DL-ATIA was 20 fold more soluble in water than DL-MATI and more stable than DL-MATI and it did not show substrate inhibition of the PpEST up to 780 mM. This result suggested that PpEST is an esterase with amidase activity which can kinetically resolve DL-ATIA to yield DAT and DL-ATIA is a better choice than DL-MATI for industrial production of DAT by the enzymatic resolution method.
In the second subject, a recombinant esterase from Pseudomonas putida IFO12996 covalently bound to magnetic nanoparticles via glutaraldehyde coupling reaction was studied. Magnetic nanoparticles Fe3O4 were prepared by coprecipitation of Fe+2 and Fe+3 ions in ammonia solution. The surface of the particles was treated with 3-aminopropyltriethoxysilane (APES) to obtain amino-silane coated particles. The amino functional group on the particle surface and the amino group of the esterase was coupled with glutaraldehyde to obtain esterase immobilized on magnetic nanoparticles. X-ray diffraction (XRD) patterns indicated the particle before and after binding to esterase were pure Fe3O4. Transmission electron microscopy (TEM) showed that the particle with mean diameter of 11.8 nm and binding to esterase didn’t significantly change its size. Fourier transform infrared (FTIR) spectroscopy confirmed the esterase bound to the particles and the measurement of protein content revealed that the weight ratio of the esterase bound to the magnetic nanoparticles was 0.058 which was about four esterase molecules per particle. The kinetic analysis data indicated that the immobilized esterase retained 63% of its original activity and it exhibited similar thermal and pH stability as free esterase. The immobilized esterase hydrolyzed DL-MATI to give DAT with enatiometric excess value of 97.2% and it retained 84% of activity after being used for 10 cycles.
In the third subject, the esterase gene of Pseudomonas putida IFO12996 was cloned and expressed in Escherichia coli which were further immobilized and retained on a packed bed bioreactor filled with Celite580. The packed bed bioreactor was used to conduct the stereoselective hydrolysis of DL-ATIA and to give DAT with yield of 34.5 %, enantiometric excess value of 97% and enantioselectivity value ＞150. The optimal pH and temperature for the reaction were 9.0 and 57℃～67℃, respectively. The kinetic constants (Km and Vmax) of immobilized cells were found to be 372.5 mM and 285.7 μmol min-1 (g cell)-1, respectively. The immobilized cells retained over 60% of the initial catalytic activity after 5 batch cycles of production. This study presented a simple, practical and economical process of immobilization of genetic engineered E. coli on a novel packed bed bioreactor for production of DAT.

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