| Summary: | 博士 === 國立成功大學 === 化學系碩博士班 === 96 === 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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