Gene expression, property characterization, and immobilization of a recombinant prolidase from Escherichia coli NovaBlue

• Main Author: 賴冠伶
• Other Authors: Lo, Huei-Fen
• Format: Others
• Language: zh-TW
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/4atk9y

碩士 === 弘光科技大學 === 食品科技所 === 106 === Abstract 　　Prolidase, a protease, has the ability to degrade Xaa-Pro dipeptide. In the past study, human prolidase was participated in the collagen synthesis, and the bacterial source of prolidase has the ability to resist organic phosphorus toxicity. The multiple amino acid sequence alignment shows the highly similarity of prolidase from eukaryotic origin and Escherichia coli prolidase (EcPepQ). Therefore, we uses EcPepQ for biochemical characterization and immobilization to explore the applicability of Prolidase. According to the comparison of multiple amino acid sequences show that the eukaryotic prolidase and EcPepQ are highly conserved in the active center and take part in the metal ion bonding. A truncated gene from Escherichia coli NovaBlue encoding a recombinant prolidase was cloned into pQE-30 to generate pQE-EcPepQ, and the overexpressed enzyme was purified from the crude extract of IPTG-induced E. coli M15 (pQE-EcPepQ) to homogeneity by nickel-chelate chromatography. The molecular weight of EcPepQ was about 57 kDa by SDS-PAGE analysis. The optimal condition for EcPepQ enzyme activity were 60°C, Tris-HCl (pH 8.0) and 0.1 mM Mn2+. The recombinant EcPepQ Tm value was 64.6°C by thermal unfolding assay. The kinetic parameters with Ala-Pro showed that the Km value and catalytic efficiency were 8.78 mM and 105.5 s-1 mM-1. Unfolding analyses using circular dichroism and tryptophan emission fluorescence revealed that [GdnHCl]0.5,N-U 1.43 M and 1.98 M, respectively. The EcPepQ also showed the high tolerance to different water / organic co-solvents. The results could be used as reference for biocatalyzation in co-solvent system. 　　In the immobilization study, the Ni2+ functionalized silica-coated magnetic nanoparticles (NiNTASiMNP) were synthesized for EcPepQ immobilization. After immobilization of the enzyme, EcPepQ was 1.5 mg/mg of nanoparticles, EcPepQ immobilization on magnetic nanoparticles was confirmed by Fourier Transform Infrared Spectroscopy. The chemical and kinetic behaviors of immobilized EcPepQ are mostly consistent with the free form enzyme. However, the immobilized EcPepQ shows the better temperature stability, pH stability and persistence than the free form enzyme. After the reused for 50 times, the immobilized enzyme could maintain 54% enzyme activity. Overall, the immobilization of enzymes on Ni2+ functionalized silica-coated magnetic nanoparticles could improve the application of EcPepQ in industry. 　　In the third part of this study, the biochemical characteristics and kinetic analysis of free and immobilized EcPepQ for the catalytic hydrolysis of organophosphorus were investigated. The temperature condition for the hydrolysis of dimethyl or diethyl nitrophenyl phosphate were 70℃. The pH condition for the free form and immobilized EcPepQ to hydrolyze the dimethyl or diethyl nitrophenyl phosphate were pH 8.0 and 9.0, respectively. The enzyme activity for the free form and immobilized EcPepQ is highly dependent on divalent metal ion, especially Mn2+, either for hydrolysis of dimethyl or diethyl nitrophenyl phosphate. The Km of the free EcPepQ hydrolyzed dimethyl and diethyl nitrophenyl phosphate was 8.49 and 3.42 mM, respectively, while the catalytic efficiencies were 0.22 and 0.03 s-1 mM-1, respectively. The Km of the immobilized EcPepQ hydrolyzed dimethyl and diethyl nitrophenyl phosphate was 1.97 and 1.05 mM, while the catalytic efficiencies were 0.17, 0.031 s-1 mM-1, respectively. In general, the results provided by the above analysis will help us to figure out the biochemical properties and the industrial application of EcPepQ. Keywords：E. coli , Prolidases, Magnetic Nanoparticles, Immobilized enzyme, dimethyl nitrophenyl phosphate, diethyl nitrophenyl phosphate