Study On The Protein Engineering Of GL-7-ACA Acylase CA130

This thesis includes three parts: (1) protein engineering of GL-7-ACA acylase CA130; (2) study on the crystallization process of labeled GL-7-ACA acylase complex; (3) Study on the RNA dependent RNA polymerase of SARS coronavirus. ⅠProtein Engineering of GL-7-ACA Acylase CA130 7-aminocephalosporinic acid (7-ACA) is a main material to half-synthesize cephalosporanic antibiotics. As the key enzyme to catalyze cephalosporin C to produce 7-ACA, glutaryl-7-amino cephalosporanic acid (GL-7-ACA) acylase is very important in the industrial production. GL-7-ACA acylase from Pseudomonas. sp130 (CA130) was engineered for improving the catalysis character to GL-7-ACA and improving its stability in application. Two series of amino acid residues were selected by computer design based on the structure of CA130 and mutated according to the aims of improving activity and stability respectively. In the first series, key residues in the substrate-binding pocket were substituted with other types residues by site-directed mutagenesis. Among these mutants constructed, two mutants, Y151αF and Q50βN, indicated improved catalysis efficiency (Kcat/Km) about 2-3 folds compared with wild CA130. Their Km values were decreased about 50% and Kcat increased to 14.4 s-1 and 16.9 s-1 from 12.3 s-1 respectively. Their activities to hydrolyze adipoyl 6-amino penicillinic acid (AD-6-APA) and GL-6-APA were also improved. In the second serie, some mutants with higher stabilities were also got. R121βA and K198βA had a 30%-58% longer half-life than CA130 (from 68.1 hours to 88.3hours and 107.5 hours). K198βA and D286βA showed an alkaline shift of optical pH about 1.0-2.0 pH units, while R121βA showed an acidic shift of optical pH about 0.8 pH units. Mutant K198βA could also retain more activity under different pH solutions (pH 7.0, 8.0, 9.0, 10.0) when kept at 37°C for 4 hours. Double mutant Q50βN/K198βA has the merits of both Q50βN and K198βA. After fermentation, it was purified and immobilized by one step. Its specific activity increased about 34.3% compared with immobilized wild CA130. ⅡStudy on the Crystallization Process of Labeled GL-7-ACA Acylase CA130 Complex 7β-bromoacetyl amino cephalosporanic acid (BA-7-ACA), an analog of glutaryl-7-amino cephalosporanic acid (GL-7-ACA), can inhibit and specifically alkylate GL-7-ACA acylase (CA130) from Pseudomonas sp.130, forming a carbon-carbon bond between BA-7-ACA and the C-2 on indole ring of Trp-β4 residue of CA130. Here we reported that BA-7-ACA labeled CA130 (BA-C130) could self-catalyze the hydrolysis of BA-7-ACA during crystallization process. The hydrolysis was confirmed to be a reaction analogous to the one of GL-7-ACA by comparative MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) spectrometry analysis. BA-C130 was inactive at room temperature, but in the process of crystallization at 18°C it catalyzed the hydrolysis of BA-7-ACA, and thus made the later become a substrate. Meanwhile, in crystals, 7-ACA was released but the acetic acid stillbound with Trp-β4, and as a result, the enzyme remained to be inactive. These results demonstrated that Trp-β4 in the αββαmotif was critical and sensitive for the activity of CA130 and also suggested that there was a conformational change induced by deacylation during the process of crystallization. ⅢStudy on the RNA Dependent RNA Polymerase of SARS Coronavirus The RNA dependent RNA polymerase (RdRp) of SARS coronavirus is very important in its genetic reproduction. The full-length gene of RdRp was cloned into pGEX4T1 and the RdRp protein was expressed in Escheria coli as a fusion protein of molecular weight about 130 KDa with glutathione-S-transferase (GST). It was mainly sequestered within inclusion bodies. The fusion protein of 130 KDa was found to be broken into three pieces of peptides: 64kD, 38.7kD and 29.9kD after the first purification by Glutathione-Sepharose 4B tag (GST) column. The dimeric polymers composed of 64kD and 38.7kD were further purified by PolyA Sepharose 4B column. It was proved that the 64kD peptide was cut from the C terminal of 130KD protein (breaking site: F↓KELLV) and the 38.7kD peptide was cut from the N terminal of 130KD protein (breaking site: M↓VPHIS) by Western blotting and N terminal sequencing analysis. The result of native-page showed that 64kD and 38.7kD formed a stable dimeric polymer after the process. This dimeric polymer had RNA-dependent DNA polymerase and RNA polymerase activities in an assay system using polyrA(dT)12-18 and polyA/oligoU as templates/primers. It may also possess the de novo initiation ability of RNA synthesis and terminal transferase (TNTase) activity (need further...