Cloning And Expression Of Two Sources Of Penicillin G Acylases

Penicillin acylase is an industrial enzyme primarily dedicated to penicillin hydrolysis for the production of 6-aminopenicillanic acid(6-APA), which is a critical starting compound for making several p-lactam antibiotics. Bacterial strains such as Arthrobacter viscosus, Bacillus megaterium, Escherichia coli, Kluyvera cryocrescens,and Providencia rettgeri,contain PGA gene. These PGAs share the same heterodimer protein structure, and their amino acid sequences reveal high homology, suggesting that they might originate from the same ancestral gene. Although E. coli PGA is the most common source for industrial application, the applicability of other bacterial PGAs has been constantly evaluated with promising results. For example, B. megaterium PGA exhibits broad substrate specificity on various cephalosporins, such as cephalexin and cefamandole. PGA from A. viscosus or B. megaterium could be produced in a large scale since most of the enzyme natively expressed or heterologously expressed in Bacillus subtilis could be extracellularly secreted. K. cryocrescens PGA has attracted interest because of many features suitable for industrial application, such as ease of immobilization and endurable stability against heat, pH, and organic solvents. Attempts based on genetic and protein engineering techniques were made to manipulate P. rettgeri PGA for acquiring cephalosporin C acylase activity, but with little success so far.In this thesis, the E. coli PGA gene and P. rettgeri PGA gene were amplified by PCR from plasmid pPA6 and P. rettgeri genomic DNA, then ligated into cloning vector pBluescript SK. Sequence analysis showed that the E. coli PGA gene was the same with the published before(Genebank AF237653), P. rettgeri PGA(ATCC31052) gene had 90.2% homology with P. rettgeri(Genebank M86533).Amino acid sequence alignment of P. rettgeri PGA demonstrated 92.8% homology with P. rettgeri PGA (ATCC29944).Expression plasmids pET24a with different genes were constructed and transformed into E.coli BL21(DE3). The study of concentration of IPTG -. grow temperature and carbon source indicated that by adding glycerol up to 5g/L and induced by 1mmol/L IPTG, the PGA recombination strains , E.coli BL21(DE)(pETePGA) and E.coli BL21(DE3)(pETrPGA), expressed highest enzyme activity up to 587.6U/L and 993.4U/L. The optimization significantly increased E.coli BL21 (DE3)(pETrPGA) expression by 65-fold compared to the native expression (15U/L) in P. rettgeri. To our knowledge, this is the highest production of P. rettgeri PGA in the E.coli expression system. The plasmid stability of pETePGA and pETrPGA indicated the loss rate of plasmids was only 8.5% and 10.8%, after cultured 50 generations in the medium without kanamycin, which showed that the plasmids were basically stable.