Expression And Immobilization Of Penicillion G Acylase In Recombinant Escherichia Coli

6-aminopenicillanic acid (6-APA) is the key precursor in the production ofβ-lactam antibiotics. For gentle reaction conditions, low pollution and high percent conversion, the enzymatic synthesis process for the production of 6-APA has been studied as a hot issue and Penicillin G Acylase (PGA) is the key enzyme in such process.The PGA gene from E.coli ATCC11105 was amplified through PCR. The whole gene sequence concludes signal peptide,αsubunit, connection peptide andβsubunit.Three different promoters were added at the 5'end of PGA gene by Overlap PCR method. These complexes were then inserted into the plasmid pGEMK-T to construct corresponding recombinant plasmids. PGA could reach relatively high activity under the conduction of tac promoter after comparing the different effects on biomass and PGA activity caused by different promoters. IPTG or phenylacetic acid was unnecessarily used as inducers for the expression of PGA.This recombinant plasmid pGEMKT-TacPGA was transformed into different E.coli strains with different genotypes. E.coli Top10F' was chosen as a better expression host cell for its rapid grow rate and high PGA activity per medium volume under the same culture conditions. Also, it was fit for industry production in a large scale.The culture conditions and medium component were optimized to enhance the PGA expression ability of the recombinant strain—E.coli Top10F'/pGEMKT-TacPGA. This recombinant strain was inoculate into 150 flask containing 90ml culture medium and incubated at 28℃for more than 30h. The PGA activity could reach 1400U/L.In the base of recombinant plasmid pGEMKT-TacPGA, a special tag sequence was added to the 3' terminal of PGA gene and pGEMKT-TacPGA-Tag was constructed. There was a special fusion peptide at the C terminal of PGA, so PGA could be specifically purified while immobilized on the carrier under the metal affinity action with Ni²⁺ which was linked on the carrier beforehand. The immobilized enzyme activity was 75U per gram carrier and it showed a fine stability during the consecutively hydrolyze sodium penicillin G for 9 times.