Screening And Culture Optimization Of Microbial Strains For Penicillin G Acylase Production

Penicillin G acylase(PGA,EC 3.5.1.11)can produce 6-aminopenicillanic acid by hydrolyzing penicillin.Penicillin G acylase can also hydrolyze cephalosporin-7-amino,an intermediate used for semi synthetic cephalosporins deacetoxy cephalosporanic acid production.The domestic and foreign literatures show that penicillin G acylase enzyme activity of natural microbial strains is generally low,almost no practical value.The highest enzyme activity reported is 40 U/mL,whith has much room for improvement.The aim of this study was to screen strains with high penicillin G acylase(PGA)activity.Induced mutation of Bacillus megaterium ATCC 14945 was performed by LiCl-ultraviolet composite mutagenesis and atmospheric and room temperature plasma(ARTP)mutagenesis and PGA activities of the strains were assayed with NIPAB method and the strain numbered 12-4 had the highest PGA activity.The study on optimization of enzyme production and purification of the strain 12-4,provide more excellent strains and enzyme producing conditions for industrial production of penicillin acylase.Using gene engineering technology and microorganism separation method for the purification and cultivation of Engineered escherichia coli strains,soil and other materials to provide high yield strains for industrial production of penicillin acylase.The main findings are as follows:1.Induced mutation of Bacillus megaterium ATCC 14945 was performed by LiCl-ultraviolet composite mutagenesis and atmospheric and room temperature plasma(ARTP)mutagenesis.The PGA activities of the strains twere assayed with NIPAB method.The strain numbered 12-4 had the highest PGA activity of 39.60 U/mL from 4.65 U/mL.After the exploration of the biological characteristics of the strain 12-4,we found that Colony surface turned slightly fold after mutation,the expression of subunit ? increased,the PGA activity could be stably passaged in strain 12-4 and was in more than 60% after successively transferring for 5 generations.After optimization of fermentation and expression conditions of strain12-4,Culturing for 6 h and adding phenylacetic acid into broth with quantity of 0.2%(w/v),continuing culture for 50 h,The PGA activity from the supernatants reached To 78.45 U/ml,which is 16.8 times than that of original strains.After preliminary purification,the yield was 64.48%.2.Using microorganism separation method for the purification and cultivation of soil to provide high yield strains.There were 64 strains,only 13 strains have penicillin acylase activity,and the activity is relatively low.The strain K3 has the highest activity of 0.398 U/mL.K3 was identified as Acinetobacter.3.The target gene fragment which synthesis in company was amplified by PCR,then inserted into PET-HGB-HA,PET-Mbp-3C,PET-GST,PET-M-3C,PET-HGB-3C,PET-32M-3C vectors respectively.The plasmid were transformed into the expression strain C+ and induced expression.The recombinant plasmid with carrier PET-HGB-3C has the highest activity,the enzyme activity was 41.49 U/mL inducing for 23 h,the enzyme activity was 58.30 U/mL inducing for 40 h.