DEVELOPMENT OF A HIGH-EXPRESSION SYSTEM FOR PENICILLIN-G ACYLASE BASED ON THE RECOMBINANT ESCHERICHIA-COLI STRAIN RE3(PKA18)

A high-expression system for penicillin G acylase (PGA) was developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chro mosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19. One particular objective for the elaboration of this expression system was the selection of a convenient host strain and modification of the growth conditions. From a total of 15 hosts, the strain RE3 cul tured in a mineral medium exhibited the highest expression of a pga-en coding gene from the recombinant plasmid and high segregational plasmi d stability. The high-expression system led to an increase in the spec ific activity of PGA up to 1,000 U g(-1) cell dry weight and a total a ctivity of about 4,500 U l(-1) of the culture in a laboratory fermento r.