PERIPLASMIC AGGREGATION LIMITS THE PROTEOLYTIC MATURATION OF THE ESCHERICHIA-COLI PENICILLIN-G AMIDASE PRECURSOR POLYPEPTIDE

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a pre cursor polypeptide by an unusual internal maturation process. We obser ved the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cell s grown at 26 degrees C. The aggregated nature of the accumulated mole cules was demonstrated using detergents and chaotropic agents in solub ilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fra ction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a conseque nce PGA production is substantially increased. Thus, periplasmic aggre gation of the PGA precursor polypeptide limits PGA production by recom binant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.