Reconstitution in vivo of penicillin G acylase activity from separately expressed subunits.

Abstract

Penicillin G acylase from Escherichia coli ATCC11105 is synthesized as a precursor polypeptide with a signal sequence for secretion into the periplasm and an endopeptide separating two subunit domains. Proteolytic processing leads to mature, heterodimeric penicillin G acylase. We have shown that the alpha- and beta-subunits of the enzyme, which have no detectable enzymatic activity on their own, can reconstitute enzyme activity when their genes are put into an E. coli host on separate plasmids. Activity is reconstituted in the cytoplasm whereas normally processing and formation of the active heterodimer occurs in the periplasm. Enzyme activity can reach levels close to wild type in the strain used. The activity recovered from a combination of alpha-subunit linked to a 54-amino-acid endopeptide and beta-subunit was lower than with the subunits alone.