GENETIC CONSTRUCTION OF CATALYTICALLY ACTIVE CROSS-SPECIES HETERODIMER PENICILLIN-G AMIDASE

A cross-species penicillin G amidase (PGA) gene (pac) coding for an alpha-peptide and a linker peptide from K. citrophila ATCC 21285 and a beta-peptide from E. coli ATCC 11105 has been constructed and cloned in E. coli. The naturally occurring PGA specific processing pathway led to the formation of a hybrid enzyme which was catalytically active. In comparison with the two wild-type enzymes the hybrid PGA was found to have higher k(cat) values for the three tested substrates benzylpenicillin, ampicillin and 6-nitro-3-phenylacetamido-benzoic acid (NIPAD). K-m was between the values of the wild-type enzymes or close to that of K. citrophila, The presented method for protein design of processed enzymes, like PGA, can be applied to combine enzyme properties from different species for special applications.