Protein engineering of penicillinase as affinity ligands for bioprocessing

Abstract

The active site and the substrate binding site of penicillinase ([beta]-lactamase) from Bacillus licheniformis were altered in this study so that the enzyme retains the specific binding capability to the [beta]-lactam antibiotics, but fails to hydrolyze them. When Lys47 in the enzyme molecule was replaced by Ala47, the mutant protein PenP(KA) lost not only its catalytic activity but also the substrate binding ability. In contrast, when Ser44 was replaced by Ala, the mutant protein PenP(SA) lost its catalytic activity but still kept the substrate binding ability. It was found that PenP(SA) exhibited the characteristic association and dissociation with penicillin G, but the dissociation constant was much larger than expected. Possible use of this mutant protein as an affinity ligand is also discussed.