Selection of metalloenzymes by catalytic activity using phage display and catalytic elution

The metallo-beta-lactamase betaLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage-bound enzyme fd-betaLII was shown to be active on benzylpenicillin as substrate; it could be inactivated by complexation of the essential zinc(II) ion with EDTA and reactivated by addition of a zinc(II) salt. A selection process was designed to extract active phage-bound enzymes from libraries of mutants in three steps: 1. inactivation of active phage-bound enzymes by metal ion complexation, 2. binding to substrate-coated magnetic beads, 3. release of phages capable of transforming the substrate into product upon zinc salt addition. The selection process was first successfully tested on model mixtures containing fd-betaLII plus either a dummy phage, a phage displaying an inactive mutant of the serine beta-lactamase TEM-1, or inactive and low-activity mutants of betaLII. The selection was then applied to extract active phage-bound enzymes from a library of mutants generated by mutagenic polymerase chain reaction (PCR). The activity of the library was shown to increase 60-fold after two rounds of selection. Eleven clones from the second round were randomly picked for sequencing and to characterize their activity and stability.