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

The metallo-beta -lactamase beta LII from Bacillus cereus 569/H/9 was displ ayed on the filamentous phage fd. The phage-bound enzyme fd-beta LII was sh own 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 ac tive phage-bound enzymes from libraries of mutants in three steps: 1. inact ivation of active phage-bound enzymes by metal ion complexation, 2. binding to substrate-coated magnetic beads, 3. release of phages capable of transf orming the substrate into product upon zinc salt addition. The selection pr ocess was first successfully tested on model mixtures containing fd-beta LI I plus either a dummy phage, a phage displaying an inactive mutant of the s erine beta -lactamase TEM-1, or inactive and few-activity mutants of beta L II. The selection was then applied to extract active phage-bound enzymes fr om a library of mutants generated by mutagenic polymerase chain reaction (P CR). The activity of the library was shown to increase 60-fold after two ro unds of selection. Eleven clones from the second round were randomly picked for sequencing and to characterize their activity and stability.