title: Selection of metalloenzymes by catalytic activity using phage display and catalytic elution.
abstract: 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.
authors.affiliation: Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Place L. Pasteur, 1, Bte 1B, 1348 Louvain-la-Neuve, Belgium.
authors.forename: I
authors.initials: I
authors.lastname: Ponsard
authors.forename: M
authors.initials: M
authors.lastname: Galleni
authors.forename: P
authors.initials: P
authors.lastname: Soumillion
authors.forename: J
authors.initials: J
authors.lastname: Fastrez
mesh_terms: D001409:Bacillus cereus / Q000201:enzymology; D002384:Catalysis; D019020:Directed Molecular Evolution / Q000379:methods*; D008667:Metalloproteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D019151:Peptide Library; D013379:Substrate Specificity / Q000235:genetics; D015032:Zinc / Q000737:chemistry / Q000494:pharmacology; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism
chemical_list: D008667:Metalloproteins; D019151:Peptide Library; D001618:beta-Lactamases; D015032:Zinc