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Selection of Metalloenzymes by Catalytic Activity Using Phage Display and Catalytic Elution
Author(s) -
Ponsard Isabelle,
Galleni Moreno,
Soumillion Patrice,
Fastrez Jacques
Publication year - 2001
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20010401)2:4<253::aid-cbic253>3.0.co;2-6
Subject(s) - phage display , chemistry , mutant , enzyme , active site , substrate (aquarium) , zinc , phagemid , bacillus cereus , stereochemistry , biochemistry , bacteriophage , biology , escherichia coli , bacteria , peptide , organic chemistry , ecology , gene , genetics
The metallo‐β‐lactamase βLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage‐bound enzyme fd‐βLII 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‐βLII plus either a dummy phage, a phage displaying an inactive mutant of the serine β‐lactamase TEM‐1, or inactive and low‐activity mutants of βLII. 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.