Selection of Metalloenzymes by Catalytic Activity Using Phage Display and Catalytic Elution

✍ Scribed by Isabelle Ponsard; Moreno Galleni; Patrice Soumillion; Jacques Fastrez

• Publisher: John Wiley and Sons
• Year: 2001
• Tongue: English
• Weight: 116 KB
• Volume: 2
• Category: Article
• ISSN: 1439-4227
• DOI: 10.1002/1439-7633(20010401)2:4<253::aid-cbic253>3.0.co;2-6

✦ Synopsis

The metallo-b-lactamase bLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage-bound enzyme fd-bLII 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 phagebound enzymes by metal ion complexation, 2. binding to substratecoated 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-bLII plus either a dummy phage, a phage displaying an inactive mutant of the serine b-lactamase TEM-1, or inactive and low-activity mutants of bLII. 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.