Related Experiment Video
Updated: Feb 7, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
Published on: March 16, 2011
E. coli surface display of streptavidin for directed evolution of an allylic deallylase
Tillmann Heinisch1, Fabian Schwizer1, Brett Garabedian1
1Department of Chemistry , University of Basel , Mattenstrasse 24a , Basel CH-4002 , Switzerland .
Abstract:
Artificial metalloenzymes (ArMs hereafter) combine attractive features of both homogeneous catalysts and enzymes and offer the potential to implement new-to-nature reactions in living organisms. Herein we present an E. coli surface display platform for streptavidin (Sav hereafter) relying on an Lpp-OmpA anchor. The system was used for the high throughput screening of a bioorthogonal CpRu-based artificial deallylase (ADAse) that uncages an allylcarbamate-protected aminocoumarin 1. Two rounds of directed evolution afforded the double mutant S112M-K121A that displayed a 36-fold increase in surface activity vs. cellular background and a 5.7-fold increased in vitro activity compared to the wild type enzyme. The crystal structure of the best ADAse reveals the importance of mutation S112M to stabilize the cofactor conformation inside the protein.
Related Concept Videos
The Evidence for Evolution
Convergent Evolution
Eukaryotic Evolution
Contrary to the endosymbiont theory, the eukaryote-first hypothesis proposes that the simpler prokaryotic and...
Synteny and Evolution
Around 80 million years ago, the human and mice lineages diverged from the common ancestor. During the course of evolution, the ancestral...
π Molecular Orbitals of the Allyl Radical
The allyl systems have identical molecular orbitals but differ in the number of π electrons....
Radical Substitution: Allylic Chlorination

