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    E. coli surface display of streptavidin for directed evolution of an allylic deallylase

    Heinisch, T. and Schwizer, F. and Garabedian, B. and Csibra, E. and Jeschek, M. and Vallapurackal, J. and Pinheiro, Vitor B. and Marlière, P. and Panke, S. and Ward, T.R. (2018) E. coli surface display of streptavidin for directed evolution of an allylic deallylase. Chemical Science , ISSN 2041-6520. (In Press)

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    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.

    Metadata

    Item Type: Article
    School: School of Science > Biological Sciences
    Depositing User: Vitor Bernardes pinheiro
    Date Deposited: 05 Jun 2018 10:04
    Last Modified: 13 Jun 2021 07:31
    URI: https://eprints.bbk.ac.uk/id/eprint/22682

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