Development of methods for the directed evolution of penicillin G acylase by in vitro compartmentalization
(2012)
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- Authors
- Supervisors
- Soumillion, Patrice
- Abstract
- (en) Enzymatic catalysts have been finely tuned for their specific function though natural evolution, by iterative mutations and selections cycles. Directed evolution mimics natural evolution at the laboratory scale and allows the selection, in large collections of variants, of enzymes bearing new properties. In vitro compartmentalization (IVC) is the directed evolution technique used in this work, and consists of aqueous microdroplets dispersed in oil acting as artificial cells. Single genes can be encapsulated in droplets together with the proteins encoded by this gene and substrates to test its enzymatic activity. These compartments can be easily manipulated using microfluidic techniques. Two fluorogenic enzymatic tests applied to the detection of the penicillin G acylase in droplets were developed. The retention of coumarin compounds in microdroplets was evaluated and a collection of substrates with a sulfonated coumarin as leaving group was synthesized. Libraries of mutants of penicillin acylase were created and screened for variants that would have acquired broader substrate specificity compared to the wild type enzyme. The second enzymatic test called AFET (for Activity Fed Enzyme Translation) is based on the in vitro translation of proteins. It consists of the linkage between the enzymatic activity of the enzyme of interest with its own in vitro translation together with the translation of a reporter gene, in our case the GFP. The presented work lays a solid foundation for future work in the directed evolution of penicillin acylase and other enzymes of interest.
- Affiliations
Citations
Woronoff, G. (2012). Development of methods for the directed evolution of penicillin G acylase by in vitro compartmentalization. https://hdl.handle.net/2078.5/156331