Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts.
Enzyme libraries displayed on the surface of microbial cells or microbeads can be screened with fluorogenic substrates that provide a physical linkage of the reaction product to the corresponding enzyme. Libraries exceeding 10(9) different variants can be quantitatively analysed and screened by flow cytometry at a rate of 30 000 cells/second. The promise of screening methods based on fluorescence-activated cell sorting for directed enzyme evolution is being realized and significantly improved enzymes have been reported recently.
Becker
Stefan
S
Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Schmoldt
Hans-Ulrich
HU
Adams
Thorsten Michael
TM
Wilhelm
Susanne
S
Kolmar
Harald
H
D001419:Bacteria / Q000201:enzymology*; D001681:Biological Assay / Q000379:methods; D002384:Catalysis; D004789:Enzyme Activation; D004798:Enzymes / Q000032:analysis* / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D019151:Peptide Library; D015202:Protein Engineering / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods
D004798:Enzymes; D008565:Membrane Proteins; D019151:Peptide Library