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.
Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, Grisebachstrasse 8, D-37077 Göttingen, Germany.
Stefan
S
Becker
Hans-Ulrich
HU
Schmoldt
Thorsten Michael
TM
Adams
Susanne
S
Wilhelm
Harald
H
Kolmar
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