Journal
CHEMBIOCHEM
Volume 11, Issue 17, Pages 2433-2447Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201000527
Keywords
asymmetric bioreduction; biocatalysis; directed evolution; enantioselectivity; robotic library screening
Funding
- UK Biotechnology and Biological Sciences Research Council (BBSRC)
- BBSRC
- Royal Society
- Biotechnology and Biological Sciences Research Council [BB/E010717/1, BB/G023581/1, BB/D01963X/1, BB/D002826/1, BB/G023581/2] Funding Source: researchfish
- BBSRC [BB/G023581/1, BB/D002826/1, BB/D01963X/1, BB/E010717/1, BB/G023581/2] Funding Source: UKRI
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This work describes the development of an automated robotic platform for the rapid screening of enzyme variants generated from directed evolution studies of pentraerythritol tetranitrate (PETN) reductase, a target for industrial biocatalysis. By using a 96-well format, near pure enzyme was recovered and was suitable for high throughput kinetic assays; this enabled rapid screening for improved and new activities from libraries of enzyme variants. Initial characterisation of several single site-saturation libraries targeted at active site residues of PETN reductase, are described. Two mutants (T26S and W102F) were shown to have switched in substrate enantiopreference against substrates (E)-2-aryl-1-nitropropene and alpha-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62% (R) for W102F). In addition, the detection of mutants with weak activity against alpha,beta-unsaturated carboxylic acid substrates showed progress in the expansion of the substrate range of PETN reductase. These methods can readily be adapted for rapid evolution of enzyme variants with other oxidoreductase enzymes.
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