Focused directed evolution of pentaerythritol tetranitrate reductase by using automated anaerobic kinetic screening of site-saturated libraries.
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 α-methyl-trans-cinnamaldehyde, respectively, with an increase in ee (62 % (R) for W102F). In addition, the detection of mutants with weak activity against α,β-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.
Faculty of Life Sciences, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK.
Martyn E
ME
Hulley
Helen S
HS
Toogood
Anna
A
Fryszkowska
David
D
Mansell
Gill M
GM
Stephens
John M
JM
Gardiner
Nigel S
NS
Scrutton
D000171:Acrolein / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000475:Alkenes / Q000737:chemistry / Q000378:metabolism; D000693:Anaerobiosis; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D016972:Enterobacter cloacae / Q000201:enzymology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019151:Peptide Library; D017434:Protein Structure, Tertiary; D013237:Stereoisomerism; D013379:Substrate Specificity
D000475:Alkenes; D001426:Bacterial Proteins; D002264:Carboxylic Acids; D019151:Peptide Library; D009249:NADP; D000171:Acrolein; C013658:propylene; D010088:Oxidoreductases; C098939:pentaerythritol tetranitrate reductase; C012843:cinnamaldehyde