Directed evolution of a type A feruloyl esterase for increased thermostability and organic solvent tolerance
Julien Durand, Victor Chaumeton, Olga Gherbovet, Mélanie Ragon, Peter Jütten, Joana L.A. Bras, Catarina I.P.D. Guerreiro, Carlos M.G.A. Fontes, Alexander Piechot, Régis Fauré, Sophie Bozonnet, Michael J. O’Donohue
Feruloyl esterases (FAE) are carboxylic esterases (EC 3.1.1) that cleave the bond between ferulic acid and L-arabinofuranosyl residues of arabinoxylans. These enzymes, in addition to helping the econstruction of lignocellulosic biomass, have a natural capacity to synthesize a wide range of bioactive molecules with interesting properties (notably anti-oxidant) in the fields of food, pharmaceuticals or cosmetics.
Within the OPTIBIOCAT European project, a methodology to express, generate and screen diversity for Aspergillus niger feruloyl esterase A was developed, tested and implemented in a high-throughput fashion, using ad hoc chromogenic probes . A total of ~10,000 mutant clones were generated and screened to isolate 13 mutants with improved thermal and/or solvent stability. The most interesting mutations were individually studied while their random recombination was performed in a second round of evolution through Staggered Extension Process (StEP) .
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