title: Directed evolution of a fungal peroxidase.
abstract: The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50 degrees C), and high peroxide concentration (5-10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.
authors: Novo Nordisk Biotech, Inc., Davis, CA 95616, USA. cherry@nnbt.com
authors: J R
authors: JR
authors: Cherry
authors: M H
authors: MH
authors: Lamsa
authors: P
authors: P
authors: Schneider
authors: J
authors: J
authors: Vind
authors: A
authors: A
authors: Svendsen
authors: A
authors: A
authors: Jones
authors: A H
authors: AH
authors: Pedersen
mesh_terms: D003302:Coprinus / Q000201:enzymology* / Q000235:genetics*; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006418:Heme / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009195:Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D006418:Heme; D006861:Hydrogen Peroxide; D009195:Peroxidase