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°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.
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Acknowledgements
The authors gratefully acknowlege the contributions of Juozas Kulys, Donna L. Moyer, Jeffery Shuster, and Birger Rostgaard Jensen.
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Cherry, J., Lamsa, M., Schneider, P. et al. Directed evolution of a fungal peroxidase. Nat Biotechnol 17, 379–384 (1999). https://doi.org/10.1038/7939
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DOI: https://doi.org/10.1038/7939
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