Directed evolution of a fungal peroxidase

The Coprinus cinereus (CIP) heme peroxidase was subjected to multiple round s of directed evolution in an effort to produce a mutant suitable for use a s a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase i s rapidly inactivated under laundry conditions due to the high pH (10.5), h igh 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 e rror-prone PCR to create random mutations. Mutants were expressed in Saccha romyces cerevisiae and screened for improved stability by measuring residua l activity after incubation under conditions mimicking those in a washing m achine. Manually combining mutations from the site-directed and random appr oaches led to a mutant with 110 times the thermal stability and 2.8 times t he oxidative stability of wild-type CiP. In the final two rounds, mutants w ere randomly recombined by using the efficient yeast homologous recombinati on system to shuffle point mutations among a large number of parents, This in vivo shuffling led to the most dramatic improvements in oxidative stabil ity, yielding a mutant with 174 times the thermal stability and 100 times t he oxidative stability of wild-type CiP.