Michael Lamsa

Directed evolution of a fungal peroxidase

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.

Screening for oxidative resistance.

Publisher Summary This chapter discusses the screening of amino acid for oxidative resistance. The oxidation of amino acid side chains in proteins has long been recognized as a primary pathway for functional inactivation. It is found that whether a protein drug targeted for intracellular action or an enzyme formulated into laundry detergent, activated oxygen species are generated that can modify protein side chains, significantly altering their hydrophobicity, charge, and size. The key to the directed evolution process is establishment of a screening system that accommodates the predicted diversity generated by the mutagenesis technique, and does not return a high rate of false positives. Growth and screening of mutants are performed using automation in 96-well microtiter plates. Assays used to screen must be validated to assure proper operation under the proposed sample conditions. An assay system must be adequately set up to investigate this effect, and in general, sufficient dilution must be performed after enzyme treatment to eliminate or minimize the effect of the chemical on the assay. It is suggested that any effect must be taken into account when setting up a control comparison in calculating the percentage remaining activity of the enzyme.