Directed evolution of a fungal peroxidase

Area of Science:

• Biotechnology
• Enzyme Engineering
• Industrial Microbiology

Background:

• Wild-type Coprinus cinereus (CiP) heme peroxidase exhibits poor stability in laundry detergents due to high pH, temperature, and peroxide concentrations.
• Peroxidases are crucial enzymes for dye-transfer inhibition in laundry applications.
• Developing robust enzymes for industrial use requires overcoming stability limitations.

Purpose of the Study:

• To engineer a stable CiP heme peroxidase mutant for effective dye-transfer inhibition in laundry detergents.
• To enhance the thermal and oxidative stability of CiP under simulated laundry conditions.

Main Methods:

• Directed evolution combining site-directed mutagenesis and error-prone PCR.
• Expression of mutants in Saccharomyces cerevisiae.
• Screening for improved stability under simulated washing machine conditions.
• In vivo homologous recombination for shuffling beneficial mutations.

Main Results:

• Initial mutants displayed moderate improvements in stability.
• Combining mutations from different approaches yielded a mutant with 110x thermal and 2.8x oxidative stability.
• Subsequent in vivo shuffling resulted in a hyper-stable mutant with 174x thermal and 100x oxidative stability compared to wild-type CiP.

Conclusions:

• Directed evolution, particularly with in vivo recombination, is effective for creating highly stable heme peroxidases.
• Engineered CiP mutants demonstrate potential for application as dye-transfer inhibitors in laundry detergents.
• The developed mutant offers superior performance under demanding laundry conditions.