Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability

Publication date: Available online 6 February 2020Source: Journal of Bioscience and BioengineeringAuthor(s): Karla Ilić Đurđić, Selin Ece, Raluca Ostafe, Simon Vogel, Ana Marija Balaž, Stefan Schillberg, Rainer Fischer, Radivoje ProdanovićLignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied fl...
Source: Journal of Bioscience and Bioengineering - Category: Biomedical Science Source Type: research