Improved degradation of azo dyes by lignin peroxidase following mutagenesis at two sites near the catalytic pocket and the application of peroxidase-coated yeast cell walls

• Published in: Frontiers of environmental science & engineering 2021-04, Vol.15 (2), p.19, Article 19
• Main Authors: Ili ur i, Karla, Ostafe, Raluca, Prodanovi, Olivera, ur evi elmaš, Aleksandra, Popovi, Nikolina, Fischer, Rainer, Schillberg, Stefan, Prodanovi, Radivoje
• Format: Article
• Language: English
• Subjects: Azo dyes; Biodegradation; Bioremediation; Catalytic activity; Cell walls; Degradation; Dyes; Earth and Environmental Science; Environment; Enzymatic activity; Enzyme activity; Enzyme immobilization; Enzymes; Fragments; Libraries; Lignin; Lignin peroxidase; Lysis; Mutagenesis; Mutants; Oxidation; Oxidoreductase; Peroxidase; Protein engineering; Research Article; Saturation mutagenesis; Toluene; Yeast; Yeast surface display; Yeasts
• ISSN: 2095-2201; 2095-221X
• DOI: 10.1007/s11783-020-1311-4

* Mutations in Lignin peroxidase Trp171 environment improved azo dyes degradation. * Expression on yeast cell surface and cell lysis allowed reusability of biocatalyst. * Aga2-LiP chimeric variants were characterized. The enzymatic degradation of azo dyes is a promising alternative to ineffective chemical and physical remediation methods. Lignin peroxidase (LiP) from Phanerochaete chrysosporium is a heme-containing lignin-degrading oxidoreductase that catalyzes the peroxide-dependent oxidation of diverse molecules, including industrial dyes. This enzyme is therefore ideal as a starting point for protein engineering. Accordingly, we subjected two positions (165 and 264) in the environment of the catalytic Trp171 residue to saturation mutagenesis, and the resulting library of 10 4 independent clones was expressed on the surface of yeast cells. This yeast display library was used for the selection of variants with the ability to break down structurally-distinct azo dyes more efficiently. We identified mutants with up to 10-fold greater affinity than wild-type LiP for three diverse azo dyes (Evans blue, amido black 10B and Guinea green) and up to 13-fold higher catalytic activity. Additionally, cell wall fragments displaying mutant LiP enzymes were prepared by toluene-induced cell lysis, achieving significant increases in both enzyme activity and stability compared to a whole-cell biocatalyst. LiP-coated cell wall fragments retained their initial dye degradation activity after 10 reaction cycles each lasting 8 h. The best-performing mutants removed up to 2.5-fold more of each dye than the wild-type LiP in multiple reaction cycles.