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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

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Abstract

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 104 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.

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Acknowledgements

We thank Professor Dane Wittrup, Massachusetts Institute of Technology, for providing the pCTCON2 vector and S. cerevisiae EBY100 competent cells, and Dr. Helga Schinkel, Fraunhofer IME (Aachen, Germany) for valuable discussions. This work was supported by funds from the Ministry of Education, Science and Technological Development of the Republic of Serbia via project numbers ON172049, ON173017 and III46010.

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Authors and Affiliations

  1. University of Belgrade-Faculty of Chemistry, Studentski trg 12–16, 11000, Belgrade, Serbia

    Karla Ilić Đurđić, Aleksandra Đurđević Đelmaš, Nikolina Popović & Radivoje Prodanović

  2. Molecular Evolution Protein Engineering and Production facility (MEPEP), Purdue University, West Lafayette, IN, 47907, USA

    Raluca Ostafe

  3. Institute of Molecular Biotechnology, RWTH Aachen University, 52074, Aachen, Germany

    Raluca Ostafe & Rainer Fischer

  4. Institute for Multidisciplinary Research, Kneza Višeslava 1, 11030, Belgrade, Serbia

    Olivera Prodanović

  5. Departments of Biological Sciences and Chemistry, Purdue University, West Lafayette, IN, 47907, USA

    Rainer Fischer

  6. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, 52074, Aachen, Germany

    Stefan Schillberg

Authors
  1. Karla Ilić Đurđić
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  2. Raluca Ostafe
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  3. Olivera Prodanović
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  4. Aleksandra Đurđević Đelmaš
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  5. Nikolina Popović
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  6. Rainer Fischer
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  7. Stefan Schillberg
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  8. Radivoje Prodanović
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Corresponding author

Correspondence to Radivoje Prodanović.

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Highlights

• 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.

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Ilić Đurđić, K., Ostafe, R., Prodanović, O. et al. 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. Front. Environ. Sci. Eng. 15, 19 (2021). https://doi.org/10.1007/s11783-020-1311-4

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  • DOI: https://doi.org/10.1007/s11783-020-1311-4

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