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

doi:10.1007/s11783-020-1311-4

Front. Environ. Sci. Eng.

2021, Vol. 15

Issue (2) : 19 https://doi.org/10.1007/s11783-020-1311-4

RESEARCH ARTICLE

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

Karla Ilić Đurđić¹, Raluca Ostafe^2,³, Olivera Prodanović⁴, Aleksandra Đurđević Đelmaš¹, Nikolina Popović¹, Rainer Fischer^3,⁵, Stefan Schillberg⁶, Radivoje Prodanović¹(

)

¹. University of Belgrade-Faculty of Chemistry, Studentski trg 12-16, 11000 Belgrade, Serbia
². Molecular Evolution Protein Engineering and Production facility (MEPEP), Purdue University, West Lafayette, IN 47907, USA
³. Institute of Molecular Biotechnology, RWTH Aachen University, 52074 Aachen, Germany
⁴. Institute for Multidisciplinary Research, Kneza Višeslava 1, 11030 Belgrade, Serbia
⁵. Departments of Biological Sciences and Chemistry, Purdue University, West Lafayette, IN 47907, USA
⁶. Fraunhofer Institute for Molecular Biology and Applied Ecology IME, 52074 Aachen, Germany

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Abstract

• 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 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.

Keywords Bioremediation Enzyme immobilization Protein engineering Yeast surface display.

Corresponding Author(s): Radivoje Prodanović

Issue Date: 11 August 2020

Cite this article:

Karla Ilić Đurđić,Raluca Ostafe,Olivera Prodanović, 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[J]. Front. Environ. Sci. Eng., 2021, 15(2): 19.

URL:

https://academic.hep.com.cn/fese/EN/10.1007/s11783-020-1311-4
https://academic.hep.com.cn/fese/EN/Y2021/V15/I2/19

Fig.1 Overview of the experimental workflow. (A) Following the insertion of the synthetic LiP gene into vector pCTCON2, the wtLiP-pCTCON2 construct was subjected to saturation mutagenesis and the resulting library expressed on the surface of yeast cells was screened for the ability to degrade azo dyes. The best-performing variants were characterized and sequenced. (B) Cell walls displaying wild-type LiP or its variants were tested in multiple cycles of azo dye degradation. Washed cell walls were incubated in a mixture of dye, H₂O₂ and veratryl alcohol for 8 h before recovery, washing and replenishment of the reaction mix. The incubation and washing steps were carried out 10 times.

Fig.2 Structure of the tryptophan environment. (A) Wild-type LiP. (B) Versatile peroxidase (VP). E= Glu, S= Ser, D= Asp, W= Trp, R= Arg. Atomic coordinates for VP were sourced from PDB file 2BOQ (^{Pérez-Boada et al., 2005}) and those for LiP were sourced from PDB file 1B82 (^{Blodig et al., 2001}).

Fig.3 Dye degradation profiles of the LiP saturation library. We tested the degradation activities of 900 randomly picked yeast colonies expressing LiP variants on the cell surface after transferring single colonies to the wells of 10 96-well microtiter plates. The remaining wells were used for the wild-type LiP and empty-vector controls. (A) Evans blue degradation profile. (B) Amido black 10B degradation profile. (C) Guinea green degradation profile.

Tab.1 Amino acids at positions 165 and 264 for wild-type LiP and 10 selected variants

Tab.2 Kinetic parameters of Aga2 fusion proteins with wild-type LiP and selected variants against three different azo dyes.

Fig.4 Multiple cycles of dye degradation by wild-type LiP and selected variants. (A) Ten cycles of Evans blue degradation by wild-type LiP (blue), ML3 (orange) and ML8 (gray) using LiP-coated yeast cell walls. (B) Ten cycles of amido black 10B degradation by wild-type LiP (blue), ML2 (orange) and ML6 (gray) using LiP-coated yeast cell walls. (C) Ten cycles of Guinea green degradation by wild-type LiP (blue), ML3 (orange) and ML9 (gray) using LiP-coated yeast cell walls. Data are means of triplicate experiments with error bars indicating standard deviations. Error bars are not visible when smaller than the symbol size.

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