# Vers l'ingéniérie des laccases pour une application environnementale

Claude Jolivalt, Pierre Briozzo, Thomas Bertrand, Catherine Madzak, Eliane Caminade, Christian Mougin

<figure><img src="image_1.png" /></figure>

## To cite this version:

Claude Jolivalt, Pierre Briozzo, Thomas Bertrand, Catherine Madzak, Eliane Caminade, et al. Vers l'ingéniérie des laccases pour une application environnementale. 2nd Séminaire d'Ecotoxicologie de l'INRA, Sep 2002, Antibes, France. 2002. [hal-01600163](https://hal.science/hal-01600163)

HAL Id: hal-01600163

https://hal.science/hal-01600163

Submitted on 7 Jun 2020

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License
# Fungal laccases: from structure-activity studies to environmental applications

<figure><img src="image_3.png" /></figure>

CLAUDE JOLIVALT¹, THOMAS BERTRAND², PIERRE BRIOZZO³, CATHERINE MADZAK⁴, AGATHE BRAULT⁵, NATHALIE JOLY³, ELIANE CAMINADE⁵, ALBERT KOLLMANN⁵, ISABELLE TOUTON⁵, CHRISTIAN MOugin⁵

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

¹Laboratoire de Synthèse Sélective et Produits Naturels, UMR CNRS 7573, ENSCP, 11 rue P. et M. Curie, 75231 Paris Cedex 05,
²Laboratoire d'Enzymologie et Biochimie Structurales, UPR CNRS 9063, 91198 Gif-sur-Yvette Cedex,
³Laboratoire de Chimie Biologique, UMR 206 INRA-INAPG, 78850 Thiverval-Grignon,
⁴Laboratoire de Génétique Moléculaire et Cellulaire, INRA-INAPG, 78850 Thiverval-Grignon,
⁵Unité de Phytopharmacie et Médiateurs Chimiques, route de Saint-Cyr, INRA, 78026 Versailles Cedex, France,
E-mail: mougin@versailles.inra.fr

## INTRODUCTION

- Fungal laccases (EC 1.10.3.2) are a group of multicopper oxidoreductive enzymes involved in lignin transformation, morphogenesis, pathogenesis and fungal virulence, as well as in the oxidation of organic compounds

- Laccases are widely used in several processes of the dye, paper (pulp bleaching) and textile industries

- A wide range of their substrates are of environmental interest, as recognized pollutants of waters and soils

- Structure-activity studies remain necessary today to highlight the high potential of laccases for environmental applications

**Our objective** is a better knowledge concerning the structural and catalytic properties of fungal laccases, as well as the mechanisms involved in the regulation of their expression, as the first step to their optimized applications

## AREAS OF RESEARCH

### 1 - Involvement of fungal laccases in the biotransformation of xenobiotics[1-3]

- Fungal laccases are involved in the metabolism of pesticides and polycyclic aromatic compounds

- According to their chemical structures, the enzymes catalyze oxidation reactions leading either to the breakdown of the substrates or to their coupling (polymerization)

- As a consequence, fungal laccases contribute to the fate of xenobiotics in the environment

### 2 - Structural data of the laccase IIIB secreted by the basidiomycete Trametes versicolor [4]

The crystal structure of the protein has been obtained with 2,5-xylidine bound in its active site

**Global fold of LacIIIB**

(atomic coordinates: pdb accession number 1KYA)
● , copper atoms; ● and ○ , glycosylation sites

<figure><img src="image_7.png" /></figure>

**T1 site and neighboring
2,5-xylidine ligand**

<figure><img src="image_8.png" /></figure>

Two charged or polar residue interact with the amino group of the xylidine:

- His (H458) coordinates the copper that functions as the primary electron acceptor

- Asp (D206) possesses acido-basic properties that could be responsible for the pH-dependent activity towards substrates

### 3 - Electrochemical studies to determine the catalytic cycle of the laccase in complex solutions[3]

- In some cases, substrate oxidation by laccases needs the presence of a redox mediator (ABTS)

- In complex media, the reaction can be disturbed by compounds such as metals

- This fact strengthens the development of recombinant enzymes with a high redox potential avoiding the use of mediator

### 4 - Molecular basis of laccase expression [5]

- The gene has been cloned in *Escherichia coli* using the pGEM®-T plasmid

- It has been sequenced and comprises 1563 bases (sequence accession number AF414109), including a peptide signal

- It was then expressed in the yeast *Yarrowia lipolytica* using the pINA1269 vector (Patent INRA/INA-PG), thus allowing the production of an active form of the recombinant enzyme

These results allow to develop a strategy of directed mutagenesis to design laccases with:

- a high efficiency in catalysis (high redox potential)

- a wider variety of substrates

- a pH of activity optimized for natural media

Additional studies need to be performed with other xenobiotics bound in the active site

**Laccase-mediated oxidation of b[a]p**

<figure><img src="image_9.png" /></figure>

## ENVIRONMENTAL APPLICATIONS

### 1 - Laccases as tools for bioremediation [1]

Laccases immobilized on a microfiltration membrane are able to reduce the amounts of phenylureas in a reactor filled with contaminated water

<figure><img src="image_10.png" /></figure>

### 2 - Laccases as biomarkers to assess the impact of chemicals [6]

Liquid cultures of *Trametes versicolor* have been treated with various xenobiotics (agrochemicals, industrial compounds) at 0.5 mM. Laccase activity is measured after 2 and 3 days of incubation

<figure><img src="image_11.png" /></figure>

**References**

[1] C. Jolivalt, S. Brenon, E. Caminade, C. Mougin, M. Pontié (2000), J. Membrane Sci. 180:103-113.

[2] C. Mougin, F.-D. Boyer, E. Caminade and R. Rama (2000), J. Agric. Food Chem. 48:4529-4534.

[3] C. Mougin, C. Jolivalt, M. Malosse, J.-C. Sigoillot J.-C., M. Asther and V. Chaplain (2002), Polycycl. Aromat. Comp. 22:673-688.

[4] T. Bertrand, C. Jolivalt, P. Briozzo, E. Caminade, N. Joly, C. Madzak and C. Mougin (2002), Biochemistry 41:7325-7333

[5] C. Jolivalt, C. Madzak and C. Mougin (2003) (in preparation).

[6] C. Mougin, A. Kollmann and C. Jolivalt (2002), Biotechnology Letters 24:139-142.