# Fungal laccases: from structure-activity studies to environmental applications

Claude Jolivalt, Pierre Briozzo, Catherine Madzak, Christian Mougin

► To cite this version:

Claude Jolivalt, Pierre Briozzo, Catherine Madzak, Christian Mougin. Fungal laccases: from structure-activity studies to environmental applications. 3rd European Meeting on Environmental Chemistry, Dec 2002, Genève, Switzerland. 2002. [hal-01601673](https://hal.science/hal-01601673).

HAL Id: hal-01601673

https://hal.science/hal-01601673

Submitted on 4 Jun 2020

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished 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_1.png" /></figure>

Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License
Fungal laccases

from structure-activity studies
to environmental applications

Claude Jolivalt, Pierre Briozzo, Catherine Madzak, Christian Mougin
What are laccases ?

oxidases produced by plants (Rhus vernicifera)

insects (Bombyx)

bacteria (Azospirillum lipoferum)

fungi (Trametes versicolor)

extracellular glycoproteins containing copper

520 to 550 aa, 60 to 80 kD

high redox potential: 0,7 V

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

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

<figure><img src="image_4.png" /></figure>
# Functions of fungal laccases

1 - natural

biodegradation of ligno-cellulosic materials
morphogenesis, pathogenesis....

2 - industrial

textile, dye and food industry
pulp paper bleaching

3 - environmental

biotransformation of xenobiotics (pollutants)

→ an increasing interest
# Direct oxidation: phenols, amines

* **oxidation of phenylurea herbicides**

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

Jolivalt C., Raynal A., Caminade E., Kokel B., le Goffic F., Mougin C., Appl. Microbiol. Biotechnol, 1999, 51:676-681

* **oxydative coupling of nonylphenol**

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

Dubroca J., Brault A., Kollmann A., Touton I., Jolivalt C., Kerhoas L., Mougin C., Springer Book, 2003 (in press)
Indirect oxidation

a redox mediator is needed:

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

$$ 
\begin{aligned}
\text{Lac} + \text{Med} &\rightarrow \text{Med}^* \\
\text{Med}^* + \text{RH} &\rightarrow \text{R}_{\text{ox}} + \text{Med}
\end{aligned}
 $$

2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid): ABTS

benzo[a]pyrene oxidation

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

Rama R., Mougin C., Boyer F.-D., Kollmann A., Malosse C., Sigoillot J.-C., Biotechnol. Letters, 1998, 20:1101-1104
# Our objectives

biotransformation mechanisms, development of tools

1

bioremediation of
polluted media

2

assessment of
ecotoxicological impact

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

studies concerning fungal laccases

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

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

structure (mode of action) gene expression
Crystal structure of the laccase from *T. versicolor*

<figure><img src="image_12.png" /></figure>

●, copper atoms

○, glycosylation sites

* the laccase is active and contains
  4 copper atoms

* a reducing substrate is bound to
  the active site (presence of a cavity)
T1 site and neighbouring of 2,5-xylidine

<figure><img src="image_13.png" /></figure>

Two aminoacids interact with the amino
group of the xylidine:

* D206, an acidic residue responsible of pH impact on the transformation rate of xenobiotics?

* H458, an electron acceptor
# Expression of the fungal laccase in the yeast *Yarrowia lipolytica*

* the gene encoding the laccase has been cloned and sequenced (Genbank AF414109), then expressed in *E. coli* and *Y. lipolytica*

<figure><img src="image_14.png" /></figure>

Y. lipolytica T-
transformants 10

8

* the recombinant enzyme is characterized by Maldi-TOF

<figure><img src="image_15.png" /></figure>

T. versicolor
4

16
# Site-directed mutation in the *T. versicolor* laccase

- wild enzyme

- **avantages**

- **limits**

- robust, extracellular

- phenols, amines

- activity

- redox mediator

- acidic pH

- recombinant enzyme

- **expected benefits**

- conserved properties

- wider substrate specifity

- increased $k_{cat}$

- increased $E^o$

- neutral pH

## 2 - vector preparation

<figure><img src="image_16.png" /></figure>

## 3 - ligation

## 1 - insert design for modified T1 site

<figure><img src="image_17.png" /></figure>

4 - expression in *E. coli* → **3 mutants**

5 - expression in *Y. lipolytica*

6 - protein analysis
A protein-grafted membrane to remove pollutants from waters

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

INRA

Institut National de la Recherche Agronomique
Laccase degradation efficiency

<figure><img src="image_20.png" /></figure>

■ Removal efficiency of 2-HF in the filtrate increases with the amount of grafted laccases

Filtration module: Minitan S
120 cm² planar membranes grafted with 4.4, 10, 50 and 220 laccase activity units.
2-HF initial concentration 0.1 g/L in acetate buffer 0.05 M, pH 5 T=22°C.
# Assessment of ecotoxicological impact on soil organisms

→ induction of fungal laccases by xenobiotics

treatment of 4-days cultures of *T. versicolor* by xenobiotics at 0.5 mM
measurement of laccase activity after 2 (●) and 3 (■) days

<figure><img src="image_21.png" /></figure>

Mougin C., Kollmann A., Jolivalt C., Biotechnol. Letters, 2002, 24:139-142
# Assessment of ecotoxicological impact on soil organisms

## Towards fungal genomics for applied environmental microbiology?

1 - measurement of enzymatic activity: some problems

2 - expression of gene encoding laccase

3 - expression of genes encoding exoenzymes (Lac, MnP, LiP, CdH...)

4 - expression of genes involved in metabolic pathways and cellular cycles

→ transcriptomics (gene profiling, DNA microarray) proteomics
Acknowledgments

V. Chaplain

Metabolic biochemistry and microbiology
C. Jolivalt, E. Caminade, A. Kollmann, A. Brault, I. Touton

Structural biochemistry and molecular biology
P. Briozzo, N. Joly, T. Bertrand, C. Madzak

Organic chemistry and mass spectrometry
C. Malosse, L. Kerhoas, F.-D. Boyer, P.-H. Ducrot, B. Kokel, J.-P. le Caer

Reactor
M. Pontié

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>