# Interference of soil contaminants with laccase activity during the transformation of complex mixtures of polycyclic aromatic hydrocarbons (PAH) in liquid media

Christian Mougin, Claude Jolivalt, Christian Malosse, Jean-Claude Sigoillot, Marcel Asther, Veronique Chaplain

► To cite this version:

Christian Mougin, Claude Jolivalt, Christian Malosse, Jean-Claude Sigoillot, Marcel Asther, et al. Interference of soil contaminants with laccase activity during the transformation of complex mixtures of polycyclic aromatic hydrocarbons (PAH) in liquid media. 18th International Symposium on Polycyclic Aromatic Compounds, Sep 2001, Cincinnati, United States. 2001. hal-01600210

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Submitted on 4 Jun 2020

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# Interference of soil contaminants with laccase activity during the transformation of complex mixtures of polycyclic aromatic hydrocarbons in liquid media

C. Mougin¹, C. Jolivalt¹, C. Malosse¹, J.-C. Sigoillot², M. Asther² and V. Chaplain¹

¹Unité de Phytopharmacie et Médiateurs Chimiques
Route de Saint-Cyr, F-78026 Versailles Cedex
email: mougin@versailles.inra.fr

²Unité de Biotechnologie des Champignons Filamenteux
163, avenue de Luminy, F-13288 Marseille Cedex 09

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INRA
# Introduction

Laccase-producing fungi are often presented as efficient tools for the bioremediation of soils and liquid wastes polluted by PAHs, but the efficiency of the enzymes has not been clearly demonstrated

**Aims of the study:**

* to determine the efficiency of fungal laccases to transform synthetic mixtures of PAHs

* to assay their use for the transformation of PAHs extracted from polluted soils

* to evidence the interference of contaminants co-extracted from the polluted soils with PAH transformation
## Properties of the fungal laccases

* Distribution: fungi, bacteria, higher plants, insects

* Function: lignin degradation, morphogenesis, pathogenesis transformation of xenobiotics

* Structure: dimeric or tetrameric glycoprotein, with 4 Cu, exocellular, 520 to 550 aa, 60 to 80 kDa secreting a N-terminal peptide

* Regulation:
  inhibitors: small anions, metals, fatty acids, chelating agents
  inducers: culture medium, lignin and related compounds, xenobiotics

* Equation: $2RH + \frac{1}{2}O_2 \rightarrow 2R^\cdot + H_2O$
Main catalyzed reactions

* RH = Phenolic compound oxidation, bond cleavage: C-C, C-N,...

$$ \text{HO-phenyl-NH-C(=O)N-} \rightarrow \text{O-phenyl-O} \quad \text{ou} \quad \text{O-phenyl-N=N(C(=O)N)-} $$

$$ \text{4-tert-butylphenol} + \text{HO}_2\text{C}6H}_4\text{CH}_3 \rightarrow \text{4-tert-butylphenol CH}_2\text{OC}_6H}_4\text{CH}_3 $$
* RH = non phenolic compound

$$ \rightarrow \text{ a redox mediator is needed: } \mathrm{Lac} + \mathrm{Med} \rightarrow \mathrm{Med}^* \\ \mathrm{Med}^* + \mathrm{RH} \rightarrow \mathrm{Rh}_{\mathrm{ox}} + \mathrm{Med} $$

<figure><img src="image_3.png" /><figcaption>2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid): ABTS</figcaption></figure>

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Experimental

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

2-liter bioreactors containing:

• 1.5 L citrate/phosphate buffer pH 3, 0.1 M

• 20 mL Tween 20

• 360 mg PAHs dissolved in 500 mL acetonitrile

• 2 mM ABTS (redox mediator)

• 4000 U laccase of Pycnoporus cinnabarinus

incubated in the dark at 30°C for 48 hours
Transformation of PAHs

<figure><img src="image_6.png" /><figcaption>Soil extract</figcaption></figure>

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

• mixture, ■ anthracene, ♦ benzo[a]pyrene, -lac, +lac
<table><thead><tr><th rowspan="2">Common names</th><th colspan="2">Amounts</th><th rowspan="2">Transformation products</th></tr><tr><th>Initial (mg L<sup>-1</sup>)</th><th>After 48 h (%)</th></tr></thead><tbody><tr><td><strong>Naphtalene</strong></td><td><strong>10.0</strong></td><td><strong>1.2</strong></td><td></td></tr><tr><td>Acenaphthylene</td><td>4.0</td><td>31.3</td><td>1,8-naphthalic acid anhydride</td></tr><tr><td><strong>Acenaphthene</strong></td><td><strong>3.0</strong></td><td><strong>17.4</strong></td><td>1,8-naphthalic acid anhydride</td></tr><tr><td>Fluorene</td><td>5.0</td><td>53.7</td><td>9-fluorenone</td></tr><tr><td>Phenanthrene</td><td>25.0</td><td>57.2</td><td>9,10-phenanthrene dione</td></tr><tr><td><strong>Anthracene</strong></td><td><strong>17.0</strong></td><td><strong>4.3</strong></td><td>9,10-anthracene dione</td></tr><tr><td>Fluoranthene</td><td>22.0</td><td>59.3</td><td></td></tr><tr><td>Pyrene</td><td>20.0</td><td>57.3</td><td>1,6- 1,8-pyrene diones</td></tr><tr><td>Benzo[a]anthracene</td><td>9.0</td><td>68.6</td><td>7,12-benzo[a]anthracene dione</td></tr><tr><td><strong>Chrysene</strong></td><td><strong>5.0</strong></td><td><strong>95.6</strong></td><td></td></tr><tr><td>Benzo[b]fluoranthene</td><td>17.0</td><td>63.5</td><td></td></tr><tr><td><strong>Benzo[k]fluoranthene</strong></td><td><strong>15.0</strong></td><td><strong>99.3</strong></td><td></td></tr><tr><td><strong>Benzo[a]pyrene</strong></td><td><strong>21.0</strong></td><td><strong>16.5</strong></td><td>1,6- 3,6- 6,12-benzo[a]pyrene diones</td></tr><tr><td>Dibenzo[ah]anthracene</td><td>1.0</td><td>73.7</td><td></td></tr><tr><td><strong>Benzo[ghi]perylene</strong></td><td><strong>1.0</strong></td><td><strong>80.0</strong></td><td></td></tr><tr><td>Indeno[123cd]pyrene</td><td>3.0</td><td>71.8</td><td></td></tr><tr><td>16 PAHs</td><td>178.0</td><td>49.7</td><td></td></tr></tbody></table>
Mechanism of laccase-mediated oxidation of BaP

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

2) $ABTS^{2+} + ABTS \rightarrow 2 ABTS^{\circ+}$
Effect of K₄Fe(CN)₆ on BaP oxidation by ABTS⁺⁺

2 mL 0.1 M citrate-phosphate buffer pH 3: 5 mM peroxodisulfate
0.1 mM ABTS, 0.1 mM BaP, 0.075 to 0.375 mM K₄Fe(CN)₆

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

* fungal laccases are able to transform 10 PAHs in a synthetic mixture of 16 compounds. The reaction needs the presence of a redox mediator

* metals, present in polluted soils, inhibit PAH transformation in withdrawing the electrons delivered by the mediator

* as the inhibition by metals is limited in time, other unknown mechanisms may explain the lack of activity of the laccases

→ Are laccases really efficient in polluted soils?
How can we enhance their potential?
Perspectives

* the results presented here have been obtained by using purified laccases from *P. cinnabarinus*. Similar data have been obtained later with enzymes from *T. versicolor*

* we have now cloned and sequenced a gene encoding for a laccase of *T. versicolor*

* we have expressed it in *E. coli*, soon in the yeast *Yarrowia lipolytica*

→ the directed evolution of the enzyme should also be performed