# Exocellular oxidases: tools to assess the exposure of soil fungi to xenobiotics?

Claude Jolivalt, Nathalie Demont-Caulet, Isabelle Touton, Agathe Brault, Albert Kollmann, Christian Mougin

► To cite this version:

Claude Jolivalt, Nathalie Demont-Caulet, Isabelle Touton, Agathe Brault, Albert Kollmann, et al. Exocellular oxidases: tools to assess the exposure of soil fungi to xenobiotics?. 4th International Symposium on Environmental Aspects of Pesticide Microbiology, SETAC, Sep 2004, Thessaloniki, Greece. 2004. [hal-01600787](https://hal.science/hal-01600787)

HAL Id: hal-01600787

https://hal.science/hal-01600787

Submitted on 4 Jun 2020

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Exocellular oxidases

*Tools to assess the exposure of soil fungi
to xenobiotics?*

Claude Jolivalt,
Biochemistry, UMR CNRS 7573, ENSCP Paris

Nathalie Demont-Caulet
Cellular Biology, INRA Versailles / Univ.-Paris VII

Isabelle Touton, Agathe Brault, Albert Kollmann, Christian Mougin
Xenobiotics and Environment, INRA Versailles
Structural and metabolic biochemistry
structural data, regulation of expression, enzyme evolution

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Biocatalysis,
bioconversion, biotransformation

Fate and impacts of
xenobiotics in soils

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bioremediation, ecotoxicology
The aims of the present project

1 - Improvement of the knowledge on fungal laccases

2 - Development of tools to assess the adverse effects of pollutants on soil ecosystems

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* Effect of xenobiotics on laccase expression and activity in liquid cultures (identification of biomarkers)

* Effect of xenobiotics on laccase expression and activity in soils inoculated with fungi (validation of biomarkers)

* Effect of xenobiotics on endogenous laccases of soils
## Ecological relevance

* Provide one of the highest biomass in the soil

* Initiate numerous trophic chains

## Functional relevance

* Responsible of the breakdown and mineralization of organic matter

* Catalyze the biotransformation of numerous xenobiotics

## Practical relevance

* Secrete exocellular oxidases: peroxidases, laccases
# The main characteristics of laccases

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oxidases mainly produced by fungi (Trametes versicolor)

and also by higher plants (Rhus vernicifera)

insects (Bombyx)

bacteria (Azospirillum lipoferum)

extracellular glycoproteins containing copper

520 to 550 aa, 60 to 80 kD

high redox potential: 0.7 V, very robust

reducing substrate

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# Functions and uses of fungal laccases

## 1 - Natural

* biodegradation of ligno-cellulosic materials

* morphogenesis, pathogenesis....

## 2 - Industrial

* textile and food industry

* pulp paper bleaching

## 3 - Environmental

* biotransformation of xenobiotics, bioremediation

* biomarkers for the assessment of adverse effects of chemicals ???
The laccases of T. versicolor

2 families of isozymes

* type A: N-terminale sequence GIGP constitutive and <u>induced</u> gene AF 414109

► lac A

* type B: N-terminale sequence AIGP constitutive, could be induced ??? gene U44430

► lac B
Effect of xenobiotics on laccase activity

* Treatment of 4-day liquid cultures of *T. versicolor* by xenobiotics

* Measurement of laccase activity after 2 and/or 3 days (ABTS)

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Screening for chemicals efficient in increasing laccase activity

Dose/response relationships
Laccase activity after 2 (●) and 3 (●) days of treatment

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Chemical concentration: 500 µM
Laccase activity after 3 days of treatment with phenylureas

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Chemical concentration: 200 µM
Laccase activity after 3 days of treatment with nonylphenols

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Effect of xenobiotics on the expression of laccase genes

Treatment of 4-day liquid cultures of *T. versicolor* with xenobiotics

* Measurement of laccase activity (ABTS)

* Extraction of mRNAs (RNeasy Mini Kit Qiagen)

* Synthesis of cDNAs by RT-PCR (Advantage RT Clontech)

* relative quantification of genes by real time PCR
  (Thermocycler Roche, ref: TEF1, gene X94913)

* detection by fluorescence (SYBR Green I)

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quantification of gene/activity expression

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Chemical concentration: 100 µM
Xylidine: dose-response relationship, lac A

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Can lac B be induced by xenobiotics?

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Chemical concentration: 100 µM

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# Effect of xenobiotics on the production of laccase proteins

* Treatment of 4-day liquid cultures of *T. versicolor* with xenobiotics

* Separation of the extracellular fluid

* Precipitation of polysaccharidic compounds with 10% acetone

* Diafiltration of the protein solution (cut off 10kD, Amicon)

* Purification on Concanavalin A-Sepharose 4B column (Sigma-Aldrich) elution with 0.2M Tris Hcl in the presence 0.5M NaCl and 0.2M o-methyl glucose

* separation by SDS-Page (12%)

* Identification of proteins by Maldi-TOF (Bruker)

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profiles of proteins
# Separation and identification of lac A isoforms

1st migration
mass

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2nd migration
pl

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Maldi-TOF

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MW Da
58463
59776

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Conclusions

* Laccase activity can be increased following fungal exposure to environmental pollutants

* Lac A (but not lac B) are induced by chemicals

* mRNA measurement is highly specific and well related to enzymatic activities, but the response is not sensitive enough to be useful (induction by 15-20)

* the proteomic approach should be developed

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Could fungal laccases be used for point contamination (heavy pollution) assessment?
Perspectives

* Understand the effects of xenobiotics at the transcriptional, traductional and post-traductional levels

* Assess the effects of metals

* Develop tools to inoculate fungi into soils and perform assays in soil

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Acknowledgements

Bertrand Dubreucq, Cellular Biology, INRA Versailles

Christian Malosse, Phytopharmacy, INRA Versailles

Agence De l'Environnement et de la Maitrise de L'Energie
(grant n° 0275048)

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www-phyto.versailles.inra.fr