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Laccases (p-diphenol:dioxygen oxidoreductases; EC 1.10.3.2) have a number of applications in the field of the food industry. These oxidative enzymes are mainly produced by white-rot fungi in copious amounts and are characterized by high substrate versatility. In addition to lignin, their natural substrate, laccases may catalyze oxidative transformation of several substrates including phenols, substituted phenols, aromatic acids, aromatic and nonaromatic alcohols, aromatic aldehydes, aromatic amines, anilines, dyes, lignin derivatives, polyaromatic hydrocarbons (PAHs), quinones, and thiols residues.Many of these components are present in various foods and beverages and their modification may result in quality upgrading: wine and beer stabilization, decreased interaction between proteins and polyphenols during fruit juice processing, increased machinability of dough, and so forth. Laccases could also be used to eliminate O2 from packaging or dissolved O2 to control odors and enhance taste of food.Relatively well documented in patent literature, laccases are described as potential actors for a large range of applications in industry. Yet current implementations in food industry remain limited due to high laccase production cost and poor control of the enzymatic reaction byproducts. However, new developments, as immobilization of laccases in nanostructured materials, might provide renewed perspectives for food industry., info:eu-repo/semantics/published

Since recent years, pollution of surface and drinking waters by chemical pollutants fromanthropogenic activities, in particular micropollutants, has become an important environmentalissue in industrialized countries. Indeed the majority of these molecules are toxic toliving organisms, even present at very low concentrations (pg.L−1 to μg.L−1). Some of themare referred as endocrine disrupting compounds. The existing conventional wastewater treatmentsare most of the time not adapted for the removal of these compounds. Among them,advanced oxidative processes give the better removal efficiencies, but with drawbacks such ashigh energetic costs, or the formation of reaction products, sometimes more toxic than theparent compounds. A promising solution for water decontamination could be bioremediation.This work is based on this technology, via the study of the use of lignolytic enzymes, such aslaccases, for the removal of micropollutants. Degradation studies of a lot of micropollutants(bisphenol A, nonylphenol, triclosan, 17-_-estradiol, etc.) have already been investigatedwith different reactor configurations, using free (solubilized) or immobilized enzymes (biocatalysts).However, a decrease in the performance of some continuous long-term degradationprocesses has been highlighted, most of the cases because of biocatalysts deactivation. Oneof the reasons to explain this deactivation was identified as being induced by the deposit ofreaction products onto the surface of the biocatalysts.The general objective of this work was to investigate the degradation of bisphenol A(BPA) by laccases oxidation in different reactor configurations. Especially, the focus wasmade on the study of BPA degradation and reaction products kinetics, and reaction productscharacterization and identification. In the continuity, a second objective was related tothe optimization of a packed-bed reactor developed in our laboratory, through the developmentof a new biocatalyst, and assessment of biocatalyst, Depuis plusieurs années, la pollution des eaux potable et de surface par des polluants chimiquesprovenant d’activités anthropogéniques, en particulier les micropolluants, est devenueune problématique environnementale importante dans les pays industrialisés. En effet, la majoritéde ces substances sont toxiques pour les organismes vivants, même présentes en trèsfaibles concentrations (de l’ordre du pg.L−1 au μg.L−1). Un grand nombre de ces substancessont considérées comme des perturbateurs endocriniens. Les techniques conventionnelles actuellesde traitement des eaux usées ne sont la plupart du temps pas adaptées à l’abattementde ces substances. Parmi ces techniques, les procédés d’oxydation avancée sont ceux quidonnent les meilleurs rendements d’abattement. Cependant, ils présentent certains inconvénients,incluant des coûts énergétiques importants, et la formation de produits de réactionparfois plus toxiques que les molécules de départ. La bioremédiation pourrait constituer unesolution prometteuse. Ce travail est basé sur cette idée, au travers de l’étude de l’utilisationd’enzymes lignolytiques, comme les laccases, pour la dégradation des micropolluants. Desétudes sur la dégradation de nombreux micropolluants (bisphénol A, nonylphénol, triclosan,17-_-estradiol, etc.) ont déjà été menées pour différentes configurations de réacteurs, avecl’usage d’enzymes libres (solubilisées) ou immobilisées (biocatalyseurs). Cependant, à longterme, une diminution des performances de dégradation lors de certains procédés continusa été mise en évidence, la plupart du temps à cause d’une désactivation des biocatalyseurs.Une raison possible pouvant expliquer cette désactivation réside dans le dépôt de produits deréaction sur la surface des biocatalyseurs.L’objectif général de ce travail a été d’étudier la dégradation d’un micropolluant, le bisphénolA (BPA), via oxydation par les laccases, dans différentes configurations de réacteurs.Plus précisément, nous nous sommes intéressés à l’étude, Doctorat en Sciences de l'ingénieur et technologie, info:eu-repo/semantics/nonPublished

Methods of monitoring of estrogenicity in water were gathered, compared, and tested within thecontext of their practical use as measurement and design tools, in the development of a process ofdegradation of estrogenic endocrine disruptors. In this work, the focus was put on in vitro assays,with the use of analytical techniques as additional analysis when possible. Practically, from aliterature review, four methods that seemed most suitable to practical use required in a processdevelopment were tested: the Yeast Estrogen Screen assay, the Lyticase-assisted Yeast EstrogenScreen assay (LYES), the MMV-LUC assay and the HPLC-UV analytical method. Dose–responsecurves in response to estrogenic standard 17β-estradiol were compared. Bisphenol Aestrogenicity was measured by the methods as well. The model for the calculation of estradiolequivalents as measurements units was adapted. The methods were assessed in terms of rangesof detection, time of experiment, cost, ease of the experiment, reproducibility, etc. Based on thatassessment, the LYES assay was selected and successfully applied to the monitoring ofestrogenicity removal from 17β-estradiol and bisphenol A. More precisely, the bioassay allowedthe acquisition of kinetic curves for a laboratory-scaled process of estrogenicity removal byimmobilized enzymes in a continuous packed-bed reactor. The LYES assay was found to have areal methodological potential for scale-up and design of a treatment process. The HPLC-UVmethod showed good complementarity with the LYES assay for the monitoring of bisphenol Aconcentrations in parallel with estrogenicity, reporting no significant estrogenicity fromdegradation byproducts, among others., SCOPUS: ar.j, info:eu-repo/semantics/published

The optimization of culture conditions for high yield laccase production by white rot fungi has been extensively studied. However, to achieve short time laccase production remains a major challenge in several cases. The present study investigated an optimal process for production of Coriolopsis gallica 1184 laccase in a high yield of 200 900 Ul-1 in 7 d by 50 L scale submerged fermentation. Coriolopsis gallica 1184 laccase appeared as a robust enzyme against downstream process; only 13.5 % of laccase activity was lost at the end of downstream procedure. The pure enzyme appeared as a one-species laccase, with a molecular mass of 66 kDa as determined by SDS-PAGE. The pH optimum for 2,2'-azino-bis-[3-ethyltiazoline-6-sulfonate] oxidation ranged between 2.5 and 3.0 in 100 mM tartrate buffer. Optimum temperature for laccase activity was determined to be around 70 °C. The kinetic of laccase was investigated with four phenolic substrates. The lowest Km values (17 and 20 μM) were found for ABTS and guaiacol, respectively. Coriolopsis gallica 1184 laccase was characterized by mass spectrometry and shows that C. gallica 1184_LacI is very likely a new member of the AA1_1 subfamily. Our results clearly show high competitive potential of the robust extracellular C. gallica 1184 laccase to use it in different industrial processes., info:eu-repo/semantics/published

Lignolytic enzymes are very promising tools for biotechnological applications in water treatment. Among them, laccases (EC 1.10.3.2: p-diphenol:dioxygen oxidoreductase) are versatile enzymes with the ability to oxidize a wide range of aromatic and non-aromatic compounds (polyphenols, aryl diamines, anilines, methoxy substituted phenols, organic/inorganic metal compounds, etc.). Laccases have confirmed their potential for the degradation of recalcitrant residual phenolic compounds in water. Typically, oxidation of a laccase substrate leads to the formation of free radical with the concomitant loss of one electron and reduction of molecular oxygen into water. The formed radicals can react via non-enzymatic reactions i.e. oxidative condensation, and oxidative phenolic coupling or oxidative coupling, leading to the formation of soluble or non-soluble reaction products, several of them of polymeric nature. As many studies have highlighted the generation of products potentially more toxic than the parent molecules with the use of tertiary treatments such as advanced oxidative processes (AOPs) applied in water treatment plans, the identification of reaction products generated by the degradation of phenolic contaminants in water by laccases, and their potential toxicity, have to be investigated. This review summarizes and discuss progress made this last decades in identification of products and their risk assessment regarding their potential toxicity for a list of the most studied recalcitrant phenolic pollutants in water, such as dyes, bisphenol A, triclosan, nonylphenol, natural and synthetic hormones, etc., SCOPUS: re.j, info:eu-repo/semantics/published

Submerged fermentation in a stirred bioreactor of the white rot fungus Cerrena unicolor C- 139 was done at a 120-L scale in the presence of wheat bran as a cheap lignocellulosic substrate for fungus growth and laccase production. Enzyme monitoring showed that laccase production started after 2 days of cultivation, attaining a maximum activity of 416.4 U·mL-1 at day 12 of fermentation. After treatment of culture liquid by successive micro- and ultrafiltration (5 kDa), a liquid concentrate containing 22203176 units of laccase was obtained. Obtaining large amount of laccase is essential for various industrial applications, including detoxification of industrial effluents, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated area, stabilization of beverages, production of cosmetics, manufacture of anti-cancer drugs, and nanobiotechnology. The cultivation method and the fungal strain used here provided a substantial amount of enzyme produced at a price lower than 0.01 € cent/unit enzyme., SCOPUS: ar.j, info:eu-repo/semantics/published

In the past decades, much concern has been expressed regarding organic micropollutants generating endocrine disruption. In particular, estrogenic endocrine disruptors, compounds that interfere with the estrogenic hormonal mechanisms, are in the center of environmental scien- tists attention. Numerous endocrine disrupting effects have been observed at concentrations corresponding to those found in aquatic environments, such as feminization of fauna, infertility, reproductive disturbance, cancer, or developmental disruption. Wastewater treatment plants ef- fluents have been identified as the main source of estrogenic endocrine disruptors in the aquatic environment, due to inappropriate treatment. Promising alternative treatment systems based on the use of ligninolytic enzymes (e.g. laccases) have recently emerged. This work falls within the framework of these new techniques. Althoughno environmentally safe threshold can clearly be set, focusing on the removal of global estrogenicity in water instead of concentrations of targeted estrogenic compounds seems a relevant research. To our knowledge, the use of these recently emerged enzymatic processes at an industrial scale (such as for the treatment of urban wastewater), oriented towards water overall estrogenicity, has not been implemented yet.The general objective of this work was to develop ans study a process of removal of estro- genicity by laccases from white-rot fungi, in laboratory, with the purpose of design and scale-up for industrial implementation. This work consisted of the conception, characterization, testing, study and modeling of this process.First, in order to enable the study and the scaling-up of a process of estrogenicity removal, op- timizing the technologies of quantification of estrogenicity in water was a real necessity. Therefore, a study of the methods of estrogenicity monitoring, within a treatment process framework, was conducted. Based on a wide literature review, existing methods were gather, Doctorat en Sciences de l'ingénieur, info:eu-repo/semantics/nonPublished

Micro pollutants (MPs) can be defined as inorganic and organic substances present atlow concentrations (pg/L to ng/L) in the environment, however having adverseconsequences for living organisms even at these low concentrations. To date, aneffective and sustainable global strategy against this insidious contamination of aquaticenvironments barely exists. Source controls and technical systems, such as wastewatertreatment plants, function only as partial barriers or not at all. The enzyme laccase wasidentified as active in the degradation of different MPs. We present data showing thatlaccase from Cerrena unicolor immobilized on mesoporous silica beads(CPC) in acontinuous column reactor, was suitable for elimination of MPs including bisphenolA(BPA), 4-nonylphenol (NP) and triclosan (TCS) .A system equipped with a 3.5 x 0.7cm column packed with immobilized laccase CPC beads (43 Units/g of enzymeactivity), and eluted at 1 mL/min with a solution containing BPA,NP and TCS eachat50 μM, was able to totally remove the three MPs present in at least 1 L of the solution.These results open the possibility to use immobilized laccase in industrial and domesticprocesses for elimination of harmful MPs., SCOPUS: ar.j, info:eu-repo/semantics/published

The evaluation of continuous elimination and transformation of toxic endocrine disruptors micropollutants (MPs) (Bisphenol A, Nonylphenol [NP] and Triclosan [TCS]) using immobilized laccase of Coriolopsis sp. showed the high potential of this laccase as a bio-agent for the elimination and transformation process of selected MPs. A toxicology test using Escherichia coli has shown that toxicity completely disappeared after transformation and elimination of Bisphenol A, while in cases of NP and TCS 95% and 90% toxicity, respectively, disappeared. As compared to initial activity, immobilized laccase activity remained practically constant at the end of several runs of elimination and transformation processes of MPs. This feature is promising for the use of immobilized Coriolopsis sp. laccase in a future industrial depollution process., SCOPUS: ar.j, info:eu-repo/semantics/published

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The white-rot fungus Cerrena unicolor C-139 produced 450 000 U l-1 of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent Km value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity. © 2012 The British Mycological Society., SCOPUS: ar.j, info:eu-repo/semantics/published

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Wastewaters produced by industry and municipalities contain numerous synthetic compounds in varying concentrations. Micro pollutants can be defined as inorganic and organic substances present at low concentrations (pg/L to ng/L) in the environment having adverse consequences for living organisms at these low concentrations. To date, an effective and sustainable global strategy against this insidious contamination of aquatic environments barely exists. Source controls and technical systems, such as wastewater treatment plants, function only as partial barriers or not at all. In this presentation, we present an efficient strain of white rot fungi producing high amounts laccase capable of transforming the tested micro pollutants (bisphenol A, nonylphenols and triclosan). The enzymes are successfully immobilized on mesoporous silica particles to form a biocatalyst. Successful and promising tests were realized with this biocatalyst in various reactors designed for the micro pollutants abatement process., info:eu-repo/semantics/nonPublished

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