Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm-1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Characterization of the CAZy Repertoire from the Marine-Derived Fungus Stemphylium lucomagnoense in Relation to Saline Conditions.

Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L-1. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.

Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus Pestalotiopsis sp.

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.

Enzyme Activities of Two Recombinant Heme-Containing Peroxidases, TvDyP1 and TvVP2, Identified from the Secretome of Trametes versicolor.

Trametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. The goal of the present work was to gain insights into the molecular biology and biochemistry of the heme-including class II and dye-decolorizing peroxidases secreted by this fungus. Proteomic analysis of the secretome of T. versicolor BRFM 1218 grown on oak wood revealed a set of 200 secreted proteins, among which were the dye-decolorizing peroxidase TvDyP1 and the versatile peroxidase TvVP2. Both peroxidases were heterologously produced in Escherichia coli, biochemically characterized, and tested for the ability to oxidize complex substrates. Both peroxidases were found to be active against several substrates under acidic conditions, and TvDyP1 was very stable over a relatively large pH range of 2.0 to 6.0, while TvVP2 was more stable at pH 5.0 to 6.0 only. The thermostability of both enzymes was also tested, and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at temperatures ranging from 30 to 50°C, the activity of TvVP2 drastically decreased, with 10 to 30% of the initial activity retained. Under the same conditions, TvDyP1 retained 20 to 80% of its enzyme activity. The two proteins were catalytically characterized, and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, with TvVP2 displaying more rapid oxidation of the two compounds. They were tested for the ability to decolorize five industrial dyes, and TvVP2 presented a greater ability to oxidize and decolorize the dye substrates than TvDyP1.IMPORTANCETrametesversicolor is a wood-inhabiting agaricomycete known for its ability to cause strong white-rot decay on hardwood and for its high tolerance of phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its ability to degrade wood, specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decolorizing peroxidase and one versatile peroxidase, were biochemically characterized in depth and compared to other fungal peroxidases.

Rheology and microstructure of gels based on wheat arabinoxylans enzymatically modified in arabinose to xylose ratio.

BACKGROUND: Arabinoxylans (AX) are polysaccharides consisting of a backbone of xyloses with arabinose substituents ester-linked to ferulic acid (FA). The arabinose to xylose ratio (A/X) in AX may vary from 0.3 to 1.1. AX form covalent gels by cross-linking of FA but physical interactions between AX chains also contribute to the network formation. The present study aimed to investigate the rheological and microstructural characteristics of gels based on AX enzymatically modified in A/X. RESULTS: Tailored AX presented A/X ranging from 0.68 to 0.51 and formed covalent gels. Dimers of FA content and elasticity (G') increased from 0.31 to 0.39 g kg-1 AX and from 106 to 164 Pa when the A/X in the polysaccharide decreased from 0.68 to 0.51. Atomic force microscopy images of AX gels showed a sponge-like microstructure at A/X = 0.68, whereas, at lower values, gels presented a more compact microstructure. Scanning electron microscopy analysis of AX gels show an arrangement of different morphology, passing from an imperfect honeycomb (A/X = 0.68) to a flake-like microstructure (A/X = 0.51). CONCLUSION: Lower A/X values favor the aggregation of AX chains resulting in an increase in di-FA content, which improves the rheological and microstructural characteristics of the gel formed. © 2017 Society of Chemical Industry.

Glyoxal oxidases: their nature and properties.

H2O2 has been found to be required for the activity of the main microbial enzymes responsible for lignin oxidative cleavage, peroxidases. Along with other small radicals, it is implicated in the early attack of plant biomass by fungi. Among the few extracellular H2O2-generating enzymes known are the glyoxal oxidases (GLOX). GLOX is a copper-containing enzyme, sharing high similarity at the level of active site structure and chemistry with galactose oxidase. Genes encoding GLOX enzymes are widely distributed among wood-degrading fungi especially white-rot degraders, plant pathogenic and symbiotic fungi. GLOX has also been identified in plants. Although widely distributed, only few examples of characterized GLOX exist. The first characterized fungal GLOX was isolated from Phanerochaete chrysosporium. The GLOX from Utilago maydis has a role in filamentous growth and pathogenicity. More recently, two other glyoxal oxidases from the fungus Pycnoporus cinnabarinus were also characterized. In plants, GLOX from Vitis pseudoreticulata was found to be implicated in grapevine defence mechanisms. Fungal GLOX were found to be activated by peroxidases in vitro suggesting a synergistic and regulatory relationship between these enzymes. The substrates oxidized by GLOX are mainly aldehydes generated during lignin and carbohydrates degradation. The reactions catalysed by this enzyme such as the oxidation of toxic molecules and the production of valuable compounds (organic acids) makes GLOX a promising target for biotechnological applications. This aspect on GLOX remains new and needs to be investigated.

Heterologous Production and Characterization of Two Glyoxal Oxidases from Pycnoporus cinnabarinus.

UNLABELLED: The genome of the white rot fungus Pycnoporus cinnabarinus includes a large number of genes encoding enzymes implicated in lignin degradation. Among these, three genes are predicted to encode glyoxal oxidase, an enzyme previously isolated from Phanerochaete chrysosporium The glyoxal oxidase of P. chrysosporium is physiologically coupled to lignin-oxidizing peroxidases via generation of extracellular H2O2 and utilizes an array of aldehydes and α-hydroxycarbonyls as the substrates. Two of the predicted glyoxal oxidases of P. cinnabarinus, GLOX1 (PciGLOX1) and GLOX2 (PciGLOX2), were heterologously produced in Aspergillus niger strain D15#26 (pyrG negative) and purified using immobilized metal ion affinity chromatography, yielding 59 and 5 mg of protein for PciGLOX1 and PciGLOX2, respectively. Both proteins were approximately 60 kDa in size and N-glycosylated. The optimum temperature for the activity of these enzymes was 50°C, and the optimum pH was 6. The enzymes retained most of their activity after incubation at 50°C for 4 h. The highest relative activity and the highest catalytic efficiency of both enzymes occurred with glyoxylic acid as the substrate. The two P. cinnabarinus enzymes generally exhibited similar substrate preferences, but PciGLOX2 showed a broader substrate specificity and was significantly more active on 3-phenylpropionaldehyde. IMPORTANCE: This study addresses the poorly understood role of how fungal peroxidases obtain an in situ supply of hydrogen peroxide to enable them to oxidize a variety of organic and inorganic compounds. This cooperative activity is intrinsic in the living organism to control the amount of toxic H2O2 in its environment, thus providing a feed-on-demand scenario, and can be used biotechnologically to supply a cheap source of peroxide for the peroxidase reaction. The secretion of multiple glyoxal oxidases by filamentous fungi as part of a lignocellulolytic mechanism suggests a controlled system, especially as these enzymes utilize fungal metabolites as the substrates. Two glyoxal oxidases have been isolated and characterized to date, and the differentiation of the substrate specificity of the two enzymes produced by Pycnoporus cinnabarinus illustrates the alternative mechanisms existing in a single fungus, together with the utilization of these enzymes to prepare platform chemicals for industry.

Activities of Secreted Aryl Alcohol Quinone Oxidoreductases from Pycnoporus cinnabarinus Provide Insights into Fungal Degradation of Plant Biomass.

Auxiliary activities family 3 subfamily 2 (AA3_2) from the CAZy database comprises various functions related to ligninolytic enzymes, such as fungal aryl alcohol oxidases (AAO) and glucose oxidases, both of which are flavoenzymes. The recent study of the Pycnoporus cinnabarinus CIRM BRFM 137 genome combined with its secretome revealed that four AA3_2 enzymes are secreted during biomass degradation. One of these AA3_2 enzymes, scf184803.g17, has recently been produced heterologously in Aspergillus niger Based on the enzyme's activity and specificity, it was assigned to the glucose dehydrogenases (PcinnabarinusGDH [PcGDH]). Here, we analyze the distribution of the other three AA3_2 enzymes (scf185002.g8, scf184611.g7, and scf184746.g13) to assess their putative functions. These proteins showed the highest homology with aryl alcohol oxidase from Pleurotus eryngii Biochemical characterization demonstrated that they were also flavoenzymes harboring flavin adenine dinucleotide (FAD) as a cofactor and able to oxidize a wide variety of phenolic and nonphenolic aryl alcohols and one aliphatic polyunsaturated primary alcohol. Though presenting homology with fungal AAOs, these enzymes exhibited greater efficiency in reducing electron acceptors (quinones and one artificial acceptor) than molecular oxygen and so were defined as aryl-alcohol:quinone oxidoreductases (AAQOs) with two enzymes possessing residual oxidase activity (PcAAQO2 and PcAAQO3). Structural comparison of PcAAQO homology models with P. eryngii AAO demonstrated a wider substrate access channel connecting the active-site cavity to the solvent, explaining the absence of activity with molecular oxygen. Finally, the ability of PcAAQOs to reduce radical intermediates generated by laccase from P. cinnabarinus was demonstrated, shedding light on the ligninolytic system of this fungus.

Essential oils and distilled straws of lavender and lavandin: a review of current use and potential application in white biotechnology.

The Lavandula genus, which includes lavender (Lavandula angustifolia) and lavandin (L. angustifolia × Lavandula latifolia), is cultivated worldwide for its essential oils, which find applications in perfumes, cosmetics, food processing and, more recently, in aromatherapy products. The chemical composition of lavender and lavandin essential oils, usually produced by steam distillation from the flowering stems, is characterized by the presence of terpenes (e.g. linalool and linalyl acetate) and terpenoids (e.g. 1,8-cineole), which are mainly responsible for their characteristic flavour and their biological and therapeutic properties. Lavender and lavandin distilled straws, the by-products of oil extraction, were traditionally used for soil replenishment or converted to a fuel source. They are mineral- and carbon-rich plant residues and, therefore, a cheap, readily available source of valuable substances of industrial interest, especially aroma and antioxidants (e.g. terpenoids, lactones and phenolic compounds including coumarin, herniarin, α-bisabolol, rosmarinic and chlorogenic acids). Accordingly, recent studies have emphasized the possible uses of lavender and lavandin straws in fermentative or enzymatic processes involving various microorganisms, especially filamentous fungi, for the production of antimicrobials, antioxidants and other bioproducts with pharmaceutical and cosmetic activities, opening up new challenging perspectives in white biotechnology applications.

A novel glucose dehydrogenase from the white-rot fungus Pycnoporus cinnabarinus: production in Aspergillus niger and physicochemical characterization of the recombinant enzyme.

Data on glucose dehydrogenases (GDHs) are scarce and availability of these enzymes for application purposes is limited. This paper describes a new GDH from the fungus Pycnoporus cinnabarinus CIRM BRFM 137 that is the first reported GDH from a white-rot fungus belonging to the Basidiomycota. The enzyme was recombinantly produced in Aspergillus niger, a well-known fungal host producing an array of homologous or heterologous enzymes for industrial applications. The full-length gene that encodes GDH from P. cinnabarinus (PcGDH) consists of 2,425 bp and codes for a deduced protein of 620 amino acids with a calculated molecular mass of 62.5 kDa. The corresponding complementary DNA was cloned and placed under the control of the strong and constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. The signal peptide of the glucoamylase prepro sequence of A. niger was used to target PcGDH secretion into the culture medium, achieving a yield of 640 mg L(-1), which is tenfold higher than any other reported value. The recombinant PcGDH was purified twofold to homogeneity in a one-step procedure with a 41 % recovery using a Ni Sepharose column. The identity of the recombinant protein was further confirmed by immunodetection using western blot analysis and N-terminal sequencing. The molecular mass of the native PcGDH was 130 kDa, suggesting a homodimeric form. Optimal pH and temperature were found to be similar (5.5 and 60 °C, respectively) to those determined for the previously characterized GDH, i.e., from Glomerella cingulata. However PcGDH exhibits a lower catalytic efficiency of 67 M(-1) s(-1) toward glucose. This substrate is by far the preferred substrate, which constitutes an advantage over other sugar oxidases in the case of blood glucose monitoring. The substrate-binding domain of PcGDH turns out to be conserved as compared to other glucose-methanol-choline (GMCs) oxidoreductases. In addition, the ability of PcGDH to reduce oxidized quinones or radical intermediates was clearly demonstrated, which raises prospects for applying this enzyme to detoxify toxic compounds formed during the degradation of lignin.

Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels.

Lignin composition and structure in young versus adult Eucalyptus globulus plants.

Lignin changes during plant growth were investigated in a selected Eucalyptus globulus clone. The lignin composition and structure were studied in situ by a new procedure enabling the acquisition of two-dimensional nuclear magnetic resonance (2D-NMR) spectra on wood gels formed in the NMR tube as well as by analytical pyrolysis-gas chromatography-mass spectrometry. In addition, milled-wood lignins were isolated and analyzed by 2D-NMR, pyrolysis-gas chromatography-mass spectrometry, and thioacidolysis. The data indicated that p-hydroxyphenyl and guaiacyl units are deposited at the earlier stages, whereas the woods are enriched in syringyl (S) lignin during late lignification. Wood 2D-NMR showed that ß-O-4' and resinol linkages were predominant in the eucalypt lignin, whereas other substructures were present in much lower amounts. Interestingly, open ß-1' structures could be detected in the isolated lignins. Phenylcoumarans and cinnamyl end groups were depleted with age, spirodienone abundance increased, and the main substructures (ß-O-4' and resinols) were scarcely modified. Thioacidolysis revealed a higher predominance of S units in the ether-linked lignin than in the total lignin and, in agreement with NMR, also indicated that resinols are the most important nonether linkages. Dimer analysis showed that most of the resinol-type structures comprised two S units (syringaresinol), the crossed guaiacyl-S resinol appearing as a minor substructure and pinoresinol being totally absent. Changes in hemicelluloses were also shown by the 2D-NMR spectra of the wood gels without polysaccharide isolation. These include decreases of methyl galacturonosyl, arabinosyl, and galactosyl (anomeric) signals, assigned to pectin and related neutral polysaccharides, and increases of xylosyl (which are approximately 50% acetylated) and 4-O-methylglucuronosyl signals.

Optimization of the Decolorization of the Reactive Black 5 by a Laccase-like Active Cell-Free Supernatant from Coriolopsis gallica.

The textile industry generates huge volumes of colored wastewater that require multiple treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from Coriolopsis gallica. Decolorization was optimized in a 1 mL reaction mixture using the response surface methodology (RSM) to test the influence of five variables, i.e., laccase-like activity, dye concentration, redox mediator (HBT) concentration, pH, and temperature, on dye decolorization. Statistical tests were used to determine regression coefficients and the quality of the models used, as well as significant factors and/or factor interactions. Maximum decolorization was achieved at 120 min (82 ± 0.6%) with the optimized protocol, i.e., laccase-like activity at 0.5 U mL−1, dye at 25 mg L−1, HBT at 4.5 mM, pH at 4.2 and temperature at 55 °C. The model proved significant (ANOVA test with p < 0.001): coefficient of determination (R²) was 89.78%, adjusted coefficient of determination (R²A) was 87.85%, and root mean square error (RMSE) was 10.48%. The reaction conditions yielding maximum decolorization were tested in a larger volume of 500 mL reaction mixture. Under these conditions, the decolorization rate reached 77.6 ± 0.4%, which was in good agreement with the value found on the 1 mL scale. RB5 decolorization was further evaluated using the UV-visible spectra of the treated and untreated dyes.

Biotransformation of the Fluoroquinolone, Levofloxacin, by the White-Rot Fungus Coriolopsis gallica.

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.

A Putative Lignin Copper Oxidase from Trichoderma reesei.

The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the ß-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ßß, SS ßO4 and GG ß5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Fungal Treatment for the Valorization of Technical Soda Lignin.

Technical lignins produced as a by-product in biorefinery processes represent a potential source of renewable carbon. In consideration of the possibilities of the industrial transformation of this substrate into various valuable bio-based molecules, the biological deconstruction of a technical soda lignin by filamentous fungi was investigated. The ability of three basidiomycetes (Polyporus brumalis, Pycnoporus sanguineus and Leiotrametes menziesii) to modify this material, the resultant structural and chemical changes, and the secreted proteins during growth on this substrate were investigated. The three fungi could grow on the technical lignin alone, and the growth rate increased when the media were supplemented with glucose or maltose. The proteomic analysis of the culture supernatants after three days of growth revealed the secretion of numerous Carbohydrate-Active Enzymes (CAZymes). The secretomic profiles varied widely between the strains and the presence of technical lignin alone triggered the early secretion of many lignin-acting oxidoreductases. The secretomes were notably rich in glycoside hydrolases and H2O2-producing auxiliary activity enzymes with copper radical oxidases being induced on lignin for all strains. The lignin treatment by fungi modified both the soluble and insoluble lignin fractions. A significant decrease in the amount of soluble higher molar mass compounds was observed in the case of P. sanguineus. This strain was also responsible for the modification of the lower molar mass compounds of the lignin insoluble fraction and a 40% decrease in the thioacidolysis yield. The similarity in the activities of P. sanguineus and P. brumalis in modifying the functional groups of the technical lignin were observed, the results suggest that the lignin has undergone structural changes, or at least changes in its composition, and pave the route for the utilization of filamentous fungi to functionalize technical lignins and produce the enzymes of interest for biorefinery applications.