Synthesis and Antifungal Activity of Norbornene Carboxamide/sulfonamide Derivatives as Potential Fungicides Targeting Laccase.

To explore more potential fungicides with new scaffolds, thirty-seven norbornene carboxamide/sulfonamide derivatives were designed, synthesized, and assayed for inhibitory activity against six plant pathogenic fungi and oomycetes. The preliminary antifungal assay suggested that the title derivatives showed moderate to good antifungal activity against six plant pathogens. Especially, compound 6 e presented excellent in vitro antifungal activity against Sclerotinia sclerotiorum (EC50=0.71 mg/L), which was substantially stronger than pydiflumetofen. In vivo antifungal assay indicated 6 e displayed prominent protective and curative effects on rape leaves infected by S. sclerotiorum. The preliminary mechanism research displayed that 6 e could damage the surface morphology and inhibit the sclerotia formation of S. sclerotiorum. In addition, the in vitro enzyme inhibition bioassay indicated that 6 e displayed pronounced laccase inhibition activity (IC50=0.63 µM), much stronger than positive control cysteine. Molecular docking elucidated the binding modes between 6 e and laccase. The bioassay results and mechanism investigation demonstrated that this class of norbornene carboxamide/sulfonamide derivatives could be promising laccase inhibitors for novel fungicide development.

Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies.

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8-233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives' interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.

Synthesis and Studies of the Inhibitory Effect of Hydroxylated Phenylpropanoids and Biphenols Derivatives on Tyrosinase and Laccase Enzymes.

The impaired activity of tyrosinase and laccase can provoke serious concerns in the life cycles of mammals, insects and microorganisms. Investigation of inhibitors of these two enzymes may lead to the discovery of whitening agents, medicinal products, anti-browning substances and compounds for controlling harmful insects and bacteria. A small collection of novel reversible tyrosinase and laccase inhibitors with a phenylpropanoid and hydroxylated biphenyl core was prepared using naturally occurring compounds and their activity was measured by spectrophotometric and electrochemical assays. Biosensors based on tyrosinase and laccase enzymes were constructed and used to detect the type of protein-ligand interaction and half maximal inhibitory concentration (IC50). Most of the inhibitors showed an IC50 in a range of 20-423 nM for tyrosinase and 23-2619 nM for laccase. Due to the safety concerns of conventional tyrosinase and laccase inhibitors, the viability of the new compounds was assayed on PC12 cells, four of which showed a viability of roughly 80% at 40 µM. In silico studies on the crystal structure of laccase enzyme identified a hydroxylated biphenyl bearing a prenylated chain as the lead structure, which activated strong and effective interactions at the active site of the enzyme. These data were confirmed by in vivo experiments performed on the insect model Tenebrio molitur.

Poly(2-oxazoline)s with a 2,2'-Iminodiacetate End Group Inhibit and Stabilize Laccase.

Poly(2-oxazoline)s (POxs) with 2,2'-iminodiacetate (IDA) end groups were investigated as inhibitors for laccase. The polymers with the IDA end groups are reversible, competitive inhibitors for this enzyme. The IC50 values were found to be in a range of 1-3 mm. Compared with IDA alone, the activity was increased by a factor of more than 30; thus indicating that attaching a polymer chain to an inhibitor can already improve the activity of the former. The enzyme activity drops to practically zero upon increasing the concentration of the most active telechelic inhibitor, IDA-PEtOx30 -IDA (PEtOx: poly(2-ethyl-2-oxazoline)), from 5 to 8 mm. This unusual behavior was investigated by means of dynamic light scattering, which showed specific aggregation above 5 mm. Furthermore, the laccase could be stabilized in the presence of POx-IDA, upon addition at a concentration of 20 mm and higher. Whereas laccase becomes completely inactive at room temperature after one week, the stabilized laccase is fully active for at least a month in aqueous solution.

Biocatalytic characterization of free and immobilized laccase from Trametes versicolor in its activation zone.

This investigation may be of interest for researchers working on the determination of several biocatalytic properties of the laccase from Trametes versicolor. So, We will treated the effects of pH, temperature, several organic components and heavy metals by performing enzyme assays in the presence of a 2,6 dimethoxyphenol (DMP) as substrate on the laccase activity. The optimum activity and temperature are 4 and 40 °C, respectively. The maximum rate of the reaction is 124.53 U/mg and the Michaelis constant is in order of 1.23 mM. The effect of metal ions on the laccase activity with a final concentrations range varying from 1 to 5 mM show that the Cu2+ ions increase the activity for concentration inferiors to 4 mM and the other metal ions have a relative influence on the laccase activity. Four tri-block copolymers based on poly(ethylene oxide) and poly(propylene oxide) and two polyethylene glycols are used to study the synthetic polymers effects on the enzymatic activity. Also, we have demonstrated that the laccase keeps 95% of its initial activity at 60 °C in the PEGDA8000 and PEGDA6000 gel matrix. The maximum rate of the immobilized laccase is approximately around 21.03 and 47.22% smaller than the free one.

A novel laccase from white rot fungus Trametes orientalis: Purification, characterization, and application.

A novel laccase (Tolacc-T) from white rot fungus Trametes orientalis was enriched to apparent homogeneity with a specific activity of 20.667U/mg protein and recovery yield of 47.33%. The SDS-PAGE gave a single band indicating that Tolacc-T appears as a monomeric protein with a molecular mass of 44.0kDa. Domain structure analysis revealed that Tolacc-T contained a typical copper II binding domain and shared three potential N-glycosylation sites, but had no copper I binding domain, demonstrating that the enzyme is really a laccase, but a novel laccase. Optimal pH and temperature of Tolacc-T was 4.0 and 80°C, respectively, and it retained more than 80% of its original activity after 2h incubation at 10°C to 50°C. The enzyme exhibited strict substrate specificity towards ABTS but showed no or trace activities towards other substrates. Among the metals tested, Mn2+ was proved to be the best activator for enhancing the laccase activity. A strongly inhibiting effect was found when NaN3, L-cysteine, and DTT were added to the enzyme. However, Tolacc-T activity was little bit inhibited in the presence of chelator EDTA. Furthermore, the enzyme was capable of degrading structurally different synthetic dyes in the absence of a redox mediator.

Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4.

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (kcat/Km) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and ß-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2'-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu2+, Co2+, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.

Poly(N-vinylpyrrolidone)-poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations.

Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants Km between 60 and 143 µM and turn over numbers kcat of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzyme's activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.

Chemical modifications of laccase from white-rot basidiomycete Cerrena unicolor.

Laccases belong to the group of phenol oxidizes and constitute one of the most promising classes of enzymes for future use in various fields. For industrial and biotechnological purposes, laccases were among the first enzymes providing larger-scale applications such as removal of polyphenols or conversion of toxic compounds. The wood-degrading basidiomycete Cerrena unicolor C-139, reported in this study, is one of the high-laccase producers. In order to facilitate novel and more efficient biocatalytic process applications, there is a need for laccases with improved biochemical properties, such as thermostability or stability in broad ranges of pH. In this work, modifications of laccase isoforms by hydrophobization, hydrophilization, and polymerization were performed. The hydrophobized and hydrophilized enzyme showed enhanced surface activity and higher ranges of pH and temperatures in comparison to its native form. However, performed modifications did not appear to noticeably alter enzyme's native structure possibly due to the formation of coating by particles of saccharides around the molecule. Additionally, surface charge of modified laccase shifted towards the negative charge for the hydrophobized laccase forms. In all tested modifications, the size exclusion method led to average 80 % inhibition removal for hydrophilized samples after an hour of incubation with fluoride ions. Samples that were hydrophilized with lactose and cellobiose showed an additional 90 % reversibility of inhibition by fluoride ions after an hour of concluding the reaction and 40 % after 24 h. The hydrophobized laccase showed higher level of the reversibility after 1 h (above 80 %) and 24 h (above 70 %) incubation with fluoride ions. The addition of ascorbate to laccase solution before a fluoride spike resulted in more efficient reversibility of fluoride inhibitory effect in comparison to the treatments with reagents used in the reversed sequence.

Biochemical characterization of laccase from hairy root culture of Brassica juncea L. and role of redox mediators to enhance its potential for the decolorization of textile dyes.

In vitro transgenic hairy root cultures provide a rapid system for physiological, biochemical studies and screening of plants for their phytoremediation potential. The hairy root cultures of Brassica juncea L. showed 92% decolorization of Methyl orange within 4 days. Out of the different redox mediators that were used to achieve enhanced decolorization, 2, 2'-Azinobis, 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) was found to be the most efficient. Laccase activity of 4.5 U mg(-1) of protein was observed in hairy root cultures of Brassica juncea L., after the decolorization of Methyl orange. Intracellular laccase produced by B. juncea root cultures grown in MS basal medium was purified up to 2.0 fold with 6.62 U mg(-1) specific activity using anion-exchange chromatography. Molecular weight of the purified laccase was estimated to be 148 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme efficiently oxidized ABTS which was also required for oxidation of the other tested substrates. The pH and temperature optimum for laccase activity were 4.0 and 40°C, respectively. The purified enzyme was stable up to 50°C and was stable in the pH range of 4.0-6.0. Laccase activity was strongly inhibited by sodium azide, EDTA, dithiothreitol and L: -cysteine. The purified enzyme decolorized various textile dyes in the presence of ABTS as an efficient redox mediator. These findings contribute to a better understanding of the enzymatic process involved in phytoremediation of textile dyes by using hairy roots.

Biochemical characteristics of a textile dye degrading extracellular laccase from a Bacillus sp. ADR.

Bacillus sp. ADR secretes an extracellular laccase in nutrient broth, and this enzyme was purified up to 56-fold using acetone precipitation and DEAE-cellulose anion exchange chromatography. The molecular weight of purified laccase was estimated to be 66 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified laccase oxidized 2,6-dimethoxy phenol, o-tolidine, hydroquinone, L-DOPA and guaiacol. The optimum pH for oxidation of o-tolidine, 2,6-dimethoxy phenol and guaiacol were 3.0, 4.0 and 5.0, respectively. The purified laccase contained 2.7 mol/mol of copper. The laccase was stable up to 40 °C and within the pH range of 7.0-9.0. Well-known inhibitors of multicopper oxidases such as, sodium azide, L-cysteine and dithiothreitol showed significant inhibition of laccase activity. The purified enzyme decolorized structurally different azo dyes with variable decolorization rates and efficiencies of 68-90%. This study is useful for understanding the precise use of Bacillus sp. ADR in the decolorization of textile dyes containing industrial wastewater.

Purification and characterization of laccase from basidiomycete Fomitella fraxinea.

A laccase was isolated from the culture filtrate of basidiomycete Fomitella fraxinea. The enzyme was purified to electrophoretical homogeneity using ammonium sulfate precipitation, anion-exchange chromatography, and gel-filtration chromatography. The enzyme was identified a monomeric protein with a molecular mass of 47 kDa sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel-filtration chromatography, and had an isoelectric point of 3.8. The N-terminal amino acid sequence for the enzyme was ATXSNXKTLAAD, which had a very low similarity to the sequences previously reported for laccases from other basidiomycetes. The optimum and temperature for 2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulfonate) (ABTS) were 3.0 and 70 degrees C, respectively. The enzyme also showed a much higher level of specific activity for ABTS and 2,6-dimethoxyphenol (DMP), where the values of the enzyme for ABTS and 2,6-DMP were 270 and 426 microM, respectively, and the Vmax values were 876 and 433.3 microM/min, respectively. The laccase activity was completely inhibited by L-cysteine, dithiothreitol (DTT), and sodium azide, significantly inhibited by Ni+, Mn+ and Ba+2, and slightly stimulated by K+ and Ca+2.

Copper dissociation as a mechanism of fungal laccase denaturation by humic acid.

Effects of humic acid on removal of hydroxy polychlorobiphenyls (PCBs) with laccase from Trametes versicolor were studied. In the absence of humic acid, hydroxy PCBs were rapidly degraded by laccase. However, the rate constants decreased with increasing humic acid concentration, the reactions being completely inhibited at 150 mg l(-1) of humic acid. Peroxidase from Arthromyces ramosus was not inhibited by the same treatment. The activity of humic acid-deactivated laccase was completely restored by copper ions (500 microM of Cu2+ in 150 mg l(-1) of humic acid), but not by other metal ions (Zn2+, Fe2+ and Hg2+). Humic acid-deactivated laccase purified by gel permeation chromatography (GPC) showed no activity against 2,2'-azino-bis(3-ethylbenzthiazoline sulfonic acid) diammonium salt and 3,5-dichloro-4-hydroxybiphenyl, but its activity was restored by copper ion treatment. Humic acid-deactivated laccase showed similar properties, such as GPC retention time and copper ion requirements for activity, to those of laccase deactivated by nitrilotriacetic acid. The extent of humic acid inhibition, expressed as activity non-recoverable by copper ion treatment, increased over time more rapidly than that of the humic acid-free control. These results suggest that short-term inactivation of laccase, i.e., less than 1 day, is attributable to a depletion of copper ion.