Understanding the biodegradation pathways of azo dyes by immobilized white-rot fungus, Trametes hirsuta D7, using UPLC-PDA-FTICR MS supported by in silico simulations and toxicity assessment.

No biodegradation methods are absolute in the treatment of all textile dyes, which leads to structure-dependent degradation. In this study, biodegradation of three azo dyes, reactive black 5 (RB5), acid blue 113 (AB113), and acid orange 7 (AO7), was investigated using an immobilized fungus, Trametes hirsuta D7. The degraded metabolites were identified using UPLC-PDA-FTICR MS and the biodegradation pathway followed was proposed. RB5 (92%) and AB113 (97%) were effectively degraded, whereas only 30% of AO7 was degraded. Molecular docking simulations were performed to determine the reason behind the poor degradation of AO7. Weak binding affinity, deficiency in H-bonding interactions, and the absence of interactions between the azo (-NN-) group and active residues of the model laccase enzyme were responsible for the low degradation efficiency of AO7. Furthermore, cytotoxicity and genotoxicity assays confirmed that the fungus-treated dye produced non-toxic metabolites. The observations of this study will be useful for understanding and further improving enzymatic dye biodegradation.

Isothermal titration calorimetric assessment of lignin conversion by laccases.

Lignin valorization may offer a sustainable approach to achieve a chemical industry that is not completely dependent on fossil resources for the production of aromatics. However, lignin is a recalcitrant, heterogeneous, and complex polymeric compound for which only very few catalysts can act in a predictable and reproducible manner. Laccase is one of those catalysts and has often been referred to as an ideal "green" catalyst, as it is able to oxidize various linkages within lignin to release aromatic products, with the use of molecular oxygen and formation of water as the only side product. The extent and rate of laccase-catalyzed lignin conversion were measured using the label-free analytical technique isothermal titration calorimetry (ITC). IITC provides the molar enthalpy of the reaction, which reflects the extent of conversion and the time-dependent power trace, which reflects the rate of the reaction. Calorimetric assessment of the lignin conversion brought about by various fungal and bacterial laccases in the absence of mediators showed marked differences in the extent and rate of conversion for the different enzymes. Kraft lignin conversion by Trametes versicolor laccase followed Michaelis-Menten kinetics and was characterized by the following thermodynamic and kinetic parameters ΔHITC = -(2.06 ± 0.06)·103 kJ mol-1 , KM = 6.6 ± 1.2 µM and Vmax = 0.30 ± 0.02 U/mg at 25°C and pH 6.5. We envision calorimetric techniques as important tools for the development of enzymatic lignin valorization strategies.

Investigation of reactive Blue 19 biodegradation and byproducts toxicity assessment using crude laccase extract from Trametes versicolor.

Crude laccase potency on biodegradation and detoxification of Reactive blue 19 (RB-19) were demonstrated, along with prediction of degradation mechanisms, pathways and byproducts analysis. Trametes versicolor, cultured on pampas grass inflorescence (Cortaderia selloana), yielded the best crude laccase activity (15.36 U/g). 10 U CLE activities demonstrated a biodegradation yield (85%) in 210 min, at pH 4, 50 °C and 200 mg/L RB-19 concentrations. Evolution of a brown color that absorbed maximally at 478 nm was observed during biodegradation. Two methods were adopted for byproducts extraction, three methods for toxicity analysis and four models for kinetic parameters (Km and Vmax) determination. 2-ethylanthracene, 2-hydroxycyclohexa-2,4-dien-1-one, 2(4-methylphenyl)-ethan-1-amine, 1-[6-hydroperoxy-4,5-bis(sulfooxy)oxan-3-yl]triaza-1,2-dien-2-ium, naphthalene-2,7-disulfonic acid and N-[(5-oxooxolan-2-yl)methyl]acetamide were detected as toxic byproducts. Brown color evolution was due to 1,1,1-triethyl-3-(methoxycarbonyl)-2,2-dioxo-2λ6-diazathian-1-ium (methoxycarbonyl sulfanyl-triethylammonium hydroxide) inner salt. Increase in color density (light to dark brown) was a function of byproduct(s) biodegradation and polymerization. RB-19 and byproduct acute toxicities were decreased significantly (98% - 6.91%). Kinetic parameters Km (18.05 mg/L) and Vmax (0.31 mg/L. min-1) from the four kinetic models demonstrated higher affinity of CLE to RB-19. CLE yielded a catalytic activity (Vmax/Km =0.017 min-1) demonstrating the flexibility of CLE active site to RB-19 binding over commercial laccase.

Bifunctional in vivo role of laccase exploited in multiple biotechnological applications.

Laccases are multicopper enzymes present in plants, fungi, bacteria, and insects, which catalyze oxidation reactions together with four electron reduction of oxygen to water. Plant, bacterial, and insect laccases have a polymerizing role in nature, implicated in biosynthesis of lignin, melanin formation, and cuticle hardening, respectively. On the other hand, fungal laccases carry out both polymerizing (melanin synthesis and fruit body formation) as well as depolymerizing roles (lignin degradation). This bifunctionality of fungal laccases can be attributed to the presence of multiple isoforms within the same as well as different genus and species. Interestingly, by manipulating culture conditions, these isoforms with their different induction patterns and unique biochemical characteristics can be expressed or over-expressed for a targeted biotechnological application. Consequently, laccases can be considered as one of the most important biocatalyst which can be exploited for divergent industrial applications viz. paper pulp bleaching, fiber modification, dye decolorization, bioremediation as well as organic synthesis. The present review spotlights the role of fungal laccases in various antagonistic applications, i.e., polymerizing and depolymerizing, and co-relating this dual role with potential industrial significance.

Laccase production optimization by response surface methodology with Aspergillus fumigatus AF1 in unique inexpensive medium and decolorization of different dyes with the crude enzyme or fungal pellets.

In the present study, alkaline pretreatment was applied for the enhanced laccase production from rice straw. Various process parameters including sodium hydroxide concentration, pH and fermentation temperature were optimized via response surface methodology (RSM) with a Box-Behnken design (BBD). Through regression analysis, it was found that laccase activity was well fitted by a quadratic polynomial equation (R(2)=0.998, Adj R(2)=0.995), and the fermentation temperature was the most significant factor influencing laccase activity. The optimized process conditions found were NaOH concentration of 0.39 mol L(-1), pH 3.12 and temperature 25.43 °C, under which laccase activity reached 142,198 ± 3586 U L(-1). Further studies were carried out to probe different dyes decolorization ability of laccase produced by Aspergillus fumigatus, A. fumigatus pellets and whole fermentation broth (WFB) using sodium hydroxide pretreated rice straw as sole carbon source. Results showed that pure laccase demonstrate limited decolorization ability to all the studied dyes, while crude laccase, A. fumigatus pellets and WFB exhibit significant decolorization ability to all the studied dyes with WFB being the most excellent one. Effectiveness of degradation was confirmed by uv-vis and phytotoxicity studies, which indicated that A. fumigatus transformed the dyes into non-toxic metabolites.

Assessment of Cryptococcus albidus for biopulping of eucalyptus.

Cryptococcus albidus shows delignification activity in nature. It was used for the biopulping of eucalyptus wood (Eucalyptus grandis) to access its potential for industrial application in the pulp and paper industry. Enzyme analysis on days 15, 30, and 60 showed the presence of laccase and xylanase as key enzymes. The production of endo-glucanase (CMCase) and exo-glucanase (FPase) was very low. Scanning electron microscopy (SEM) showed the surface colonization of wood and loosening of wood fibers in C. albidus-treated samples. Fourier-transformation infrared spectroscopy (FT-IR) indicated the chemical modification of eucalyptus wood. Denaturing gradient gel electrophoresis (DGGE) analysis on days 15, 30, and 60 confirmed the presence of C. albidus throughout the experiments. Cryptococcu albidus was able to suppress the growth of a native population. Further, after 60 days both the control and treated eucalyptus wood chips were given kraft pulping treatment. The kappa number of pulp of control wood was 21 and for treated wood was 17. Kappa number is considered a measure of lignin content in wood; hence the treatment of eucalyptus by C. albidus (biopulping) was effective in reducing its lignin content and can be used for biopulping in the pulp and paper industry.

Prediction model based on decision tree analysis for laccase mediators.

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (E(i)), pK(a), enthalpy of formation of radical (Δ(f)H), and OH bond dissociation energy (D(O-H)). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Potential applications of laccase-mediated coupling and grafting reactions: a review.

Many industries are currently pursuing enzymatic approaches for developing green chemistry technologies mainly due to shortcomings of physico-chemical methods, growing environmental concerns, legal restrictions, and increasing scientific knowledge. Laccase-assisted reactions, in particular, are being intensively investigated as they are generally eco-friendly and have wide application potential. Laccases only require oxygen as co-substrate, they release water as the only by-product and have a wide substrate range which can be further extended by use of laccase-mediator systems. Consequently, research covering various applications of laccase has been rapidly increasing in recent years, particularly in the areas of coupling and grafting reactions. This review summarizes the advances that have been made in developing technologies based on laccase-mediated coupling and grafting reactions for potential application in areas such as environmental pollution control, modification of lignocellulose materials, food industry, biosensors, textile industry, pharmaceutical industry, and in organic synthesis.

Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes.

Fungal laccases have been used in various fields ranging from processes in wood and paper industries to environmental applications. Although a few bacterial laccases have been characterized in recent years, prokaryotes have largely been neglected as a source of novel enzymes, in part due to the lack of knowledge about the diversity and distribution of laccases within Bacteria. In this work genes for laccase-like enzymes were searched for in over 2,200 complete and draft bacterial genomes and four metagenomic datasets, using the custom profile Hidden Markov Models for two- and three-domain laccases. More than 1,200 putative genes for laccase-like enzymes were retrieved from chromosomes and plasmids of diverse bacteria. In 76% of the genes, signal peptides were predicted, indicating that these bacterial laccases may be exported from the cytoplasm, which contrasts with the current belief. Moreover, several examples of putatively horizontally transferred bacterial laccase genes were described. Many metagenomic sequences encoding fragments of laccase-like enzymes could not be phylogenetically assigned, indicating considerable novelty. Laccase-like genes were also found in anaerobic bacteria, autotrophs and alkaliphiles, thus opening new hypotheses regarding their ecological functions. Bacteria identified as carrying laccase genes represent potential sources for future biotechnological applications.

Industrial and biotechnological applications of ligninolytic enzymes of the basidiomycota: a review

Ligninolytic enzymes of the basidiomycetes play a crucial role in the global carbon cycle. The demand for application of ligninolytic enzymes complexes of white-rot fungi in industry and biotechnology is ever increasing due to their use in a variety of processes. Ligninolytic enzymes have potential applications in a large number of fields, including the chemical, fuel, food, agricultural, paper, textile, cosmetic industrial sectors and more. This ligninolytic system of white-rot fungi is also directly involved in the degradation of various xenobiotic compounds and dyes. Their capacities to remove xenobiotic substances and produce polymeric products make them a useful tool for bioremediation purposes. This paper reviews the applications of ligninolytic enzymes of basidiomycetes within different industrial and biotechnological area.

Kinetic and theoretical comprehension of diverse rate laws and reactivity gaps in Coriolus hirsutus laccase-catalyzed oxidation of acido and cyclometalated Ru(II) complexes.

The reactivity of the acido Ru(II) complexes cis-[RuCl(2)(LL)(2)], [RuCO(3)(LL)(2)], cis-[RuCO(3)-(bquin)(2)] (LL = 2,2'-bipyridine (bpy) and 1,10-phenanthroline (phen); bquin = 2,2'-biquinoline) and cyclometalated Ru(II) derivatives of 2-phenylpyridine and 4-(2-tolyl)pyridine [Ru(o-C(6)H(4)-2-py)(phen)(2)]PF(6) (1), [Ru(o-C(6)H(3)-p-R-2-py)(bpy)(MeCN)(2)]PF(6) (2), and [Ru(o-C(6)H(3)-p-R-2-py)(phen)(MeCN)(2)]PF(6) (3) (R = H (a), Me (b)) toward laccase from Coriolus hirsutus has been investigated by conventional UV-vis spectroscopy at pH 3-7 and 25 degrees C. The acido and cyclometalated complexes are readily oxidized into the corresponding Ru(III) species, but the two types of complexes differ substantially in reactivity and obey different rate laws. The acido complexes are oxidized more slowly and the second-order kinetics, first-order in laccase and Ru(II), holds with the rate constants around 5 x 10(4) M(-1) s(-1) at pH 4.5 and 25 degrees C. The cyclometalated complexes 1-3 react much faster and the hyperbolic Michaelis-Menten kinetics holds. However, it is not due to formation of an enzyme-substrate complex but rather because of the ping-pong mechanism of catalysis, viz. E(ox) + Ru(II) --> E(red) + Ru(III) (k(1)); E(red) + 1/4O(2) --> E(ox) (k(2)), with the rate constants k(1) in the range (2-9) x 10(7) M(-1) s(-1) under the same conditions. The huge values of k(1) move the enzymatic oxidation toward a kinetic regime when the dioxygen half-reaction becomes the rate-limiting step. Cyclometalated compounds 1-3 can therefore be used for routine estimation of k(2), that is, the rate constant for reoxidation for laccases by dioxygen. The mechanism proposed was confirmed by the direct stopped-flow measurements of the k(2) rate constant (8.1 x 10(5) M(-1) s(-1) at 26 degrees C) and supported by the theoretical modeling of interaction between the bpy analogue of 1 and Coriolus hirsutes laccase using Monte Carlo simulations.

Laccases and their applications: a patent review.

Laccases are an interesting group of multi copper enzymes, which have received much attention of researchers in last decades due to their ability to oxidize both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants. This makes these biocatalysts very useful for their application in several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, polymer synthesis, bioremediation of contaminated soils, wine and beverage stabilization. Laccases are also used as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. Recently, the utility of laccases has also been applied to nanobiotechnology. This paper reviews recent and important patents related to the properties, heterologous production, molecular cloning, and applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates.

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.

Stereochemical determination and bioactivity assessment of (S)-(+)-curcuphenol dimers isolated from the marine sponge Didiscus aceratus and synthesized through laccase biocatalysis.

Electrospray ionization mass spectrometry-guided isolation of extracts from Didiscus aceratus led to the discovery of several new derivatives of the bioactive bisabolene-type sponge metabolite (S)-(+)-curcuphenol (1). The compounds obtained by this method included a mixture of known (2) and new (3) dihydroxylated analogs as well as a novel family of dimeric derivatives, dicurcuphenols A-E (4-8), and dicurcuphenol ether F (9). Dimers 4-9 were also subsequently obtained through a hemisynthetic method in which 1 was incubated with the enzyme laccase. Atropisomeric dimers 5 and 6 were subjected to vibrational circular dichroism analysis thereby establishing their absolute biaryl axial chirality as P and M, respectively. In contrast to 1, metabolites 2-9 exhibited weak or no cytotoxic or lipoxygenase inhibitory effects.

[Comparative stability assessment of laccases from the basidiomycetes Coriolus hirsutus and Coriolus zonatus in the presence of effectors]. / Sravnitel'noe izuchenie stabil'nosti lakkaz iz bazidiomitsetov Coriolus hirsutus i Coriolus zonatus v prisutstvii éffectorov.

Stability characteristics of the laccases of the basidiomycetes Coriolus hirsutus and Coriolus zonatus were measured comparatively at temperatures 25 and 40 degrees C in the presence of various effectors (proteins, salts, polyalcohols, polyacids, and polyelectrolytes). Stabilization effects of cations on the laccases from C. hirsutus and C. zonatus decreased in the descending series Cu2+ > Mg2+ > Ca2+ and Ca2+ > Mg2+ > Mn2+, respectively. Tween 20 caused insignificant stabilization of the two enzymes. The C. zonatus laccase was also insignificantly stabilized as a result of treatment with bovine serum albumin. The enzymatic activity of the laccase preparations from C. hirsutus and C. zonatus was conserved virtually completely after vacuum drying (84 and 93%, respectively). The most effective stabilizer of the C. hirsutus laccase was found to be dextran (17 kD). Dry preparations treated with this agent conserved up to 95% of the enzymatic activity. The most effective stabilizer of the C. zonatus was polyacrylic acid (102% of the initial activity).