Laccase Research Papers - Academia.edu (original) (raw)

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very... more

Laccases have received much attention from researchers in last decades due to their ability to oxidise both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Such applications include the detoxification of industrial effluents, mostly from the paper and pulp, textile and petrochemical industries, use as a tool for medical diagnostics and as a bioremediation agent to clean up herbicides, pesticides and certain explosives in soil. Laccases are also used as cleaning agents for certain water purification systems, as catalysts for the manufacture of anti-cancer drugs and even as ingredients in cosmetics. In addition, their capacity to remove xenobiotic substances and produce polymeric products makes them a useful tool for bioremediation purposes. This paper reviews the applications of laccases within different industrial fields as well as their potential extension to the nanobiotechnology area.

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. In fungi, laccases... more

Laccases (benzenediol:oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases that catalyze the oxidation of an array of aromatic substrates concomitantly with the reduction of molecular oxygen to water. In fungi, laccases carry out a variety of physiological roles during their life cycle. These enzymes are being increasingly evaluated for a variety of biotechnological applications due to their broad substrate range. In this review, the most recent studies on laccase structural features and catalytic mechanisms along with analyses of their expression are reported and examined with the aim of contributing to the discussion on their structure–function relationships. Attention has also been paid to the properties of enzymes endowed with unique characteristics and to fungal laccase multigene families and their organization.

Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth... more

Pleurotus ostreatus is a white rot basidiomycete that produces several extracellular laccase isoenzymes, including phenol oxidase A1b (POXA1b), POXA2, and POXC. POXC was the most abundant isoenzyme produced under all of the growth conditions examined in this study. Copper was the most efficient inducer of laccase activity among the putative inducers tested. The amounts of all of the previously described laccase isoenzymes increased substantially in copper-supplemented cultures. Under these conditions expression of POX isoenzymes was regulated at the level of gene transcription. It is worth noting that poxa1b mRNA was the most abundant induced transcript at all of the growth times analyzed, and the amount of this mRNA increased until day 7. The discrepancy between thepoxa1b transcript and protein amounts can be explained by the presence of a high level of the protein in P. ostreatuscellular extract, which indicated that the POXA1b isoenzyme could be inefficiently secreted and/or that...

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of pathogenic bacteria, fungi and helminths. The study of melanin is difficult because these pigments defy complete... more

Melanins are enigmatic pigments that are produced by a wide variety of microorganisms including several species of pathogenic bacteria, fungi and helminths. The study of melanin is difficult because these pigments defy complete biochemical and structural analysis. Nevertheless, the availability of new reagents in the form of monoclonal antibodies and melanin-binding peptides, combined with the application of various physical techniques, has provided insights into the process of melanization. Melanization is important in microbial pathogenesis because it has been associated with virulence in many microorganisms. Melanin appears to contribute to virulence by reducing the susceptibility of melanized microbes to host defence mechanisms. However, the interaction of melanized microbes and the host is complex and includes immune responses to melanin-related antigens. Production of melanin has also been linked to protection against environmental insults. Interference with melanization is a ...

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was... more

The oxidation of polycyclic aromatic compounds was studied in systems consisting of laccase from Trametes versicolor and so-called mediator compounds. The enzymatic oxidation of acenaphthene, acenaphthylene, anthracene, and fluorene was mediated by various laccase substrates (phenols and aromatic amines) or compounds produced and secreted by white rot fungi. The best natural mediators, such as phenol, aniline, 4-hydroxybenzoic acid, and 4-hydroxybenzyl alcohol were as efficient as the previously described synthetic compounds ABTS [2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] and 1-hydroxybenzotriazole. The oxidation efficiency increased proportionally with the redox potentials of the phenolic mediators up to a maximum value of 0.9 V and decreased thereafter with redox potentials exceeding this value. Natural compounds such as methionine, cysteine, and reduced glutathione, containing sulfhydryl groups, were also active as mediator compounds.

Sugarcane bagasse hydrolysis with 2.5% (v/v) HCl yielded 30.29g/L total reducing sugars along with various fermentation inhibitors such as furans, phenolics and acetic acid. The acid hydrolysate when treated with anion exchange resin... more

Sugarcane bagasse hydrolysis with 2.5% (v/v) HCl yielded 30.29g/L total reducing sugars along with various fermentation inhibitors such as furans, phenolics and acetic acid. The acid hydrolysate when treated with anion exchange resin brought about maximum reduction in furans (63.4%) and total phenolics (75.8%). Treatment of hydrolysate with activated charcoal caused 38.7% and 57.5% reduction in furans and total phenolics, respectively. Laccase reduced total phenolics (77.5%) without affecting furans and acetic acid content in the hydrolysate. Fermentation of these hydrolysates with Candida shehatae NCIM 3501 showed maximum ethanol yield (0.48g/g) from ion exchange treated hydrolysate, followed by activated charcoal (0.42g/g), laccase (0.37g/g), overliming (0.30g/g) and neutralized hydrolysate (0.22g/g).

The ability of peroxidases and laccases enzymes to treat organic pollutants is reviewed. Enzymatic methods generally have low energy requirements, are easy to control, can operate over a wide range of conditions and have a minimal... more

The ability of peroxidases and laccases enzymes to treat organic pollutants is reviewed. Enzymatic methods generally have low energy requirements, are easy to control, can operate over a wide range of conditions and have a minimal environmental impact. Peroxidases and laccases have broad substrate specificities and can catalyze the oxidation of a wide range of toxic organic compounds. The results show that an enzymatic oxidation can diminish the toxicity of some polycyclic aromatic hydrocarbons (PAHs), phenols, organophosphorus pesticides and azo dyes in laboratory and some field conditions. Due to the hydrophobicity and low aqueous solubility of these substrates, reactions are usually performed in the presence of organic solvents. However, it was detected that organic solvents can provoke enzyme denaturation, unfavorable substrate partition, inhibition or stabilization of enzyme–substrate complexes, depending on the enzyme, substrate and organic solvent used. Strategies to overcome these problems are proposed. Additionally, the low stability of heme-containing peroxidases to hydrogen peroxide, the low reaction rates of laccases, the mediators toxicity, the limited availability and high costs of these enzymes are other limitations detected for commercial applications. Due to field reaction conditions are more complex than laboratory conditions efforts have to be made to achieve the cheap overproduction of these biocatalysts in heterologous hosts and also their modification by chemical means or protein engineering to obtain more robust and active enzymes.

The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel... more

The major laccase isoenzyme LAP2 secreted by the white-rot basidiomycete Trametes pubescens in response to high copper concentrations was purified to apparent electrophoretic homogeneity using anion-exchange chromatography and gel filtration. The monomeric protein has a molecular mass of 65 kDa, of which 18% is glycosylation, and a pI value of 2.6. The pH optima of the laccase depend on the substrates oxidized and show bell-shaped pH activity profiles with an optimum of 3-4.5 for phenolic substrates such as 2,6-dimethoxyphenol or syringaldazine, while the non-phenolic substrates ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and ferrocyanide show a monotonic pH profile with a rate increasing with decreasing pH. The catalytic efficiencies k(cat)/K(m) determined for some of its substrates were 48 x 10(6), 47 x 10(6), 20 x 10(6) and 7 x 10(6) M(-1) s(-1) for ABTS, syringaldazine, ferrocyanide and oxygen, respectively. Furthermore, the gene lap2 encoding the purified l...

The evolution of lignin biosynthesis was critical in the transition of plants from an aquatic to an upright terrestrial lifestyle. Lignin is assembled by oxidative polymerization of two major monomers, coniferyl alcohol and sinapyl... more

The evolution of lignin biosynthesis was critical in the transition of plants from an aquatic to an upright terrestrial lifestyle. Lignin is assembled by oxidative polymerization of two major monomers, coniferyl alcohol and sinapyl alcohol. Although two recently discovered laccases, LAC4 and LAC17, have been shown to play a role in lignin polymerization in Arabidopsis thaliana, disruption of both genes only leads to a relatively small change in lignin content and only under continuous illumination. Simultaneous disruption of LAC11 along with LAC4 and LAC17 causes severe plant growth arrest, narrower root diameter, indehiscent anthers, and vascular development arrest with lack of lignification. Genome-wide transcript analysis revealed that all the putative lignin peroxidase genes are expressed at normal levels or even higher in the laccase triple mutant, suggesting that lignin laccase activity is necessary and nonredundant with peroxidase activity for monolignol polymerization during...

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese... more

Panaeolus sphinctrinus, Panaeolus papilionaceus, and Coprinus friesii are described as producers of ligninolytic enzymes. P. papilionaceus and P. sphinctrinus both produced a laccase. In addition, P. sphinctrinus produced a manganese peroxidase. C. friesii secreted a laccase and two peroxidases similar to the peroxidase of Coprinus cinereus. The purified laccases and peroxidases were characterized by broad substrate specificities, significant enzyme activities at alkaline pH values, and remarkably high pH optima. The two peroxidases of C. friesii remained active at pH 7.0 and 60 degrees C for up to 60 min of incubation. The peroxidases were inhibited by sodium azide and ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas the laccases were inhibited by sodium azide and N,N-diethyldithiocarbamic acid. As determined by native polyacrylamide gel electrophoresis and isoelectric focusing, all three fungi produced laccase isoenzymes.

1-Hydroxybenzotriazole, violuric acid, andN-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons.... more

1-Hydroxybenzotriazole, violuric acid, andN-hydroxyacetanilide are three N-OH compounds capable of mediating a range of laccase-catalyzed biotransformations, such as paper pulp delignification and degradation of polycyclic hydrocarbons. The mechanism of their enzymatic oxidation was studied with seven fungal laccases. The oxidation had a bell-shaped pH-activity profile with an optimal pH ranging from 4 to 7. The oxidation rate was found to be dependent on the redox potential difference between the N-OH substrate and laccase. A laccase with a higher redox potential or an N-OH compound with a lower redox potential tended to have a higher oxidation rate. Similar to the enzymatic oxidation of phenols, phenoxazines, phenothiazines, and other redox-active compounds, an “outer-sphere” type of single-electron transfer from the substrate to laccase and proton release are speculated to be involved in the rate-limiting step for N-OH oxidation.

Olive-mill wastewater (OMW), an effluent of olive oil extraction process, is annually produced in huge amounts in olive growing areas. An interesting option for its disposal is the spreading on agricultural land, provided that phytotoxic... more

Olive-mill wastewater (OMW), an effluent of olive oil extraction process, is annually produced in huge amounts in olive growing areas. An interesting option for its disposal is the spreading on agricultural land, provided that phytotoxic effects are neutralized. The objective of the present investigation was to evaluate the potential of an enzyme-based treatment in removing OMW phytotoxicity. To this aim, germinability experiments on durum wheat (Triticum durum Desf. cv. Duilio) were conducted in the presence of different dilutions of raw or enzyme-treated OMW. OMW treatment with laccase resulted in a 65% and 86% reduction in total phenols and ortho-diphenols respectively, due their polymerization as revealed by size-exclusion chromatography. Raw OMW exerted a significant concentration-dependent inhibition on the germinability of durum wheat seeds which was evident up to a dilution rate of 1:8. When the effluent was treated with a fungal laccase, germinability was increased by 57% at a 1:8 dilution and by 94% at a 1:2 dilution, as compared to the same dilutions using untreated OMW. The treatment with laccase also decreased the mean germination time by about 1 day as compared to untreated controls. These results show that germinability inhibition due to OMW can be reduced effectively using fungal laccase, suggesting that phenols are the main determinants of its phytotoxicity.

The ability of several Pleurotus spp. strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied. All strains tested in this work were able to grow in OMW without any addition of nutrients and any... more

The ability of several Pleurotus spp. strains to remove phenolic compounds from an olive oil mill wastewater (OMW) was studied. All strains tested in this work were able to grow in OMW without any addition of nutrients and any pre-treatment, except sterilization. High laccase activity was measured in the growth medium, while 69–76% of the initial phenolic compounds were removed. The black color of OMW became yellow-brown and brighter as the strains grew. The lowest phenolic concentrations were reached after 12/15 days. A decrease of the phytotoxicity, as described by the parameter Germination Index, was noticed in the OMW treated with some Pleurotus spp strains, although this decrease was not proportional to the phenolic removal. A new parameter, namely Phenol-toxicity Index, was considered in the present paper. Using this parameter it was found that the remaining phenolics and/or some of the oxidation products of the laccase reaction in the treated OMW were more toxic than the original phenolic compounds.

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila... more

A genomic DNA segment encoding an extracellular laccase was isolated from the thermophilic fungus Myceliophthora thermophila, and the nucleotide sequence of this gene was determined. The deduced amino acid sequence of M. thermophila laccase (MtL) shows homology to laccases from diverse fungal genera. A vector containing the M. thermophila laccase coding region, under transcriptional control of an Aspergillus oryzae alpha-amylase gene promoter and terminator, was constructed for heterologous expression in A. oryzae. The recombinant laccase expressed in A. oryzae was purified to electrophoretic homogeneity by anion-exchange chromatography. Amino-terminal sequence data suggests that MtL is synthesized as a preproenzyme. The molecular mass was estimated to be approximately 100 to 140 kDa by gel filtration on Sephacryl S-300 and to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carbohydrate analysis revealed that MtL contains 40 to 60% glycosylation. The laccase ...

The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is... more

The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is performed by the oxidised form of the mediator (Med(ox)), generated on its interaction with laccase. The Med(ox) can follow either an electron transfer (ET) or a radical hydrogen atom transfer (HAT) route of oxidation of the substrates. Experimental evidence is reported that enables unambiguous assessment of the occurrence of either one the oxidation routes with each of the investigated mediators, namely, ABTS, HBT, HPI and VLA. Support to the conclusions is provided by (i) investigating the intermolecular selectivity of oxidation with appropriate substrates, (ii) attempting Hammett correlations for the oxidation of a series of 4-X-substituted benzyl alcohols, (iii) measuring the kinetic isotope effect, (iv) investigating the product pattern with suitable ...

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of... more

We studied the metabolism of polycyclic aromatic hydrocarbons (PAHs) by using white rot fungi previously identified as organisms that metabolize polychlorinated biphenyls. Bran flakes medium, which has been shown to support production of high levels of laccase and manganese peroxidase, was used as the growth medium. Ten fungi grown for 5 days in this medium in the presence of anthracene, pyrene, or phenanthrene, each at a concentration of 5 microg/ml could metabolize these PAHs. We studied the oxidation of 10 PAHs by using laccase purified from Coriolopsis gallica. The reaction mixtures contained 20 microM PAH, 15% acetonitrile in 60 mM phosphate buffer (pH 6), 1 mM 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), and 5 U of laccase. Laccase exhibited 91% of its maximum activity in the absence of acetonitrile. The following seven PAHs were oxidized by laccase: benzo[a]pyrene, 9-methylanthracene, 2-methylanthracene, anthracene, biphenylene, acenaphthene, and phenanthrene...