Use of laccase as a novel, versatile reporter system in filamentous fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2'-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (Km = 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

Molecular docking and dynamics simulation analyses unraveling the differential enzymatic catalysis by plant and fungal laccases with respect to lignin biosynthesis and degradation

Laccase, widely distributed in bacteria, fungi, and plants, catalyzes the oxidation of wide range of compounds. With regards to one of the important physiological functions, plant laccases are considered to catalyze lignin biosynthesis while fungal laccases are considered for lignin degradation. The present study was undertaken to explain this dual function of laccases using in-silico molecular docking and dynamics simulation approaches. Modeling and superimposition analyses of one each representative of plant and fungal laccases, namely, Populus trichocarpa and Trametes versicolor, respectively, revealed low level of similarity in the folding of two laccases at 3D levels. Docking analyses revealed significantly higher binding efficiency for lignin model compounds, in proportion to their size, for fungal laccase as compared to that of plant laccase. Residues interacting with the model compounds at the respective enzyme active sites were found to be in conformity with their role in lignin biosynthesis and degradation. Molecular dynamics simulation analyses for the stability of docked complexes of plant and fungal laccases with lignin model compounds revealed that tetrameric lignin model compound remains attached to the active site of fungal laccase throughout the simulation period, while it protrudes outwards from the active site of plant laccase. Stability of these complexes was further analyzed on the basis of binding energy which revealed significantly higher stability of fungal laccase with tetrameric compound than that of plant. The overall data suggested a situation favorable for the degradation of lignin polymer by fungal laccase while its synthesis by plant laccase.     

Comparative Characterization of Methods for Removal of Cu(II) from the Active Sites of Fungal Laccases

Copper-containing sites of laccases isolated from the Basidiomycetes Coriolus hirsutus and Coriolus zonatus were characterized by optical methods and EPR spectroscopy. Methods for preparation of fungal laccase derivatives free from type 2 copper ions were compared. The data of EPR spectroscopy and spectrophotometric titration of copper sites showed that only a modified method based on the use of bathocuproine as a chelator for type 2 copper yielded laccase derivatives completely free from type 2 copper. The original enzymes can be reconstituted from the derivatives by dialysis under anaerobic conditions, resulting in complete recovery of native conformation of the protein molecule and the structure of the copper-containing site.     

Electrochemical Studies of a Truncated Laccase Produced in Pichia pastoris

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI→LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (k_het for LCCIa = 1.3 × 10⁻⁴ cm s⁻¹). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.     

Electrochemical studies of a truncated laccase produced in Pichia pastoris

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI-->LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (k(het) for LCCIa = 1.3 x 10(-4) cm s(-1)). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.     

Crystal structure of an ascomycete fungal laccase from Thielavia arenaria--common structural features of asco-laccases

Laccases are copper-containing enzymes used in various applications, such as textile bleaching. Several crystal structures of laccases from fungi and bacteria are available, but ascomycete types of fungal laccases (asco-laccases) have been rather unexplored, and to date only the crystal structure of Melanocarpus albomyces laccase (MaL) has been published. We have now solved the crystal structure of another asco-laccase, from Thielavia arenaria (TaLcc1), at 2.5 ? resolution. The loops near the T1 copper, forming the substrate-binding pockets of the two asco-laccases, differ to some extent, and include the amino acid thought to be responsible for catalytic proton transfer, which is Asp in TaLcc1, and Glu in MaL. In addition, the crystal structure of TaLcc1 does not have a chloride attached to the T2 copper, as observed in the crystal structure of MaL. The unique feature of TaLcc1 and MaL as compared with other laccases structures is that, in both structures, the processed C-terminus blocks the T3 solvent channel leading towards the trinuclear centre, suggesting a common functional role for this conserved 'C-terminal plug'. We propose that the asco-laccases utilize the C-terminal carboxylic group in proton transfer processes, as has been suggested for Glu498 in the CotA laccase from Bacillus subtilis. The crystal structure of TaLcc1 also shows the formation of a similar weak homodimer, as observed for MaL, that may determine the properties of these asco-laccases at high protein concentrations.     

Differential regulation by organic compounds and heavy metals of multiple laccase genes in the aquatic hyphomycete Clavariopsis aquatica

To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete Clavariopsis aquatica) for the first time. Five putative laccase genes (lcc1 to lcc5) identified in C. aquatica were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc. lcc4 is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laccases of terrestrial fungi, none of the C. aquatica laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in C. aquatica, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 μM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats.     

Properties of a laccase produced by<Emphasis Type="Italic">Phanerochaete flavido-alba</Emphasis> induced by vanillin

Phanerochaete flavido-alba is able to remove simple and polymeric phenols from the recalcitrant wastes of the olive oil industry, in a process in which a laccase is involved. This report describes the characterization of a laccase produced by P. flavido-alba and induced by vanillin. Although the amino acid composition of the purified enzyme is typical for laccases, other molecular characteristics show that it is quite different from fungal laccases. The purified laccase oxidized preferably o- and p-biphenols.     

Yellow laccase from the fungus Pleurotus ostreatus D1: purification and characterization

Yellow laccase was isolated from a solid-phase culture of the fungus Pleurotus ostreatus D1 and characterized. It is a copper-containing enzyme with molecular weight 64 kDa. Its absorption spectrum lacks the maximum at 610 nm, characteristic of fungal laccases and corresponding to type I copper atom. The optimum pH values for the enzyme were determined. They proved to be: 7.0 for syringaldazine, 8.0 for pyrocatechol, and 4.0 for 2,2'-azine-bis-(3-ethylbenzothiazoline-6-sulfonate and 2,6-dimethoxyphenol. Kinetic parameters (Km and Vmax) for oxidation of these substrates were determined. The effect of inhibitors (SDS, 2-mercaptoethanol, and EDTA) on the activity of the enzyme was studied. It was shown that yellow laccase from Pleurotus ostreatus D1 oxidized anthracene to anthraquinone by 95% without any mediator.     

Novel enzymatic oxidation of Mn2+ to Mn3+ catalyzed by a fungal laccase.

Fungal laccases are extracellular multinuclear copper-containing oxidases that have been proposed to be involved in ligninolysis and degradation of xenobiotics. Here, we show that an electrophoretically homogenous laccase preparation from the white rot fungus Trametes versicolor oxidized Mn2+ to Mn3+ in the presence of Na-pyrophosphate, with a Km value of 186 microM and a Vmax value of 0.11 micromol/min/mg protein at the optimal pH (5.0) and a Na-pyrophosphate concentration of 100 mM. The oxidation of Mn2+ involved concomitant reduction of the laccase type 1 copper site as usual for laccase reactions, thus providing the first evidence that laccase may directly utilize Mn2+ as a substrate.     

Laccase of Cyathus bulleri: structural, catalytic characterization and expression in Escherichia coli

Cyathus bulleri, a ligninolytic fungus, produces a single laccase the internal peptides (3) of which bear similarity to laccases of several white rot fungi. Comparison of the total amino acid composition of this laccase with several fungal laccases indicated dissimilarity in the proportion of some basic and hydrophobic amino acids. Analysis of the circular dichroism spectrum of the protein indicated 37% alpha-helical, 26% beta-sheet and 38% random coil content which differed significantly from that in the solved structures of other laccases, which contain higher beta-sheet structures. The critical role of the carboxylic group containing amino acids was demonstrated by determining the kinetic parameters at different pH and this was confirmed by the observation that a critical Asp is strongly conserved in both Ascomycete and Basidiomycete laccases. The enzyme was denatured in the presence of a number of denaturing agents and refolded back to functional state with copper. In the folding experiments under alkaline conditions, zinc could replace copper in restoring 100% of laccase activity indicating the non-essential role of copper in this laccase. The laccase was expressed in Escherichia coli by a modification of the ligation-anchored PCR approach making it the first fungal laccase to be expressed in a bacterial host. The laccase sequence was confirmed by way of analysis of a 435 bp sequence of the insert.     

Cell thermolysis – A simple and fast approach for isolation of bacterial laccases with potential to decolorize industrial dyes

Laccases belong to multicopper oxidases and are widespread in nature. Currently, mainly fungal laccases are applied in biotechnological processes. One reason for this is that fungal laccases are much better studied. Compared to fungal laccases, bacterial laccases possess some advantageous characteristics like high stability at elevated temperatures and alkaline pH values. Intracellular recombinant expression of bacterial laccases in E. coli makes however downstream processing more complex and time-consuming compared to extracellular expression of fungal enzymes. Here, we demonstrate that cell disruption by cell thermolysis is an efficient and simple method for the isolation and partial purification of recombinant bacterial laccases. Three different laccases, Tth from Thermus thermophilus, CotA from Bacillus subtilis and Ssl1 from Streptomyces sviceus, were used to compare cell disruption by cell thermolysis with sonication and high-pressure homogenization, with and without subsequent heat treatment. Cell thermolysis resulted in high laccase activities per gram of cell wet weight and in the highest specific activities of the laccases. For example, specific activity of Tth laccase after cell thermolysis was 469-fold higher than after sonication. Furthermore, high decolorization activity towards indigo carmine and alizarin red S of these laccases, isolated via cell thermolysis, demonstrate their potential for technical applications.     

Use of Laccase as a Novel, Versatile Reporter System in Filamentous Fungi

Laccases are copper-containing enzymes which oxidize phenolic substrates and transfer the electrons to oxygen. Many filamentous fungi contain several laccase-encoding genes, but their biological roles are mostly not well understood. The main interest in laccases in biotechnology is their potential to be used to detoxify phenolic substances. We report here on a novel application of laccases as a reporter system in fungi. We purified a laccase enzyme from the ligno-cellulolytic ascomycete Stachybotrys chartarum. It oxidized the artificial substrate 2,2′-azino-di-(3-ethylbenzthiazolinsulfonate) (ABTS). The corresponding gene was isolated and expressed in Aspergillus nidulans, Aspergillus niger, and Trichoderma reesei. Heterologously expressed laccase activity was monitored in colorimetric enzyme assays and on agar plates with ABTS as a substrate. The use of laccase as a reporter was shown in a genetic screen for the isolation of improved T. reesei cellulase production strains. In addition to the laccase from S. charatarum, we tested the application of three laccases from A. nidulans (LccB, LccC, and LccD) as reporters. Whereas LccC oxidized ABTS (K_m= 0.3 mM), LccD did not react with ABTS but with DMA/ADBP (3,5-dimethylaniline/4-amino-2,6-dibromophenol). LccB reacted with DMA/ADBP and showed weak activity with ABTS. The different catalytic properties of LccC and LccD allow simultaneous use of these two laccases as reporters in one fungal strain.     

细菌漆酶的生物信息学分析

漆酶是一种含铜的多酚氧化酶。本研究利用生物信息学分析工具对细菌的漆酶蛋白序列的基本性质、保守结构域、系统发育树以及结构等进行了分析。所分析细菌主要分布在变形菌门、放线菌和厚壁菌门。细菌漆酶的物理化学性质相似,但不同细菌来源的漆酶之间序列相似性较低,但其仍然具有容易识别的保守结构域特征。二级结构及三级结构具有明显的相似性。细菌漆酶的生物信息分析为其功能研究及在其他微生物中研究该类酶提供了基础。     

Structure,functionality and tuning up of laccases for lignocellulose and other industrial applications

Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and involves a series of electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be expanded by means of supplementary mediators that essentially function as vehicles for electron transfer. Comparisons of amino acid sequences and structural traits of selected laccases reveal conservation of the active site trinuclear center geometry but differences in loop conformations. We also evaluate the features and regions of laccases in relation to modification and evolution of laccases for various industrial applications including lignocellulosic biomass processing.     

Screening for novel laccase-producing microbes

AIMS: To discover novel laccases potential for industrial applications. METHODS AND RESULTS: Fungi were cultivated on solid media containing indicator compounds that enabled the detection of laccases as specific colour reactions. The indicators used were Remazol Brilliant Blue R (RBBR), Poly R-478, guaiacol and tannic acid. The screening work resulted in isolation of 26 positive fungal strains. Liquid cultivations of positive strains confirmed that four efficient laccase producers were found in the screening. Biochemical characteristics of the four novel laccases were typical for fungal laccases in terms of molecular weight, pH optima and pI. The laccases showed good thermal stability at 60 degrees C. CONCLUSIONS: Plate-test screening based on polymeric dye compounds, guaiacol and tannic acid is an efficient way to discover novel laccase producers. The results indicated that screening for laccase activity can be performed with guaiacol and RBBR or Poly R-478. SIGNIFICANCE AND IMPACT OF THE STUDY: Laccases have many potential industrial applications including textile dye decolourization, delignification of pulp and effluent detoxification. It is essential to find novel, efficient enzymes to further develop these applications. This study showed that relatively simple plate test screening method can be used for discovery of novel laccases.     

Comparative Study of Substrates and Inhibitors of Azospirillum lipoferum and Pyricularia oryzae Laccases

Azospirillum lipoferum and Pyricularia oryzae laccases were compared, using several substrates and inhibitors. Sixteen phenolic or nonphenolic compounds were found to be substrates of both fungal and bacterial laccases. In the presence of different phenol oxidase inhibitors, P. oryzae and A. lipoferum laccase activities had similar properties.     

The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight.

In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.     

Role of laccase from Coriolus versicolor MTCC-138 in selective oxidation of aromatic methyl group

Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations are expensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.