Melanocarpus Albomyces Laccase Research Articles

Comparative modeling and molecular docking analysis of white, brown and soft rot fungal laccases using lignin model compounds for understanding the structural and functional properties of laccases

Extrinsic catalytic properties of laccase enable it to oxidize a wide range of aromatic (phenolic and non-phenolic) compounds which makes it commercially an important enzyme. In this study, we have extensively compared and analyzed the physico-chemical, structural and functional properties of white, brown and soft rot fungal laccases using standard protein analysis software. We have computationally predicted the three-dimensional comparative models of these laccases and later performed the molecular docking studies using the lignin model compounds. We also report a customizable rapid and reliable protein modelling and docking pipeline for developing structurally and functionally stable protein structures. We have observed that soft rot fungal laccases exhibited comparatively higher structural variation (higher random coil) when compared to brown and white rot fungal laccases. White and brown rot fungal laccase sequences exhibited higher similarity for conserved domains of Trametes versicolor laccase, whereas soft rot fungal laccases shared higher similarity towards conserved domains of Melanocarpus albomyces laccase. Results obtained from molecular docking studies showed that aminoacids PRO, PHE, LEU, LYS and GLN were commonly found to interact with the ligands. We have also observed that white and brown rot fungal laccases showed similar docking patterns (topologically monomer, dimer and trimer bind at same pocket location and tetramer binds at another pocket location) when compared to soft rot fungal laccases. Finally, the binding efficiencies of white and brown rot fungal laccases with lignin model compounds were higher compared to the soft rot fungi. These findings can be further applied in developing genetically efficient laccases which can be applied in growing biofuel and bioremediation industries.

Mediated electron transfer of cellobiose dehydrogenase and glucose oxidase at osmium polymer-modified nanoporous gold electrodes

Nanoporous and planar gold electrodes were utilised as supports for the redox enzymes Aspergillus niger glucose oxidase (GOx) and Corynascus thermophilus cellobiose dehydrogenase (CtCDH). Electrodes modified with hydrogels containing enzyme, Os-redox polymers and the cross-linking agent poly(ethylene glycol)diglycidyl ether were used as biosensors for the determination of glucose and lactose. Limits of detection of 6.0 (±0.4), 16.0 (±0.1) and 2.0 (±0.1) μM were obtained for CtCDH-modified lactose and glucose biosensors and GOx-modified glucose biosensors, respectively, at nanoporous gold electrodes. Biofuel cells composed of GOx- and CtCDH-modified gold electrodes were utilised as anodes, together with Myrothecium verrucaria bilirubin oxidase (MvBOD) or Melanocarpus albomyces laccase as cathodes, in biofuel cells. A maximum power density of 41 μW/cm(2) was obtained for a CtCDH/MvBOD biofuel cell in 5 mM lactose and O2-saturated buffer (pH 7.4, 0.1 M phosphate, 150 mM NaCl).

On the reactivity of the Melanocarpus albomyces laccase and formation of coniferyl alcohol dehydropolymer (DHP) in the presence of ionic liquid 1-allyl-3-methylimidazolium chloride

Abstract Some ionic liquids are able to dissolve wood, including lignin and lignocellulose, and thus they provide an efficient reaction media for modification of globally abundant wood-based polymers. Lignin can be modified with laccases (EC 1.10.3.2), multicopper oxidases, which selectively catalyze the oxidation of phenolic hydroxyl to the phenoxy radical in lignin by using oxygen as the co-substrate and an electron acceptor. Many enzymes, including laccases, retain their catalytic activity in the presence of ionic liquids. However, the enzyme activity is usually decreased in the presence of ionic liquids, and the most deactivating ionic liquids have been observed to be those dissolving wood most efficiently. In the present study the activity, pH optimum and catalyzed oxidation of coniferyl alcohol by the laccase from the ascomycete Melanocarpus albomyces was investigated in the ionic liquid 1-allyl-3-methyl-imidazolium chloride ([Amim]Cl), known to dissolve wood and expected to affect the laccase activity. Indeed, with an increasing concentration of [Amim]Cl, the activity of M. albomyces laccase decreased, and the pH range of the enzyme activity was narrowed. The pH optimum, using 2,6-dimethoxyphenol as the substrate, was shifted from 6.5 to 6.0 when the amount of [Amim]Cl was increased to 60% (m-%). It was also found that the inhibition of laccase with NaN 3 was not as severe in the ionic liquid as in water. The insoluble fraction of the dehydropolymer (DHP) formed in the presence of [Amim]Cl had clearly higher molecular weight compared to the one formed in water. DHPs formed in the absence and presence of [Amim]Cl both contained β -5, β – β , β - O -4, α -C O/ β - O -4 and α - O -4/ β - O -4 structures. However, in the presence of [Amim]Cl, less β - O -4, slightly less β -5 and more β – β structures were formed.

A glucose/oxygen enzymatic fuel cell based on redox polymer and enzyme immobilisation at highly-ordered macroporous gold electrodes

Highly ordered macroporous electrodes are prepared by electro-deposition of gold through a polystyrene sphere template. Drop-coating redox polymer and either glucose oxidase, for the anode, or Melanocarpus albomyces laccase, for the cathode on the macroporous gold provides film-coated electrodes for assembly of membrane-less glucose/oxygen enzymatic fuel cells (EFC) in pH 7.4 buffer containing 10 mM glucose and 0.15 M NaCl. Under these conditions the maximum power density of 17 μW cm(-2) for EFCs using films adsorbed to planar gold electrodes increased to 38 μW cm(-2) for films adsorbed to 2½ sphere gold macroporous electrodes.

A mediated glucose/oxygen enzymatic fuel cell based on printed carbon inks containing aldose dehydrogenase and laccase as anode and cathode

Enzyme electrodes show great potential for many applications, as biosensors and more recently as anodes and cathodes in biocatalytic fuel cells for power generation. Enzymes have advantages over metal catalysts, as they provide high specificity and reaction rates, while operating under mild conditions. Here we report on studies related to development of mass-producible, completely enzymatic printed glucose/oxygen biofuel cells. The cells are based on filter paper coated with conducting carbon inks containing mediators and laccase, for reduction of oxygen, or aldose dehydrogenase, for oxidation of glucose. Mediator performance in these printed formats is compared to relative rate constants for the enzyme–mediator reaction in solution, for a range of anode and cathode mediators. The power output and stability of fuels cells using an acidophilic laccase isolated from Trametes hirsuta is greater, at pH 5, than that for cells based on Melanocarpus albomyces laccase, that shows optimal activity closer to neutral pH, at pH 6. Highest power output, although of limited stability, was observed for ThL/ABTS cathodes, providing a maximum power density of 3.5μWcm−2 at 0.34V, when coupled to an ALDH glucose anode mediated by an osmium complex. The stability of cell voltage above a threshold of 200mV under a moderate 75kΩ load is used to benchmark printed fuel cell performance. Highest stability was obtained for a printed fuel cell using osmium complexes as mediators of glucose oxidation by aldose dehydrogenase, and oxygen reduction by T. hirsuta laccase, maintaining cell voltage above 200mV for 137h at pH 5. These results provide promising directions for further development of mass-producible, completely enzymatic, printed biofuel cells.

Oxidation of lignans and lignin model compounds by laccase in aqueous solvent systems

The stability and activity of the low redox potential Melanocarpus albomyces laccase (MaL) in various aqueous organic (acetone, ethanol, propylene glycol, diethylene glycol monomethyl ether) solvent systems was studied spectrophotometrically using 2,6-dimethoxyphenol (2,6-DMP) as substrate. In addition, reactivity of the enzyme with two lignans; matairesinol (MR) and 7-hydroxymatairesinol (HMR), was examined by oxygen consumption measurements in the most potential aqueous organic solvent systems. Polymerization of the lignans by MaL was verified by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC). Polymerization of the higher molecular weight lignin model compound, dehydrogenation polymers (DHPs), was studied by SEC. The solubilities of industrial softwood and hardwood kraft lignins were evaluated as parameters for investigation of enzymatic modification in aqueous organic solvent systems. The functioning of MaL in different aqueous organic media was excellent. Propylene glycol and diethylene glycol monomethyl ether were better solvents than ethanol or acetone in enzymatic oxidations. Even though they were the best solvents for enzyme oxidation, ethanol and propylene glycol were selected for further tests because of their different physicochemical properties. The results obtained in this study for the use of laccase-catalysed reactions in organic solvents to improve the efficiency of lignin oxidation may be exploited in several applications and areas in which the solubility of the reactants or products is a limiting factor.

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 Å 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.

Probing the Dioxygen Route in Melanocarpus albomyces Laccase with Pressurized Xenon Gas

Laccases catalyze the oxidation of phenolic substrates and the concominant reduction of dioxygen to water. We used xenon as an oxygen probe in search of routes for the entry of dioxygen into the catalytic center. Two xenon-pressurized crystal structures of recombinant Melanocarpus albomyces laccase were determined, showing three hydrophobic Xe-binding sites located in domain C. The analysis of hydrophobic cavities in other laccase structures further suggested the preference of domain C for binding of hydrophobic species such as dioxygen, thus suggesting that the hydrophobic core of domain C could function as a channel through which dioxygen can enter the trinuclear copper center.

Essential role of the C‐terminus in Melanocarpus albomyces laccase for enzyme production, catalytic properties and structure

The C-terminus of the fungal laccase from Melanocarpus albomyces (MaL) is processed during secretion at a processing site conserved among the ascomycete laccases. The three-dimensional structure of MaL has been solved as one of the first complete laccase structures. According to the crystal structure of MaL, the four C-terminal amino acids of the mature protein penetrate into a tunnel leading towards the trinuclear site. The C-terminal carboxylate group forms a hydrogen bond with a side chain of His140, which also coordinates to the type 3 copper. In order to analyze the role of the processed C-terminus, site-directed mutagenesis of the MaL cDNA was performed, and the mutated proteins were expressed in Trichoderma reesei and Saccharomyces cerevisiae. Changes in the C-terminus of MaL caused major defects in protein production in both expression hosts. The deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL(559) was practically inactive. Detailed characterization of the purified L559A mutant expressed in S. cerevisiae showed the importance of the C-terminal plug for laccase activity, stability, and kinetics. Moreover, the crystal structure of the L559A mutant expressed in S. cerevisiae showed that the C-terminal mutation had clearly affected the trinuclear site geometry. The results in this study clearly confirm the critical role of the last amino acids in the C-terminus of MaL.

On the Reactions of Two Fungal Laccases Differing in Their Redox Potential with Lignin Model Compounds: Products and Their Rate of Formation

Laccases (EC 1.10.3.2) are multicopper oxidases able to oxidize phenolic compounds such as lignin-related polyphenols. Since the discovery that so-called mediators effectively extend the family of laccase substrates, direct interactions between lignin-like materials and laccase have gained much less attention. In this work, the aim was to characterize oxidation products formed in direct laccase-catalyzed oxidation of different guaiacylic and syringylic lignin model compounds with two different laccases: a low redox potential Melanocarpus albomyces laccase and a high redox potential Trametes hirsuta laccase. By following the formation of different, mainly biphenylic (5-5) and benzylic oxidation products, it was found that although both of these enzymes generated practically the same pattern of products with particular types of syringyl and guaiacyl compounds, in some cases a clear difference in the rates of their formation was observed. The results also confirm further to the suggestions that syringylic compounds are able to act as mediators in their own oxidation reactions and also that in some instances acetylation of phenolic material may produce altered, unexpected structures.

Structure–Function Studies of a Melanocarpus albomyces Laccase Suggest a Pathway for Oxidation of Phenolic Compounds

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

Evaluation of performance and stability of biocatalytic redox films constructed with different copper oxygenases and osmium-based redox polymers

We are interested in investigating the applications of biocatalytic mediated reduction of oxygen by oxygenases in films on electrode surfaces, as such reactions can form the basis for biosensors or biocatalytic fuel cell development. Here we present approaches aimed at improving the stability and signal output of such films. These include selection of oxygen reducing biocatalysts which are active under physiological conditions and development of redox mediators which offer the opportunity to tailor the mediator to each enzyme. It was found that for each enzyme Melanocarpus albomyces laccase (MaL), Trametes hirsutus laccase (ThL) or bilirubin oxidase (MvBOD) it was the biocatalytic films mediated by Os(2,2′-bipyridine) 2Cl·PVI that not only generated the highest current densities compared to Os(4,4′-dimethyl-2,2′-bipyridine) 2Cl·PVI and Os(4,4′-dichloro-2,2′-bipyridine) 2Cl·PVI, but also proved to be the most stable over 48 h. Under physiological conditions electrodes constructed from MvBOD generated the highest initial current densities for each of the osmium redox polymers, however these films proved to be the least stable over 48 h. Stability could be improved using surface pre-treatment.

Performance of a Glucose/O2 Enzymatic Biofuel Cell Containing a Mediated Melanocarpus albomyces Laccase Cathode in a Physiological Buffer

AbstractWe report on the performance of a prototype glucose/O2 biofuel cell in a physiological buffer. The cell consists of cathode based on Melanocarpus albomyces laccase (with a reported T1 copper redox potential of + 0.26 V vs. Ag/AgCl) co‐immobilised with an osmium redox polymer mediating film on glassy carbon (GC) separated by a Nafion 117 membrane from a GC anode in anolyte of glucose oxidase, N,N,N′,N′‐tetramethyl‐p‐phenylenediamine (TMPD) as a mediator, all immersed in pH 7.4 phosphate buffer solution containing 0.15 M NaCl and dissolved oxygen, thermostated at 37 °C, mimicking physiological conditions. Two osmium redox polymers are employed for cathodic mediation: [Os(2,2′‐bipyridine)2(polyvinylimidazole)10Cl]+/2+ (E°′ 0.22 V vs. Ag/AgCl) and [Os(4,4′‐dichloro‐2,2′‐bipyridine)2(polyvinylimidazole)10Cl]+/2+ (E°′ 0.35 V vs. Ag/AgCl). Power outputs of 52 μW cm–2 at 0.21 V and 17 μW cm–2 at 0.34 V were obtained for assembled fuel cells containing the respective redox polymer‐mediated laccase cathodes, illustrating the increased power obtained as a result of higher biocatalytic efficiency using a redox polymer with redox potential tailored for the enzyme.

Electroreduction of O 2 at a mediated Melanocarpus albomyces laccase cathode in a physiological buffer

We report on oxygen reduction in a physiological buffer solution (0.05 M phosphate buffer containing dissolved O 2, 0.1 M NaCl, pH 7.4, 37 °C) by Melanocarpus albomyces laccase, co-immobilized with [Os(2,2’-bipyridine) 2(polyvinylimidazole) 10Cl] +/2+ as a mediator, on glassy carbon electrodes. Such oxygen cathodes yielded current densities of 3.8 mA cm −2 at 0.2 V vs. Ag/AgCl, the largest current density reported to date for a mediated laccase cathode in physiological buffer solutions, showing promise for development of biocatalytic fuel cell prototypes.

A near atomic resolution structure of a Melanocarpus albomyces laccase

We have solved a crystal structure from Melanocarpus albomyces laccase expressed in the filamentous fungus Trichoderma reesei (rMaL) at 1.3A resolution by using synchrotron radiation at 100K. At the moment, this is the highest resolution that has been attained for any multicopper oxidase. The present structure confirmed our earlier proposal regarding the dynamic behaviour of the copper cluster. Thermal ellipsoids of copper atoms indicated movements of trinuclear site coppers. The direction of the type-3 copper motion was perpendicular to the type-2 copper. In addition, the structure at 1.3A resolution allowed us to describe important solvent cavities of the enzyme and the structure is also compared with other known multicopper oxidases. T2 and T3 solvent cavities, and a putative SDS-gate, formed by Ser142, Ser510 and the C-terminal Asp556 of rMaL, are described. We also observed a 2-oxohistidine, an oxidized histidine, possibly caused by a metal-catalysed oxidation by the trinuclear site coppers. To our knowledge, this is the first time that 2-oxohistidine has been observed in a protein crystal structure.

Physiological evaluation of the filamentous fungus Trichoderma reesei in production processes by marker gene expression analysis

BackgroundBiologically relevant molecular markers can be used in evaluation of the physiological state of an organism in biotechnical processes. We monitored at high frequency the expression of 34 marker genes in batch, fed-batch and continuous cultures of the filamentous fungus Trichoderma reesei by the transcriptional analysis method TRAC (TRanscript analysis with the aid of Affinity Capture). Expression of specific genes was normalised either with respect to biomass or to overall polyA RNA concentration. Expressional variation of the genes involved in various process relevant cellular functions, such as protein production, growth and stress responses, was related to process parameters such as specific growth and production rates and substrate and dissolved oxygen concentrations.ResultsGene expression of secreted cellulases and recombinant Melanocarpus albomyces laccase predicted the trends in the corresponding extracellular enzyme production rates and was highest in a narrow "physiological window" in the specific growth rate (μ) range of 0.03 – 0.05 h-1. Expression of ribosomal protein mRNAs was consistent with the changes in μ. Nine starvation-related genes were found as potential markers for detection of insufficient substrate feed for maintaining optimal protein production. For two genes induced in anaerobic conditions, increasing transcript levels were measured as dissolved oxygen decreased.ConclusionThe data obtained by TRAC supported the usefulness of focused and intensive transcriptional analysis in monitoring of biotechnical processes providing thus tools for process optimisation purposes.

A crystallographic and spectroscopic study on the effect of X-ray radiation on the crystal structure of Melanocarpus albomyces laccase

Laccases ( p-diphenol dioxygen oxidoreductases) belong to the family of blue multicopper oxidases, which catalyse the four-electron reduction of dioxygen to water concomitantly through the oxidation of substrate molecules. Blue multicopper oxidases have four coppers, a copper (T1) forming a mononuclear site and a cluster of three coppers (T2, T3, and T3′) forming a trinuclear site. Because X-rays are known to liberate electrons during data collection and may thus affect the oxidation state of metals, we have investigated the effect of X-ray radiation upon the crystal structure of a recombinant laccase from Melanocarpus albomyces through the use of crystallography and crystal absorption spectroscopy. Two data sets with different strategies, a low and a high-dose data set, were collected at synchrotron. We have observed earlier that the trinuclear site had an elongated electron density amidst coppers, suggesting dioxygen binding. The low-dose synchrotron structure showed similar elongated electron density, but the high-dose X-ray radiation removed the bulk of this density. Therefore, X-ray radiation could alter the active site of laccase from M. albomyces. Absorption spectra of the crystals (320, 420, and 590 nm) during X-ray radiation were measured at a home laboratory. Spectra clearly showed how that the band at 590 nm had vanished, resulting from the T1 copper being reduced, during the long X-ray measurements. The crystal colour changed from blue to colourless. Absorptions at 320 and 420 nm seemed to be rather permanent. The absorption at 320 nm is due to the T3 coppers and it is proposed that absorption at 420 nm is due to the T2 copper when dioxygen or a reaction intermediate is close to this copper.

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme.

Previous studies on Melanocarpus albomyces laccase have shown that this enzyme is very interesting for both basic research purposes and industrial applications. In order to obtain a reliable and efficient source for this laccase, it was produced in the filamentous fungus Trichoderma reesei. Two approaches were used: production of a non-fused laccase and a hydrophobin-laccase fusion protein. Both proteins were expressed in T. reesei under the cbh1 promoter, and significantly higher activities were obtained with the non-fused laccase in shake-flask cultures (corresponding to about 230 mg l(-1)). Northern blot analyses showed rather similar mRNA levels from both expression constructs. Western analysis indicated intracellular accumulation and degradation of the hydrophobin-laccase fusion protein, showing that production of the fusion was limited at the post-transcriptional level. No induction of the unfolded protein response pathway by laccase production was detected in the transformants by Northern hybridization. The most promising transformant was grown in a fermenter in batch and fed-batch modes. The highest production level obtained in the fed-batch culture was 920 mg l(-1). The recombinant laccase was purified from the culture supernatant after cleaving the major contaminating protein, cellobiohydrolase I, by papain. The recombinant and wild-type laccases were compared with regard to substrate kinetics, molecular mass, pH optimum, thermostability, and processing of the N- and C-termini, and they showed very similar properties.