Purification of Pycnoporus sanguineus CS 2 laccase
Laccases (benzenediol: oxygen oxidoreductases, EC 188.8.131.52) are enzymes that catalyze the oxidation of phenolic compounds and aromatic amines with the simultaneous reduction of molecular oxygen to water . They are widely distributed in many plants and fungi, some insects and bacteria, being particularly abundant in white-rot basidiomycetes . Typical fungal laccases are described as glycosylated multicopper proteins, which are produced as extracellular monomeric forms of around 60-80 kDa, containing four copper atoms and 15-20% carbohydrates. Operatively, they are moderately thermotolerant, showing optima activity at 50-55 °C, and under acidic conditions (pH 3-5); although their maxima stability occurs in the alkaline zone (pH 8-9) . Their copper atoms are distributed in three different sites bringing unique spectroscopic properties: The type 1 copper (CuT1) atom, is responsible of the intense blue color of enzymes by light absorption around 610 nm; The type 2 copper (CuT2) atom exhibits a weak absorption in the visible region; and the two type 3 copper (CuT3) atoms are present as a binuclear center, which has an absorption maximum about 330 nm. Moreover, CuT2 and CuT3 copper atoms are structural and functionally arranged as a trinuclear cluster. The four copper atoms form part of the active site of enzyme contributing directly to reaction. CuT1 is involved in the initial electron subtraction from reducer substrates, while trinuclear CuT2 and CuT3 cluster is responsible of the electron transference, from CuT1 to diatomic oxygen .
According to their redox potential, most of blue laccases belong to class II (-500 to -600 mV) or class III (-700 to -800 mV) laccases . This is a disadvantage when compared to the ability of lignin peroxidase (LiP) and manganese peroxidase (MnP) to attack compounds with higher redox potential, including non-phenolic lignin units. To overcome this limitation laccases have evolutively developed a synergistic catalytic strategy, which combines a flexible ability to recognize a great variety of chemical compounds, with an extended capability to act at the distance through the activation of diffusible low molecular substances which serve as redox mediators. From a biological stand point this strategy let laccases to become one of the most versatile enzymes in nature, adaptable to multiple functions in plants, insects, fungi and bacteria. Another interesting possibility arises from the properties of atypical “yellow” and “white” laccases, which have shown the ability to catalyze the direct oxidation of high redox potential non-phenolic lignin model substrates or polyaromatic hydrocarbons [6, 7]. It has been proposed that the improved redox capabilities of these laccases come up either, by substituting some copper atoms for zinc, iron, or manganese in the metal clusters or by a change of the redox state of the CuT1(due to the interaction with a lignin-derived ligand) at the active site of, otherwise normal laccase protein structures. So, evolution and prevalence of laccases as a part of the lignin modifying enzyme (LME) system in white-rot basidiomycetes could also be the result of a “biochemical spring-up” mechanism acting under a short term ecophysiological selective pressure.
Whether directly or by mediation, laccases are able to oxidize a broad range of natural or xenobiotic compounds, including: mono, poly or methoxy- amine- and chloro-substituted phenols as well as aromatic heterocyclic and inorganic/organometallic substances; some of them recognized among the most recalcitrant industrial pollutants, for example; polycyclic aromatic hydrocarbons (PAH), pentachlorophenols (PCP), polychlorinated biphenyls (PCB), 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT), trinitrotoluene (TNT), and many azo, triarylmethane, anthraquinonic, indigoid and heterocyclic textile dyes [8,9]. Therefore, laccases are considered enzymes with a great potential for the development of environmental and industrial applications. Current and potential laccase applications include biobleaching of pulp and bioremediation of pulp and paper industries, bioremediation of olive mill wastewater, bioremediation of effluents of the textile and dye manufacturing industries, biocatalytic synthesis of antibiotics and novel polymeric materials, development of biosensors, clarification and stabilization of beer, juices and wines, and panification [1, 10, 11]. Some laccase formulations have already reached a commercial significance, but general thought is that their biotechnological applications and performances could be greatly improved or expanded with the development and finding of new enzyme variants with desirable functional properties, such as higher redox potential, optimum activity at neutral or alkaline pH and thermal stability [12, 13, 14]. It has been proposed that these new laccases could be obtained by protein engineering or through the exploration of the natural biodiversity. The importance of prospective studies in natural biodiversity applying an ecophysiological approach is illustrated by reports about isolation of new thermostable laccases from fungi, either from thermophilic compost  or tropical environments [13,16], or by the finding of novel laccases with improved ability to oxidize substrates with a higher than normal redox potential culturing under solid phase conditions [6, 7,17]. Northeast Mexico shelters a high diversity of white-rot basidiomycetes as a result of its particular combination of physiography and climate, including species associated to pine, oak and mixed forests, sub-mountain and semi-desert scrublands, and grass-land. In this work we first present information on the isolation, identification and selection of a northeast Mexico native strain of Pycnoporus sanguineus CS2, as a potential producer of thermostable laccases. Results on the purification and partial characterization of its laccase are then exposed, stressing on its thermal stability and ability to attack high redox potential substrates and recalcitrant dyes without the participation of redox mediators.
2. Materials and methods
All chemicals used as buffers, enzyme substrates, culture media ingredients and electrophoresis reagents, were reactive grade and commercially available through local distributors of Difco, Sigma-Aldrich and Fluka, or BioRad products: PDB (potato dextrose broth), bacteriological agar, yeast extract, malt extract, peptone and dextrose, were from Difco. Acrylamide, bis-acrylamide, TEMED (N,N,N’,N’- tetramethylethylenediamine), 2-mercaptoethanol, SDS (sodium dodecyl sulfate), trizma-base, glycine, Coomassie blue, and low range markers kit, were from Bio Rad. Enzyme substrates and dyes: 2,6-dimethoxyphenol (2,6-DMP); o-dianisidine (3,3′-dimethoxybenzidine); ABTS (2,2’–azino-bis(3-ethylbenzthiazolin-6-sulphonic acid); syringaldazine (4-hydroxy-3,5-dimethoxybenzaldehyde azine); DMAB (3-dimethylaminobenzoic acid), MBTH (3-methyl-2-benzothiazolinone hydrazone); Methyl Red (Acid Red 2; CI 13020); Reactive Black 5 (RB 5; CI 20505), were from Sigma, Fluka or Aldrich. Chromatographic matrices; DEAE-Sepharose and Q-Sepharose from Sigma-Aldrich, and Biogel P-100 from BioRad. All other chemicals, including solvents, inorganic salts, acids and bases, were from Reactivos Químicos Monterrey, S.A. or CTR-Scientific S.A. de C.V. Solutions and culture media were prepared with bidistilled water from Laboratorios Monterrey, S. A.
2.2. Isolation and identification of fungal strain
The Pycnoporus strain used in our experiments was isolated from fruit bodies developing on decayed logs that were gathered in a man-disturbed sub-mountain scrubland around Monterrey, N.L. (Northeast México). Mycelia cultures were obtained by standard mycological techniques, according to the procedure previously described . Briefly, small flesh sections were aseptically removed from inside the carpophores, and transferred to YMGA (0.4% Yeast extract, 1.0% Malt extract, 0.4% Glucose, 1.5% agar) plates, supplemented with 10% Tartaric Acid and 0.004% Benomyl. Plates were incubated at 28 °C, and those with extensive mycelia growth were analyzed under the microscope to confirm a successful isolation. Stock cultures were maintained by periodic transfers every two or three months on YMGA plates and kept refrigerated at 4 °C. Carpophore morphologic features, measurements and photographs were registered previous to dissections for microscopic examination, and identification was done by following the taxonomical keys in reference , and in  for genera of polypores and the most common macromycetes from Mexico, respectively.
2.3. Enzyme and protein assays
Laccase activity was determined by triplicate at 25 °C in 3 ml cuvettes, monitoring the increase in absorbance at A468 (ε=49,600 M-1cm-1), using a Shimadzu UV-VIS mini 1240 spectrophotometer and 2,6-DMP as substrate. The assay mixture contained 0.01ml enzymatic extract, 0.1 ml of 60 mM 2.6-DMP in 2.89 ml of 200 mM citrate-phosphate buffer at pH 4.0 . One unit of laccase activity was defined as the amount of enzyme required to oxidize 1 μmol of 2, 6-DMP per minute at 25 °C. In some cases, it was necessary to assess the presence of lignin peroxidase (LiP) and manganese peroxidase (MnP). LiP activity was estimated by the H2O2-dependant veratryl alcohol oxidation to veratraldehyde as in reference  MnP activity was measured by the formation of Mn3+-tartrate complex during the oxidation of MnSO4 in tartrate buffer as in . The protein concentration was estimated by the Bradford assay (Protein Assay Bradford of BioRad) with bovine serum albumin as standard.
2.4. Strain selection and enzyme production
Isolated Pycnoporus strain was selected as a potential source of thermostable laccases in a preliminary screening with crude enzyme preparations. 250 ml Erlenmeyer flask, containing 50 ml of natural LME inducers containing Bran Flakes (BF) media (2% Bran Flakes® in 60 mM potassium phosphate pH 6.0) , were inoculated with three 0.5 cm diameter cylinders of mycelia taken from the border of a YMGA growing colony and incubated at 28 °C under agitation at 150 rpm. Aliquots (200 μl) were removed from cultures and the extracellular fluid was separated by centrifugation at 14 K (Eppendorf 5415 C). Enzyme activity in supernatants was determined with 2 mM 2, 6-DMP final concentration, as described above, after sample incubation at 60 °C during different times in a four hour period. This phase of study included four different Pycnoporus sp. (CS 2, CS 20, CS 43 and LE 90) strains from the native basidiomycete collection of our laboratory. Among them, Pycnoporus sanguineus CS 2 was selected on the basis of its ability to produce a thermostable 2,6-DMP oxidizing activity, and for showing apparently a single band of activity when incubated with 2,6-DMP and SGZ in a parallel native PAGE analysis of crude supernatants.
Enzyme production was evaluated in submerged liquid cultures on the natural containing laccase-inductors BF or a modified Kirk medium (MK) , with the following composition: 10 g l-1 dextrose, 1.0 g l-1 yeast extract, 5.0 g l-1 peptone, 2.0 g l-1 ammonium tartrate, 1.0 g l-1 KH2PO4, 0.5 g l-1 MgSO4, 0.5 g l-1 KCl, and 1.0 ml of 100 X trace element solution (0.5 g EDTA, 0.2 g FeSO4, 0.01 g ZnSO4, 0.003 g MnCl2, 0.03 g H3BO4, 0.02 g CoCl2, 0.001 g CuCl2, and 0.003 g NaMoO4 in 100 ml); amended with 350 μM CuSO4 and 3% ethanol . Cultures were performed at 28 °C and 150 rpm for 14 days. 50 μL aliquots were taken every two days to determine the laccase activity.
2.5. Laccase purification
All the procedures were performed at 4 °C, unless otherwise stated. Extracellular liquid from 14 day-old submerged cultures was separated from mycelium by filtration through a cotton-polyester 50:50% cloth. Then, water-soluble polysaccharides were removed from sample solution by freezing (- 20 °C for 24 h), thawing and filtration (Whatman # 1). Culture filtrate was concentrated to approximately 200 ml by 10 kDa ultrafiltration (Millipore prep/scale TFF cartridge). The obtained fluid was further reduced to 20 ml by using a stirred ultrafiltration system equipped with an YM10 membrane (Amicon, Millipore). The reddish-brown enzyme concentrate was equilibrated by diafiltration with 20 mM potassium phosphate, pH 6.0, and applied to a pre-equilibrated anion-exchange DEAE-Sepharose column (2.5 × 17 cm). Once on the column, unadsorbed protein and most of the pigment were removed by washing with two volumes of equilibrium buffer. Retained proteins were eluted with a linear gradient of potassium phosphate pH 6.0 from 20 to 300 mM, and the eluted fractions were assayed for laccase activity and the A280 nm monitored. Fractions with laccase activity were pooled, concentrated, equilibrated by diafiltration with 100 mM potassium phosphate, and applied on a pre-equilibrated Biogel P-100 column (2.6 x 65 cm). The loaded proteins were eluted with the same buffer. Active fractions were pooled, concentrated and diafiltrated against 20 mM potassium phosphate buffer pH 6.0. Enzyme was further purified by anion-exchange on a pre-equilibrated Q-Sepharose column (2.5 x 17 cm). Once set the sample, active fractions were eluted with a lineal gradient of potassium phosphate from 20 to 300 mM. These fractions were pooled, concentrated and diafiltrated against water, and stored at - 20 °C.
2.6. Electrophoresis analysis
Protein purity and molecular mass were evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as in reference  with 4% stacking gel and 12% resolving gel. Protein bands were stained with Coomassie brilliant blue and the molecular mass (Mr) of purified laccase was determined by calculating the relative mobility of standard protein markers: phosphorylase b 97.4 kDa; serum albumin 66.2 kDa; ovalbumin 45 kDa; carbonic anhydrase 31 kDa; lysozyme 14.4 kDa; aprotinin 6.5 kDa (SDS-PAGE molecular weight standards low range, BioRad). Native PAGE was carried out as described at reference . Activity staining of laccase was performed by incubating with 2, 6-DMP or the pair MBTH + DMAB in 200 mM sodium acetate buffer, pH 4.5. For identification and comparing proposes, the corresponding laccase band was removed from a parallel gel and submitted to the Proteomic Unit of IBT-UNAM at Cuernavaca, Morelos for aminoacid sequencing; resulting in the sequencing of six internal peptides.
2.7. UV-Vis absorbance spectra
As a part of the characterization of the physicochemical properties of the laccase, its absorbance spectrum from 200 to 800 nm was obtained in a UV-Vis Shimadzu-Mini 1240 spectrophotometer. The assay was performed with 25 μM of protein diluted in 1 ml of bidistilled water.
2.8. Effect of pH on enzymatic activity
Optimum pH of activity was determined in McIlvine buffer (consisting of a combination of 100 mM citrate/50 mM potassium phosphate) adjusted in a range from 3.0 to 7.0. Activity determination was made according to described method with 2,6-DMP using 0.2 M citrate phosphate buffer, at pH 4.0.
2.9. Effect of temperature on enzyme activity and stability
The effect of temperature on reaction rate was determined using 2, 6-DMP in 0.2 M citrate/phosphate buffer, at pH 4.0. The temperature of reaction mixture was adjusted to indicate value and then the reaction was started by the addition of enzyme. The assays were done by triplicate and data in graphics appear as relative activity as a function of temperature, considering as 100% the average of maxima obtained. The activation energy of the system was calculated by the Arrhenius model, according to the expression: Log k = [-Ea / 2.303R (1/T)] + Log A, where: k is the rate constant (it depends of temperature); A is preexponential factor or frecuency factor. Ea the activation energy (expressed in J/mol); R is the gas universal constant (8.314 J K-1 mol-1), and T the absolute temperature (°K). In the thermostability assays, the enzyme was pre-incubated at 50, 60 and 70 °C for the indicated periods of time and activity was measured at 25 °C on 2, 6-DMP in 0.2 M citrate/phosphate buffer, at pH 4. The assays were carried out by triplicate and data are expressed as percent of remaining activity as a function of incubation time; taking as 100% the average value of activity at time zero for each temperature. Inactivation process was adjusted to an exponential decay model, from which the constants of heath inactivation (k) and the half-life times were calculated according to the expression: ln (No/N) = kt, where; No is the activity at the starting of incubation; N is remaining activity after a certain incubation time (t); k correspond to the first-order inactivation constant, and t1/2 is the half-life time, calculated as ln 2/k = 0.693/k
2.10. Determination of kinetic parameters
Kinetic analysis was performed on some common substrates of laccase: 2, 6-DMP, ABTS, o-dianisidine and SGZ. Reaction mixtures were prepared in 0.2 M citrate/phosphate buffer at pH 4.0 and the change in optical density by minute was measured by triplicate at different substrate concentrations (0.05, 0.1, 0.5, 1.0, 5.0, 10.0 mM) or those indicated for each assay. The assays were performed at 468 nm for 2, 6-DMP (ε = 49,600 M-1 cm-1), 436 nm for ABTS (29,400 M-1cm-1), 460 nm for o-dianisidine (11,000 M-1 cm-1) and 525 nm for SGZ (ε= 65 000 M-1 cm-1). The values of the Michaelis constant (Km), maximum velocity (Vmax), turnover number (Kcat) and specificity constant Kcat/Km were estimated according to the Lineweaver-Burk method.
2.11. Decolorization assays
The decolorizing ability of laccase was evaluated with two recalcitrant dyes, the non-phenolic azo Methyl Red (MR), and the diazo reactive black 5 (RB 5). The reaction mixture consisted of 0.890 ml of 0.2 M citrate/phosphate buffer, pH 4.0, 0.1 ml of 250 μM MR or RB5 (final concentration 25 μM), and 0.01 ml of pure laccase (final concentration 5 U/ml). Assays were performed at 25 °C and reaction was initiated with the addition of enzyme. Decolorization was estimated by the decreasing of absorbance at 530 nm for MR or 597 nm for RB5. The results are expressed as the percent of remaining color as a function of incubation time according to the relationship: remaining color (%) = [(Abs final/ Abs initial)]*100, where: Abs final correspond to the absorbance value at the indicated incubation times, and Abs initial: is the initial (t = 0) absorbance value.
3. Results and discussion
3.1. Strain identification
In this study an autochthonous strain of Pycnoporus sp (CS 2) was selected as a potential source of thermostable laccases for its ability to produce a thermotolerant 2, 6-DMP oxidizing activity in preliminary assays with crude filtrates from submerged cultures. This basidiomycete was initially isolated from fruit bodies, growing on decayed logs in a man disturbed sub-mountain scrubland around Monterrey, N.L. México (Figure 1), and identified by its morphological and microscopic traits. According to their morphological and microscopic characteristics, the carpophores corresponded to the species Pycnoporus sanguineus (L.) Murrill, for their bright orange to orange-red, red or cinnabar-red shelf-like basidiomes, which are nearly round to elongated or fan-shaped in outline, have a dry surface, smooth or finely hairy, wrinkled or warty and attain 2-12 cm diameter and 0.2 to 0.5 cm thick. Their margins are thin and the under surfaces are covered by small pores (3-4 per mm), bright orange to orange red or red ranging from 0.5-1.5 mm long. Their white spores are smooth and oblong-elliptical in shape and range from 4.2 to 5.2 microns long by 2 to 3.5 microns width and the flesh is tough, red to yellowish red, staining black with KOH. On the bases of these features, we assigned the strain under study as Pycnoporus sanguineus CS 2.
3.2. Production and purification of laccase
Guzmán (2003), considers that P. sanguineus is a tropical variant of the temperate zone species P. cinnabarinus, adapted to man disturbed sites, where it is common in fallen logs and fences, always in sunny places . As its closely related species, P. cinnabarinus and P. coccineus, P. sanguineus is recognized as an efficient lignin decomposer, in spite of its relatively simple LME system composed of laccases . These features make Pycnoporus species an attractive group of white-rot basidiomycetes for the production and purification of unusual laccases [16, 30]. In this study, laccase production was carried out in submerged liquid cultures on a modified Kirk basal medium (MK), amended with 3.5 mM CuSO4 and 3 % ethanol, as chemical laccase inducers and on Bran-Flakes medium (BF), containing natural LME inducers. Under these conditions maxima volumetric productions were reached in both media after 14-16 days (Figure 2). As laccase titers on MK media were about thrice higher than that on BF media (7.5 U ml-1 vs 2.3 U min-1), it was selected for enzyme production in purification assays. Consistently with other reports on LME production by Pycnoporus species, LiP and MnP were not detected [5, 16, 30, 31, 32].
Laccase purification was started from about 1850 ml of mycelium-free filtrates from 14 day-old submerged cultures. After 10K ultraconcentration and sequential steps of anionic exchange chromatography on DEAE- Sepharose, gel filtration on Biogel P-100, and anionic exchange on Q-Sepahrose, laccase activity eluted as an apparently single protein peak with 100-140 mM phosphate (Figure 3). When aliquots of pooled laccase from this last chromatographic step were analyzed by denaturing SDS-PAGE, multiple protein bands were detected by Coomassie staining (not shown). A similar effect was reported in a work with a Fusarium proliferum laccase. As the multiband effect persisted after SDS substitution by other detergents, but disappeared in the absence of SDS, this phenomenon was associated to the presence of detergent on denaturing PAGE . However, when we applied both a heat denatured sample and a non-boiled sample in parallel using the same SDS gel, a protein multiband and a single band were detected by Coomassie staining, respectively. Furthermore, the simple band pattern was also obtained in duplicates by activity staining using laccase substrates. This indicates that thermal treatment could be responsible of the observed multiband effect on denaturing conditions, instead of the SDS by itself. A summary of purification data is shown in Table 1. By this procedure a 16.7-fold purification and activity recovery of 25.5%, with specific activity of 69 U mg-1 protein was achieved. Concentrated purified enzyme showed the blue color characteristic of multi-cupper oxidases.
|Purification step||Protein (mg)||Enzyme Activity (IU)||Specific Activity (U/mg)||Recovery(%)||Purification(fold)|
|Ultraconcentration 10 K||422||3724||8.8||57.4||2.1|
3.2. Biochemical properties
Electrophoresis analysis indicated that Pycnoporus sanguineus CS 2 produced only one laccase under the conditions used in this study. According to non-denaturing SDS-PAGE this laccase is a monomeric protein with a molecular weight of 64.4 kDa, and activity staining of native gels exhibited a single broad band when incubated with both, 2,6-DMP and the pair MBTH + DMAB, showing the same migration as the Coomassie blue stained band (Figure 4). Molecular mass of purified laccase was very similar to those reported for different Pycnoporus sanguineus strains [30, 31, 32], and it was consistent with the reported for most of basidiomycetes laccases [2, 3]. As expected for its visual appearance described above, the UV-Vis spectrum of purified enzyme was characteristic of the typical blue laccases, displaying the absorbance peak near to 600 nm related to the Cu-T1 centers, and the shoulder at 330 nm of Cu-T3 binuclear centers (Figure 5). Nonetheless, the oxidative coupling of MBTH and DMAB in the absence of mediators was indicative that Pycnoporus sanguineus CS2 laccase has the capability to catalyze reactions requiring a higher than usual redox potential for typical laccases .
In addition to blue laccases, other “atypical” forms of the enzyme named “yellow” laccases and “white” laccases have been reported. In the first case, it has been proposed that a variation in the redox state of Cu-T1 centers, by the presence of endogenous ligands, decreases the absorbance at 600 nm, without altering the spectral characteristics of the Cu-T2 and Cu-T3 centers, resulting in a yellow color . In white laccases, like the one produced by Pleurotus ostreatus, it has been informed the presence of a single copper atom, which is accompanied by two of zinc and one of iron, instead of the regular four copper atoms . In both, yellow and white laccases, the protein structure is similar to that found in blue laccases, but the changes in the redox state of the active site (whereas by the presence of the endogenous mediator or by the substitutions in the Cu centers), enables them to oxidize directly substrates of higher redox potential. According to all the above, the laccase produced by P. sanguineus CS 2 corresponds to a blue laccase, most likely containing the regular composition of Cu in its catalytic centers, but like atypical laccases is capable of acting on substrates of higher redox potential.
3.3. pH and temperature dependence
Enzyme was further characterized for its pH and temperature dependence. The effect of pH on laccase activity was studied using some of the most common laccase substrates, including the phenolic 2, 6-DMP, o-dianisidine and SGZ, as well as the non-phenolic ABTS. In general, laccase exhibited optima activity in the zone of pH between 3.0 and 4.5, depending on the particular substrate, then it declined in a gradual way towards the neutral zone of pH, and was completely lost at pH 6.5. Optimal pH values were 3.5, 3.5, 4.5 and 3.0 for 2, 6-DMP, o-dianisidine, SGZ and ABTS, respectively (Figure 6). These results were similar to those reported in literature for most of the fungal laccases . It is known that biphasic pH-activity profiles with phenolic substrates (as the one showed by SGZ), are a consequence of two opposite effects: one generated by the difference in the redox potential between the reducer substrate and the Cu-T1 centers, when changing from acidic to neutral conditions. The other one is directly associated to the inhibitory action of OH- ions over the activity of Cu-T2/T3 centers. For non-phenolic substrates as ABTS, the first effect should be minimal and the inhibition by OH- reflects the monotonic decrease in the enzyme activity [12, 34].
The influence of temperature on P. sanguineus CS 2 laccase activity was investigated with 2, 6-DMP (2 mM), at pH 4.0, in the zone, from 20–80 °C. The profile temperature-activity showed a gradual increase from the lower limit at 20 °C to achieve an optimal value at 65 °C, and declined as temperature approached 80 °C. However, the enzymatic activity in these conditions remained relatively high compared to the value showed under optimal conditions (with a level close to 85%) (Figure 7). Indicating that P. sanguineus CS 2 laccase is a thermotolerant enzyme . These data were evaluated according to the Arrhenius model in order to estimate the energy of activation (Ea) for the system. This parameter has been relatively little studied in thermotolerant laccases. The calculated Ea value (16.2 kJ/mol) for P. sanguineus CS 2 laccase is similar to the values reported for other thermotolerant laccases, as that for Myceliophora thermophila (19 kJ/mol)  and for the recombinant laccase from Coprinus cinereus (14 kJ/mol) , but smaller than those calculated in this report for other laccases, which apparently did not show a direct relationship between thermotolerance and the magnitude of Ea. On the other hand, the function showed a change in slope in the high temperature zone (50-70 °C) before the enzyme denaturing breaking zone. This effect could correspond to a decrease in the Ea of the system, caused by a thermotropic transition of the protein conformation, which should facilitate the limiting step of the reaction. Other possibility would be the coexistence of two enzyme populations, one of them showing an increased activity by temperature and the other being totally inactivated by thermal denaturing. These alternatives should be further explored.
Thermostability is a desirable property of industrial enzymes. Curves of temperature-stability of P. sanguineus CS 2 laccase showed that enzyme retained practically all of its activity after incubation for 8 h at 50 and 60 °C. Moreover, when incubations at 60 °C were extended to 24 h, the laccase retained 98% of its original activity (not shown). The enzyme also retained almost 50% of its activity after 4h at 70 °C. Inactivation curve showed a first-order decaying behavior (correlation > 0.96), with a calculated half-life (t1/2) of 3.85 h [corresponding to a constant of thermal inactivation (k) of 0.187 h-1]. To the best of our knowledge, this is one of the highest t1/2 values found in laccases from mesophilic fungi. It is known that most of typical fungal laccases lose their activity in a few minutes at 60 °C [3, 15, 30, 38].
In comparison to laccases isolated from other Pycnoporus species, t1/2 value at 70 °C here described is higher than those reported for laccase I (0.13 h) and laccase II (2.06 h) from Pycnoporus sp SYBC-L1 [13, 30], and for the laccase from the thermotolerant P. sanguineus CeIBMD001 (0.21 h) . Native laccase also seems to be more resistant to thermal inactivation than P. sanguineus SCC 108 laccase (t1/2 = 3.33 h at 65 °C) reported by  and the P. sanguineus CCT- 4518 laccase studied in , which lost 60% of its initial activity after 2 h at 70 °C. Interestingly, three laccases from tropical or subtropical strains of Pycnoporus species (P. sanguineus BRFM 902, P. sanguineus BRFM 66, and P. coccineus BRFM 938) of different geographic regions (French Guinea, China and Australia, respectively) with remarkable thermal resistance have been recently reported by a research group in France . A relationship between P. sanguineus CS 2 laccase with these and other Pycnoporus laccases already described was established by comparing the aminoacid sequences of an internal protein fragment (peptides 2+3+4, Table 2) from the native laccase with those sequences deposited at GenBank. Aminoacid sequence of P. sanguineus CS 2 laccase showed 99 % similarity to P. sanguineus BRFM 902 laccase, 93% to P. coccineus BRFM 938 , P. cinnabarinus PM laccases , and P. sanguineus BRFM 66 laccase , but only 84 % to Trametes cinnabarina  and 71% to P sanguineus CeIBMD001 laccase . These results highlight the importance of Pycnoporus species biodiversity for the prospection for new thermostable laccases.
3.4. Kinetic properties
The kinetic properties of enzyme were studied with some typical substrates. The values of the Michaelis constant (Km), catalytic constant (Kcat) and specificity constant (Kcat/Km), were calculated by the Lineweaver-Burk method. Laccase showed the highest affinity and molecular activity, on ABTS (Km = 23 mM, Kcat = 221 s-1) compared to o-dianisidine (Km = 44 mM, Kcat = 197 s-1), and 2, 6-DMP (Km =41 mM, Kcat = 88 s-1). So, in terms of catalytic efficiency the best substrate resulted ABTS (Kcat/Km = 9.4 x 106 s-1 M-1) followed by o-dianisidine (Kcat/Km = 4.5 x 106 s-1 M-1) and 2, 6-DMP (Kcat/Km = 2.2 x 106 s-1 M-1). These results are summarized in Table 3, comparing the values of specificity constants (Kcat/Km) for these substrates with those reported for other Pycnoporus laccases, the native enzyme showed higher values for all assayed substrates except for the reported laccase II from Pycnoporus sp SYBC-L1 . Like typical laccases, the enzyme of P. sanguineus CS 2, showed activity on a variety of substrates, such as the phenolic 2, 6-DMP, o-dianisidine and SGZ, as well as the non-phenolic ABTS.
|Peptide||Amino acid sequences|
|Substrate||K m (μM)||V max|
|k cat /K m|
|2,6-DMP||41||500||88||2.16 x 106|
|o-dianisidine||44||1111||197||4.49 x 106|
|ABTS||23||1250||221||9.38 x 106|
Among these substrates this laccase showed preference for ABTS and this characteristic was consistent with most of fungal laccases [32, 41, 42]. Unexpectedly the substrate saturation graphics with SYR showed a sigmoidal-like behavior instead of the common hyperbolic one (not shown). This result could be explained considering a kinetic mechanism of positive cooperativity as that described for monomeric mnemonical enzymes , where a conformational change of interacting enzyme at the end of the first catalytic cycle, reacts more readily with a second substrate molecule than other free-enzyme. Other factor contributing to this result could be the presence of ethanol in routinely SYR assay affecting the substrate solubility and/or enzyme activity. Whether mechanistic on phenomenological, this observation must be taken into account in future works, considering the relevance of this substrate in laccase characterization.
3.5. Dye decolorization
As revealed by the activity staining of native gels shown above, P. sanguineus CS 2 laccase was also able to promote the oxidative coupling between MBTH and DMAB. This reaction has been considered as indicative of the ability of some laccases to catalyze reactions requiring a higher redox potential, as in the case of the enzymatic decolorization of many synthetic dyes. The non-phenolic azo MR [44, 45] and diazo RB 5 dyes  have been used as models for studying the ability of laccases to degrade recalcitrant compounds (Figure 9). Although general consensus is that laccases require meditators for acting over these dyes, P. sanguineus CS 2 laccase showed the capability to decolorize directly both compounds, but with different ability. Decolorization of MR and RB 5 reached a level of 70 %, and 15% respectively, after 4 h at 25 °C.
While Ganoderma lucidum , Trametes trogii  and Lentinula edodes  laccases were only able to decolorize RB 5 in the presence of mediators, a recent report state that three Pycnoporus laccases , were able to perform this decolorization in the absence of mediators, under similar conditions used in this work, with decolorization reaching from 29 to 45% after 52 h, at room temperature. The recalcitrance of RB 5 to laccase decolorization has been explained by its high redox potential or steric hindrances limiting accessibility of enzyme to –OH and –NH2 groups in dye. As in this study native laccase attained around 70% decolorization after 20 h at room temperature, decolorization assays were performed at 60 °C taking advantage of its thermostability trying to overcome limiting factors. As expected, decolorization process was faster under the influence of temperature, reaching around 50% after 4 h, although it also seems to be limited faster (Figure 10). While MR decolorization fitted an exponential first order decay model, RB 5 decolorization changes rapidly from this behavior to fit a polynomial second order model. This effect could be related to several factors as an increased enzyme inactivation by endogenous generated reaction intermediates and/or dead-end transformation products. This relationship must be investigated in future work. Nonetheless these results illustrate the potential of the thermostable Pycnoporus sanguineus CS2 laccase for practical applications.
4. Conclusion and future prospects
Its thermostability and ability for acting on high redox substrates and recalcitrant dyes, makes Pycnoporus sanguineus CS 2 laccase a good prospect for its application in industrial and environmental processes. This laccase could also be interesting as a model in studies associating structure-function of thermotolerant proteins from mesophilic microorganisms.