Kinetics of laccase-catalysed TEMPO oxidation

The oxidation of TEMPO (2,2,6,6-tetramethyl-piperidine-1-oxyl radical) has been studied in the presence of recombinant laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) from Polyporus pinsitus (rPpL), Myceliophthora thermophila (rMtL), Coprinus cinereus (rCcL) and Rhizoctonia solani (rRsL) in buffer solution pH 4.5–7.3 and at 25 °C. At pH 5.5 the oxidation constant calculated from the initial rate of TEMPO oxidation was 1.7 × 10⁴, 1.4 × 10³, 7.8 × 10² and 5.2 × 10² M⁻¹ s⁻¹ for rPpL, rRsL, rCcL and rMtL, respectively. The maximal activity of rPpL-catalysed TEMPO oxidation was at pH 5.0. The pK_a obtained in neutral pH range was 6.2. The reactivity of laccases is in a good agreement with laccases copper type I redox potential.

TEMPO oxidation rate increased 541 times in the presence of 10-(3-propylsulfonate) phenoxazine (PSPX). The model of synergistic TEMPO and PSPX oxidation was proposed. Experimentally obtained rate constants for rPpL-catalysed PSPX oxidation were in a good agreement with those calculated from the synergistic model, therefore confirming the feasibility of the model. The acceleration of TEMPO oxidation with high reactive laccase substrates opens new possibilities for TEMPO application as a mediator.     

Kinetics of N-substituted phenothiazines and N-substituted phenoxazines oxidation catalyzed by fungal laccases

Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and 25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (k_ox) values of the PI formation are (6.1±3.1)×10⁵ M⁻¹s⁻¹ for rPpL and (2.5±0.9)×10⁴ M⁻¹s⁻¹ for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval from 2.2×10⁵ to 2.1×10⁷ M⁻¹s⁻¹ for rPpL and from 1.3×10² to 1.8×10⁵ M^-1s⁻¹ for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu of rPpL. The variation of k_red values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus) electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are 5.0×107 M⁻¹s⁻¹ and 0.29 eV, respectively.     

Amperometric biosensors based on recombinant laccases for phenols determination

Graphite (GE) or printed graphite electrode (PGE) based biosensors containing recombinant fungal laccase Polyporus pinsitus (rPpL), and Myceliophthora thermophila (rMtL) were developed. The enzymes were immobilized using bovine serum albumin and glutaraldehyde. At pH 5.5 and -0.1 V, the calibration graphs of GE based biosensors were hyperbolic if pyrocatechol was used. The concentration of substrate that results in 50% of steady-state response (EC(50)) was 0.7 mM and sensitivity (S) was 3.8 mA/M. The sensitivity increased up to 4 A/M if larger amount of rPpL was used. The sensitivity of biosensors changed little during 9 days of exploitation, but decreased at longer time. The PGE based biosensors were mounted into the flow-through cell and calibrated under kinetic regime. EC(50) of the biosensors containing rPpL varied from 0.6 to 4.0 mM and sensitivity varied from 0.11 to 1.9 mA/M. The response of biosensor containing thermostable laccase rMtL was less, but response saturated at larger pyrocatechol concentration. The sensitivity changed little during 6 days. Both type of biosensors responded also to 1-naphthol, o-phenylenediamine, guaiacol, o-anizidine, benzidine. The experiments demonstrate recombinant laccases application to biosensor engineering and their use to phenol and related compound determination under steady-state and flow-through regimes.     

Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn2+ oxidation.

Two laccase isoenzymes produced by Pleurotus eryngii were purified to electrophoretic homogeneity (42- and 43-fold) with an overall yield of 56.3%. Laccases I and II from this fungus are monomeric glycoproteins with 7 and 1% carbohydrate content, molecular masses (by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 65 and 61 kDa, and pIs of 4.1 and 4.2, respectively. The highest rate of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) oxidation for laccase I was reached at 65 degrees C and pH 4, and that for laccase II was reached at 55 degrees C and pH 3.5. Both isoenzymes are stable at high pH, retaining 60 to 70% activity after 24 h from pH 8 to 12. Their amino acid compositions and N-terminal sequences were determined, the latter strongly differing from those of laccases of other basidiomycetes. Antibodies against laccase I reacted with laccase II, as well as with laccases from Pleurotus ostreatus, Pleurotus pulmonarius, and Pleurotus floridanus. Different hydroxy- and methoxy-substituted phenols and aromatic amines were oxidized by the two laccase isoenzymes from P. eryngii, and the influence of the nature, number, and disposition of aromatic-ring substituents on kinetic constants is discussed. Although both isoenzymes presented similar substrate affinities, the maximum rates of reactions catalyzed by laccase I were higher than those of laccase II. In reactions with hydroquinones, semiquinones produced by laccase isoenzymes were in part converted into quinones via autoxidation. The superoxide anion radical produced in the latter reaction dismutated, producing hydrogen peroxide. In the presence of manganous ion, the superoxide union was reduced to hydrogen peroxide with the concomitant production of manganic ion. These results confirmed that laccase in the presence of hydroquinones can participate in the production of both reduced oxygen species and manganic ions.     

Mediator-assisted laccase-catalyzed oxidation of 4-hydroxybiphenyl

The kinetics of oxidation of 4-hydroxybiphenyl (4-HBP) catalyzed by laccase from Polyporus pinsitus was studied in the presence of methyl syringate (MS), which acts as an electron-transfer mediator. Measurements were performed in 0.05 M acetate buffer, pH 5.5, in the presence of 4-HBP, MS, and laccase. It is shown that the oxidation rate of the lowly reactive substrate 4-HBP significantly increases during synergistic action of the highly reactive substrate MS. Bimolecular kinetic constants of interaction between the oxidized form of laccase and MS, the former and 4-HBP, and the oxidized form of MS and 4-HBP were calculated. A kinetic scheme of the synergistic substrate action is suggested; based on this scheme, the dependence of the initial rate on reagent concentration is derived. Analyzing experimental data, we obtained kinetic constants close to those obtained by modeling the processes.     

Influence of nutrients on enhancing laccase production by Botryosphaeria rhodina MAMB-05.

The physiological requirements needed to enhance the production of laccases by the ascomycete Botryosphaeria rhodina MAMB-05 in submerged cultivation were examined under non-induced and induced (veratryl alcohol, VA) conditions. Under non-induced conditions (-VA), the initial pH, C:N ratio, and inorganic N source did not influence laccase production, in contrast to Tween 80, soybean oil, and copper, which significantly increased laccase production, and proline and urea, which suppressed laccase formation. In addition, Tween 60 could serve as the sole carbon source for the production of these enzymes. Under VA-induced conditions of fungal growth, factors such as inoculum type, time-point of addition of inducer, initial pH, C:N ratio, and type of N source, influenced the production of laccases; however, unlike the non-induced conditions, proline and urea did not act as suppressors. Each of these physiological conditions exerted different effects on biomass production. The nutritional conditions examined for B. rhodina MAMB-05 are discussed in relation to their influence on fungal growth and laccase production.     

Purification, characterization, molecular cloning, and expression of two laccase genes from the white rot basidiomycete Trametes villosa.

Two laccases have been purified to apparent electrophoretic homogeneity from the extracellular medium of a 2,5-xylidine-induced culture of the white rot basidiomycete Trametes villosa (Polyporus pinsitus or Coriolus pinsitus). These proteins are dimeric, consisting of two subunits of 63 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and have typical blue laccase spectral properties. Under nondenaturing conditions, the two purified laccases have different pIs; purified laccase forms 1 and 3 have pIs of 3.5 and 6 to 6.5, respectively. A third purified laccase form 2 has the same N terminus as that of laccase form 3, but its pI is in the range of 5 to 6. The laccases have optimal activity at pH 5 to 5.5 and pH < or = 2.7 with syringaldazine and ABTS [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid)] as substrates, respectively. The genes lcc1 and lcc2 coding for the two purified laccases (forms 1 and 3) have been cloned, and their nucleotide sequences have been determined. The genes for lcc1 and lcc2 have 8 and 10 introns, respectively. The predicted proteins are 79% identical at the amino acid level. From Northern (RNA) blots containing total RNA from both induced and uninduced cultures, expression of lcc1 is highly induced, while the expression of lcc2 appears to be constitutive. Lcc1 has been expressed in Aspergillus oryzae, and the purified recombinant protein has the same pI, spectral properties, stability, and pH profiles as the purified native protein.     

Isolation and Purification of Enzymes from Ligninolytic Complex of the Basidial Fungus <Emphasis Type="Italic">Trametes pubescens</Emphasis> (Schumach.) Pilat and Study of Their Properties

A method for purification of enzymes from the ligninolityc complex of the basidiomycete Trametes pubescens (Schumach.) Pilat has been elaborated. Two homogeneous isoforms of laccases (laccase 1 and laccase 2) as well as a homogeneous preparation of lignin peroxidase were isolated. Basic biochemical parameters of the enzymes were determined, such as the molecular weights (67, 67, and 45 kD, respectively), isoelectric points (5.3, 5.1, and 4.2, respectively), as well as content and composition of the carbohydrate moiety of the laccases (N-acetylglucosamine, mannose, and xylose). The pH dependences and thermal stabilities of the laccases were investigated. The kinetic parameters of the enzymatic reactions catalyzed by the laccases were determined using different substrates, such as catechol, hydroquinone, 2,2 -azinobis-(3-ethylbenzthiazoline-6-sulfonate), and K4Fe(CN)6. The structure of the active sites of both laccases and the lignin peroxidase were studied by EPR, CD, and UV-VIS spectroscopy, as well as using fluorescence analysis. Our studies showed similarity of the spectral characteristics of the two laccases, whereas their kinetic properties were found to be different.     

Comparative Studies of Extracellular Fungal Laccases

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.     

Purification and characterization of two laccase isoenzymes from a ligninolytic fungus Trametes gallica

Constant laccase activities were detected in culture supernatant of newly isolated basidiomycete Trametes gallica. Tryptone and glucose have great effects on the production of laccase. Two laccase isoenzymes (Lac I and Lac II) produced by T. gallica were purified to homogeneity (51- and 50-fold, respectively) by gel filtration chromatography, anion exchange chromatography, and improved native PAGE, with an overall yield of 24.8%. Lac I and Lac II from this fungus are glycoproteins with 3.6% and 4% carbohydrate content, the same molecular masses (by SDS-PAGE) of 60 kDa, and the pI of 3.1 and 3.0, respectively. Native gel electrophoresis indicates that the two laccases have different migration ratios. Lac I and Lac II have the same optimal pH of 3.0 on 2,6-dimethoxyphenol (DMP), pH 2.2 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and of pH 4.0 on guaiacol. The highest rate of ABTS oxidation for both laccases was reached at 70 degrees C. Both laccases are stable from pH 6 to 9, retaining 88-90% activity after 24 hr incubation, and show good stability when incubated at temperatures lower than 40 degrees C. The Km values of Lac I for ABTS, DMP, and guaiacol are 0.118 x 10(-2), 0.420, and 0.405 mM, respectively; the Km values of Lac II for ABTS, DMP, and guaiacol are 0.086 x 10(-2), 0.41, and 0.40 mM, respectively. Their N-terminal sequences are determined and show strong similarity with those from other basidiomycetes. Graphite-furnace atomic absorption analysis revealed that both laccases have four copper atoms per protein molecule, but they have no type I copper signal at around 600 nm and a type III copper signal near 330 nm. Cyanide, azide, and halides completely inhibit the enzyme activity, whereas EDTA has less inhibition.     

Effect of inducers and process parameters on laccase production by Streptomyces psammoticus and its application in dye decolourization

The process parameters influencing the production of extracellular laccases by Streptomyces psammoticus MTCC 7334 were optimized in submerged fermentation. Coffee pulp and yeast extract were the best substrate and nitrogen source respectively for laccase production by this strain. The optimization studies revealed that the laccase yield was maximum at pH 7.5 and temperature 32 degrees C. Salinity of the medium was also observed to be influencing the enzyme production. An agitation rate of 175 rpm and 15% inoculum were the other optimized conditions for maximum laccase yield (5.9 U/mL). Pyrogallol and para-anisidine proved to be the best inducers for laccase production by this strain and the enzyme yield was enhanced by 50% with these inducers. S. psammoticus was able to decolourize various industrial dyes at different rates and 80% decolourization of Remazol Brilliant Blue R (RBBR) was observed after 10 days of incubation in dye based medium.     

Combined effect of copper and initial pH of the culture on production of laccase and manganese peroxidase by Stereum hirsutum (Willd) Pers

Stereum hirsutum is a white-rot fungus which produces laccase and manganese peroxidase as part of its ligninolytic system. Maximal ligninases production (under the conditions studied) was obtained in the media containing 250 microM Cu++ along with a pH value of 5.5. The fitting of ligninase production to a linear equation showed negative interaction between increasing values of pH and concentration of copper.     

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.     

Certain biochemical and physicochemical properties of the inducible form of extracellular laccase from basidiomycetes Coriolus hirsutus

An inducible form of extracellular laccase (EC 1.14.18.1) was isolated from the basidiomycete Coriolus hirsutus. The induction was performed with 0.11 microM syringaldazine, a substrate of laccase. The inducible form of the enzyme consisted of two isoforms, laccase I1 and laccase I2, whose molecular weights were 69 +/- 2 and 67 +/- 2 kDa, respectively. The isoelectric points of these isoenzymes were found to be 3.5 and 4.2, respectively. The optimum pH range for both laccases was 4.4-4.6, and the optimum temperature was 50 degrees C. The thermal stability of these isoenzymes was examined, and KM values for the substrates syringaldazine and pyrocatechol were determined. Our biochemical and physicochemical studies demonstrated that inducible laccase isoforms differed from constitutive forms in molecular weight, IP, KM, and thermal stability. However, their optimum pH ranges and temperatures were identical.     

Induction, isolation, and characterization of two laccases from the white rot basidiomycete Coriolopsis rigida

Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH(2)). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH(2) extended the reactions catalyzed by these enzymes to the production of H(2)O(2), the oxidation of Mn(2+), the reduction of Fe(3+), and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.     

Purification and characterisation of a novel laccase from the ascomycete Melanocarpus albomyces

A novel laccase from the ascomycete Melanocarpus albomyces was purified and characterised. The enzyme was purified using anion exchange chromatography, hydrophobic interaction chromatography and gel filtration, and the purified laccase was biochemically characterised. It had activity towards typical substrates of laccases including 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate), dimethoxyphenol, guaiacol, and syringaldazine. The laccase showed good thermostability and it had a pH optimum at neutral pH, both unusual properties for most known fungal laccases. The activity of the laccase from M. albomyces was highest at 60-70 degrees C. With guaiacol and syringaldazine the pH optima were rather broad: 5-7.5 and 6-7, respectively. It retained 50% of its activity after 5 h incubation at 60 degrees C. The molecular weight of the laccase was about 80 kDa and the isoelectric point 4.0. The ultraviolet-visible absorption and electron paramagnetic resonance spectra of the purified laccase indicated that the typical three types of copper were present.     

Molecular and enzymatic characterisation of extra- and intracellular laccases from the acidophilic ascomycete Hortaea acidophila

The pigmented ascomycete Hortaea acidophila is able to grow at a pH as low as 0.6 and produces laccases that are involved in melanin synthesis. We now present data on an extracellular and an intracellular laccase which exhibit a high stability at low pH. Furthermore, the optimum for enzyme acitivity is extraordinarily low with pH 1.5 for the intracellular laccase with 2,6-dimethoxyphenol (DMOP) as substrate. Two complete laccase gene sequences of H. acidophila were amplified by inverse polymerase chain reaction (PCR). Whereas the deduced protein laccase I contains an predicted N-terminal signal sequence for protein export, laccase II does not and thus may represent the intracellular laccase. The acidophilic character of both laccases seems to be reflected in their primary structure.     

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68·7–97·5% similarity with other Polyporale laccases. The three laccases (59·5–62·9 kDa with 7–10% carbohydrate content) had high redox potentials (0·72–0·75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase‐1‐hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo‐ and pH stability, catalytic efficiency towards 2,2′‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale‐up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor‐made enzymes.