Characterization of two new multiforms of Trametes pubescens laccase

Electrochemical properties of two multiforms of laccase from Trametes pubescens basidiomycete (LAC1 and LAC2) have been studied. The standard redox potentials of the T1 sites of the enzymes were found to be 746 and 738 mV vs. NHE for LAC1 and LAC2, respectively. Bioelectroreduction of oxygen based on direct electron transfer between each of the two forms of Trametes pubescens laccase and spectrographic graphite electrodes has been demonstrated and studied. It is concluded that the T1 site of laccase is the first electron acceptor, both in solution (homogeneous case) and when the enzymes are adsorbed on the surface of the graphite electrode (heterogeneous case). Thus, the previously proposed mechanism of oxygen bioelectroreduction by adsorbed fungal laccase was additionally confirmed using two forms of the enzyme. Moreover, the assumed need for extracellular laccase to communicate directly and electronically with a solid matrix (lignin) in the course of lignin degradation is discussed. In summary, the possible roles of multiforms of the enzyme based on their electrochemical, biochemical, spectral, and kinetic properties have been suggested to consist in broadening of the substrate specificity of the enzyme, in turn yielding the possibility to dynamically regulate the process of lignin degradation according to the real-time survival needs of the organism.     

Perturbations of the T1 copper site in the CotA laccase from Bacillus subtilis: structural, biochemical, enzymatic and stability studies

Site-directed mutagenesis has been used to replace Met502 in CotA laccase by the residues leucine and phenylalanine. X-ray structural comparison of M502L and M502F mutants with the wild-type CotA shows that the geometry of the T1 copper site is maintained as well as the overall fold of the proteins. The replacement of the weak so-called axial ligand of the T1 site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme (E ⁰=455 mV). However the M502L mutant exhibits a twofold to fourfold decrease in the k _cat values for the all substrates tested and the catalytic activity in M502F is even more severely compromised; 10% activity and 0.15–0.05% for the non-phenolic substrates and for the phenolic substrates tested when compared with the wild-type enzyme. T1 copper depletion is a key event in the inactivation and thus it is a determinant of the thermodynamic stability of wild-type and mutant proteins. Whilst the unfolding of the tertiary structure in the wild-type enzyme is a two-state process displaying a midpoint at a guanidinium hydrochloride concentration of 4.6 M and a free-energy exchange in water of 10 kcal/mol, the unfolding for both mutant enzymes is clearly not a two-state process. At 1.9 M guanidinium hydrochloride, half of the molecules are in an intermediate conformation, only slightly less stable than the native state (approximately 1.4 kcal/mol). The T1 copper centre clearly plays a key role, from the structural, catalytic and stability viewpoints, in the regulation of CotA laccase activity.     

产漆酶菌株筛选及一株产酶菌株的优化与鉴定

【目的】从26株真菌菌株中筛选高产漆酶菌株。【方法】采用愈创木酚法进行产漆酶菌株的筛选,通过正交实验对筛选出的高产菌株进行优化,并通过形态学和分子系统学对菌株进行鉴定。【结果】26株真菌菌株中有4株可产生漆酶,其中菌株H52.1为产漆酶最好菌株;菌株H52.1产漆酶优化培养基碳源为可溶性淀粉,氮源为硝酸铵,pH为8,金属离子为Ca2+;经鉴定,该菌株为大孢戴氏霉。【结论】大孢戴氏霉在产漆酶方面值得进一步研究开发。     

Systematic screening strategy for fungal laccase activity of endophytes from Otoba gracilipes with bioremediation potential

Fungal laccases are promising for biotechnological applications, including bioremediation and dye biotransformation, due to their high redox potential and broad substrate specificity. However, current bioprospecting methods for identifying laccase-producing fungi can be challenging and time-consuming. For early detection, it was developed a three-step, multi-criteria weighting system that evaluates fungal strains based on: First, the biotransformation capacity of three dyes (i.e., Congo red, brilliant blue G-250, and malachite green), at three different pH values, and with a relative weighting supported for the redox potential of each colorant. The relative decolorization coefficient (RDC), used as th2e first classification criterion, expressed their potential performance. Second, under the same conditions, laccase activity was estimated by observing the different degrees of oxidation of a given substrate. The selection criterion was the relative oxidation coefficient (ROC). Finally, laccase activity was quantified in submerged fermentations using three inducers (i.e., loofah sponge, Tween 80, and veratyl alcohol). This multicriteria screening strategy evaluated sixteen isolated endophytic fungal strains from Otoba gracilipes. The system identified Beltraniopsis sp. ET-17 (at pH values of 5.00 and 5.50) as a promising strain for dye biotransformation, and Phlebia floridensis as the best laccase producer, achieving a high activity of 116 μmol min⁻¹ L⁻¹ with loofah sponge as an inducer. In-vitro testing confirmed the efficacy of P. floridensis, with 53.61 % decolorization of a dye mixture (brilliant blue-Congo red. ratio 1:1) after 15 days of incubation. Thus, with the proposed screening strategy it was possible to highlight two species of interest at an early bioprospecting stage on a Colombian native tree poorly explored.     

Effect of solvent phase transitions on enzymatic activity and structure of laccase from <Emphasis Type="Italic">Coriolus hirsutus</Emphasis>

The effect of solvent phase transitions on catalytic activity and structure of the active site of laccase produced by the Basidiomycetes Coriolus hirsutus 072 was studied. As shown by small-angle X-ray scattering, laccase exists in solution as a mixture of monomeric and aggregated particles in the percent ratio 85: 15. This ratio did not change on phase transitions. A complex nature of laccase activity dynamics during thawing and further heating to 20°C was shown. Spontaneous oxidation of T1 copper center in the temperature range 12–20°C was not observed. According to spectral data, the structure of laccase active sites including all copper centers of types T1, T2, and T3 changes during the phase transition.     

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.     

Amelioration of the ability to decolorize dyes by laccase: relationship between redox mediators and laccase isoenzymes in Trametes versicolor

The effect of redox mediators in the dye decolorization by two laccase isoenzymes from Trametes versicolor cultures supplemented with barley bran has been investigated. All the redox mediators tested, 1-hydroxybenzotriazole (HBT), promazine (PZ), para-hydroxybenzoic acid (pHBA) and 1-nitroso-2-naphthol-3,6-disulfonic acid (NNDS), led to higher dye decolorization than those obtained without mediator addition. Among the different tested mediators, PZ was the most effective one at a low range of concentration (0.5–50 μM) and the natural mediator employed, pHBA did not improve significantly the degree of decolorization, and was slightly inhibitory.The two laccase isoenzymes, LacI and LacII, showed different decolorization capability depending on the mediator used. No significant differences were detected for NNDS, however LacII was more effective than LacI in the presence of PZ, while in the presence of HBT LacI was the fastest and the most effective isoenzyme.     

Heterologous expression of lcc1 from Lentinula edodes in tobacco BY-2 cells results in the production an active, secreted form of fungal laccase

Laccase (Lcc) is a lignin-degrading enzyme produced by white-rot fungi and has been the subject of much interest in the field of bioremediation due to its ability to oxidize phenolic compounds. In this report, we describe the isolation and characterization of lcc1, a novel gene of Lentinula edodes that encodes Lcc1, and demonstrate that recombinant Lcc1 is expressed in an active, secreted form in tobacco BY-2 cells in culture. The open reading frame of lcc1 was 1,557 base pairs in length and encoded a putative protein of 518 amino acids. We introduced a chimeric form of lcc1 (CaMV35Sp:clcc1) into tobacco BY-2 cells and obtained several stable clcc1 transformants that expressed active Lcc1. Lcc1 activity in BY-2 culture media was higher than in cellular extracts, which indicated that recombinant Lcc1 was produced in a secreted form. Recombinant Lcc1 had a smaller apparent molecular weight and exhibited a different pattern of posttranslational modification than Lcc1 purified from L. edodes. The substrate specificity of purified recombinant Lcc1 was similar to L. edodes Lcc1, and both enzymes were able to decolorize the same set of dyes. These results suggest that heterologous expression of fungal Lcc1 in BY-2 cells will be a valuable tool for the production of sufficient quantities of active laccase for bioremediation.     

Early attack and subsequent changes produced in an industrial lignin by a fungal laccase and a laccase-mediator system: an analytical approach

An industrial kraft pine lignin (Indulin AT, KL) was characterized and treated in both aqueous-buffered media and dioxane to water, either with a partially purified laccase from Fusarium proliferatum or with the laccase plus 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic-acid (ABTS) as mediator. The changes in the lignin after different incubation periods were analyzed through the application of high performance liquid chromatography (HPLC), UV–visible (Vis) spectroscopy and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS). At the onset of incubation, laccase-treated samples showed a slight polymerization and strong modifications in UV–Vis spectra. Through Py-GC/MS, a decrease in phenolic and methoxy-bearing pyrolysis products was observed, in contrast to an increase in the more oxidized products. After longer incubation periods (48 h) a substantial polymerization was detected by HPLC, along with a decrease in the guaiacyl (G) units. In contrast, the analysis by HPLC of the samples recovered from the laccase-ABTS system (LMS) showed an intense depolymerization, accompanied by a sizeable loss in G units and a decrease in the methyl and ethyl side-chain phenolic compounds. These results provide conclusive evidence of a rapid initial attack of the industrial lignin by laccase and notable modifications in the KL after longer incubation periods with laccase or LMS.     

Nuclear amination catalyzed by fungal laccases: Comparison of laccase catalyzed amination with known chemical routes to aminoquinones

Laccases are able to initiate nuclear amination of p-hydroquinones with primary aromatic amines, resulting in the formation of the corresponding monoaminated and diaminated quinones. Two laccase catalyzed reactions are compared with established synthetic routes to aminoquinones, showing that formation of products from laccase catalyzed reaction is comparable with reaction using sodium iodate as oxidant. Advantages and disadvantages of laccase catalyzed amination are discussed. It is concluded that laccase catalysis is less suitable than sodium iodate oxidation for the amination of simple p-hydroquinones with simple amines.     

Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest K_cat and K_cat/K_m were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn²⁺, Cd²⁺, Fe²⁺, Cu²⁺ and Na⁺ activated laccase while Ca²⁺ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co²⁺, Hg²⁺, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: Purification and characterization

An unusual halotolerant-alkaline laccase from Streptomyces psammoticus has been purified to homogeneity through anion exchange and gel filtration chromatography steps with an overall purification fold of 12.1. The final recovery of the enzyme was 22.1%. The molecular mass of the purified laccase was about 43 kDa. The enzyme was active in the alkaline pH range with pH optima at 8.5 and 97% activity retention at pH 9.0. The optimum temperature was 45 °C. The enzyme was stable in the pH range 6.5–9.5 and up to 50 °C for 90 min. The enzyme was tolerant to NaCl concentrations up to 1.2 M. It was inhibited by all the putative laccase inhibitors while the enzyme was activated by metal ions like Fe, Zn, Cu, Na and Mg. Fe enhanced the enzyme activity by twofold (204%). The enzyme showed lowest K_m value with pyrogallol (0.25 mM) followed by ABTS (0.39 mM). The purified enzyme was a typical blue laccase with an absorption peak at 600 nm.     

Expression and characterization of a recombinant multi-copper oxidase: laccase IV from Trametes versicolor

A cDNA encoding LccIV, a previously uncharacterized laccase isozyme of the white-rot basidiomycete Trametes versicolor, was expressed in the methylotrophic yeast Pichia pastoris. The LccIV isozyme is not expressed by T. versicolor under normal culture conditions and the enzyme was, therefore, investigated to determine whether it had any unusual properties. The native signal peptide of LccIV directed efficient secretion and correct proteolytic processing of LccIV to the mature form, whereas, substitution with the Saccharomyces cerevisiae α-mating factor signal peptide led to retention of an additional tetrapeptide at the amino-terminus of the secreted enzyme and ∼25% lower specific activity in fermentor medium. Active LccIV was purified to homogeneity by sequential steps of ion-exchange, size-exclusion and hydrophobic interaction chromatography. The enzyme contains ∼25% N-linked glycans (∼40% total carbohydrate) and has an apparent molecular mass of ∼85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and ∼100 kDa by size-exclusion chromatography, indicating a monomeric structure. A pH of 5.5 was optimal for oxidation of 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Thus, the LccIV isozyme appears to be similar in these respects to the laccase isozymes constitutively expressed by T. versicolor.     

平菇漆酶基因在毕赤酵母中的分泌表达及酶学性质研究

采用RTPCR技术克隆到一个平菇(Pleurotusostreatus)漆酶基因的全长cDNA,命名为lccPo1,其序列提交GenBank,登录号为AY450404。将其ORF克隆到毕赤酵母表达载体pHBM906,转化3株毕赤酵母GS115、KM71和SMD1168,该漆酶基因在3种毕赤酵母菌株中均实现了分泌表达。3种摇瓶培养条件①25℃,1.0%(VV)甲醇;②20℃,1.0%(VV)甲醇;③20℃,0.5%(VV)甲醇,进行比较研究后发现适当提高甲醇浓度有利于漆酶在低温条件下表达,而降低培养温度到20℃则可以提高漆酶的产量2～6倍。3株重组毕赤酵母在其最适反应条件下测得三者粗酶液最高漆酶酶活分别为3.19UmL[GS115(pHBM565)]、2.56UmL[KM71(pHBM565)]和2.49UmL[SMD1168(pHBM565)]。对重组酶进行相关的酶学性质分析表明,三者的最适反应pH值约为4.2,最适反应温度约为60℃。重组毕赤酵母GS115(pHBM565)所产酶的热稳定性稍好,在pH稳定性方面三者没有太大差异。     

Enzymatic and spectroscopic studies on the activation or inhibition effects by substituted phenolic compounds in the oxidation of aryldiamines and catechols catalyzed by Rhus vernicifera laccase

The effect of various phenolic compounds on the activity of Rhus vernicifera laccase (Lc) has been evaluated using two different substrates, N,N-dimethyl-p-phenylenediamine and p-tert-butylcatechol. The observed effect strongly depends on the phenol employed and involves either a moderate activation, by halophenols, or inhibition, by acidic phenols. The collective data are consistent with an open active site in Lc, which is capable of accommodating more than one substrate or phenol molecule. According to NMR relaxation experiments, a phenol molecule binds at an average distance from type 1 Cu of about 6 Å, while evidence from electron paramagnetic resonance (EPR) experiments shows that binding of another phenol molecule induces a change, and probably occurs close to, the type 2/type 3 cluster. The effect of phenolic compounds on Lc reactivity is related to a modification of the substrate affinity for the enzyme. This affinity can either be increased, probably through π-stacking or other types of interactions, or decreased, due to competition for the same site. In addition, the alteration induced in the trinuclear copper cluster has a marked effect on the enzyme reactivity. The inhibition observed with acidic phenols is probably due to the protonation of an enzyme intermediate produced at the trinuclear site, e.g. the peroxy intermediate, that causes the release of hydrogen peroxide and prevents the reaction of this intermediate with the substrate.     

Azo dye decolorization by a laccase/mediator system in a membrane reactor: enzyme and mediator reusability

This paper presents the use of a membrane-integrated reactor system with recycling of laccase and mediator for azo dye decolorization. From initial screening of different laccases and mediators, Trametes versicolor laccase and syringaldehyde provided the best system for decolorization. Decolorization yields of 98, 88, 80 and 78% were obtained for Red FN-2BL, Red BWS, Remazol Blue RR and Blue 4BL, respectively. The reaction parameters were optimized and a membrane reactor was set up for dye decolorization in batch mode with reuse of the enzyme. Between 10 and 20 batches could be run with decolorization yields from 95 to 52% depending on the dye type. To study the possibility of reusing both enzyme and mediator, the reactor was run using 2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPO) coupled to polyethylene glycol (PEG). Nine batches were run for the treatment of Remazol Blue RR, providing decolorization yields of 96-78%. Cost analysis of the processes showed that the costs of laccase/syringaldehyde or laccase/TEMPO were almost equal when running 20 batches, but the cost for the PEG-TEMPO was higher. However, the advantages associated with reuse of the mediator should motivate further development of the concept.     

Degradation of diclofenac,trimethoprim, carbamazepine,and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent

Abstract

The degradation of diclofenac (DCF), trimethoprim (TMP), carbamazepine (CBZ), and sulfamethoxazole (SMX) by laccase from Trametes versicolor was investigated. Experiments were conducted using the pharmaceuticals individually, or as a mixture at different initial concentrations (1.25 and 5?mg/L each). The initial enzymatic activity of all the treated samples was around 430–460?U_(DMP)/L. The removal of the four selected pharmaceuticals tested individually was more effective than when tested in mixtures under the same conditions. For example, 5?mg DCF/L was completely removed to below its detection limit (1?µg/L) within 8?h in the individual experiment vs. after 24?h when dosed as a mixture with the other pharmaceuticals. A similar trend was visible with other three pharmaceuticals, with 95 vs. 39%, 82 vs. 34% and 56 vs. 49% removal after 48?h with 5?mg/L of TMP, CBZ, and SMX tested individually or as mixtures, respectively. In addition, at the lower initial concentration (1.25?mg/L each), the removal efficiency of TMP, CBZ, and SMX in mixtures was lower than that obtained at the higher initial concentrations (5?mg/L each) during both the individual and combined treatments. Four enzymatic transformation products (TPs) were identified during the individual treatments of DCF and CBZ by T. versicolor. For TMP and SMX, no major TPs were observed under the experimental conditions used. The toxicity of the solution before and after enzymatic treatment of each pharmaceutical was also assessed and all treated effluent samples were verified to be non-toxic.     

Switching from blue to yellow: altering the spectral properties of a high redox potential laccase by directed evolution

Abstract

During directed evolution to functionally express the high redox potential laccase from the PM1 basidiomycete in Saccharomyces cerevisiae, the characteristic maximum absorption at the T1 copper site (Abs₆₁₀T1Cu) was quenched, switching the typical blue colour of the enzyme to yellow. To determine the molecular basis of this colour change, we characterized the original wild-type laccase and its evolved mutant. Peptide printing and MALDI-TOF analysis confirmed the absence of contaminating protein traces that could mask the Abs₆₁₀T1Cu, while conservation of the redox potential at the T1 site was demonstrated by spectroelectrochemical redox titrations. Both wild-type and evolved laccases were capable of oxidizing a broad range of substrates (ABTS, guaiacol, DMP, synapic acid) and they displayed similar catalytic efficiencies. The laccase mutant could only oxidize high redox potential dyes (Poly R-478, Reactive Black 5, Azure B) in the presence of exogenous mediators, indicating that the yellow enzyme behaves like a blue laccase. The main consequence of over-expressing the mutant laccase was the generation of a six-residue N-terminal acidic extension, which was associated with the failure of the STE13 protease in the Golgi compartment giving rise to alternative processing. Removal of the N-terminal tail had a negative effect on laccase stability, secretion and its kinetics, although the truncated mutant remained yellow. The results of CD spectra analysis suggested that polyproline helixes were formed during the directed evolution altering spectral properties. Moreover, introducing the A461T and S426N mutations in the T1 environment during the first cycles of laboratory evolution appeared to mediate the alterations to Abs₆₁₀T1Cu by affecting its coordinating sphere. This laccase mutant is a valuable departure point for further protein engineering towards different fates.