Polymeric microspheres as support to co-immobilized Agaricus bisporus and Trametes versicolor laccases and their application in diazinon degradation

The laccase enzymes of Agaricus bisporus and Trametes versicolor were successfully covalently co-immobilized on poly(glycidyl methacrylate) microspheres. The enzyme load reached after the co-immobilization of both enzymes was 6.75 U g⁻¹ carrier. The resulting biocatalyst showed the combined properties of both immobilized enzymes, increasing their optimum pH and temperature ranges. The storage and operational stabilities were also improved after co-immobilization. In presence of mediator (ABTS) the organophosphate pesticide diazinon was 100% biodegraded after 48 h of reaction with 0.2 U/mL of co-immobilized enzymes (at the two maximum activity pH values: 2.0 and 3.0). In the absence of a mediator, the degradation percentages were above 88%. Data showed that, compared with single enzymatic immobilization, the co-immobilization of the two laccases is an easy, efficient, and low cost alternative to expanding the range of work of the biocatalyst, thereby improving the stability and some biochemical properties to generate a powerful alternative for pesticide degradation in a wide range of conditions.     

C-N coupling of 3-methylcatechol with primary amines using native and recombinant laccases from Trametes versicolor and Pycnoporus cinnabarinus

Five laccase genes from Pycnoporus cinnabarinus and Trametes versicolor encoding for different isoenzymes have been cloned, recombinantly expressed and characterized. Following C-N coupling of primary linear, branched-chained and cyclic amines to 3-methylcatechol was mediated by native and recombinant laccases yielding the corresponding secondary amines. Formation of C5-monoaminated ortho-methylquinones occurred within 1-2 h; prolonged incubation led to the formation of high-molecular mass products. No difference between the use of native or recombinant isoenzymes from P. cinnabarinus or T. versicolor was observed. Optimization of the reaction conditions included variation of amine donor ratios, pH, amount and type of enzyme preparations. The formation of by-products could be suppressed at pH values corresponding to the enzymes optima (pH 4-5). A total of 10 secondary amines were synthesized with product formations of up to 80%. Furthermore, all purified secondary amines were characterized by NMR-, LC-MS- and HRMS-analysis and log P values were determined.     

Ligninolytic Fungal Laccases and Their Biotechnological Applications

Lignin is amorphous in nature, lacks stereoregularity, and is not susceptible to hydrolytic attack. Despite its resistant nature, it is however degraded by various microorganisms, particularly, white-rot fungi. Such fungi are capable of extracellular production of lignin peroxidase, manganese peroxidase, and laccase, the three major enzymes associated with ligninolysis. Though all white-rot fungi do not produce all the three enzymes, laccase occupies an important place in ligninolysis. Laccase belongs to a diverse group of enzymes called oxidoreductases and is also known as benzenediol: oxygen oxidoreductase. They have low substrate specificity. The copper-containing enzyme laccase has been detected in a variety of organisms such as bacteria, fungi, plants, and insects. Mostly, these are extracellular proteins, although intracellular laccases have also been detected in some fungi and insects. Fungal laccases are believed to play a variety of roles, such as, morphogenesis, pathogenesis, and lignin degradation. As an oxidase, laccase is used in many agricultural, industrial, and medicinal applications. Current investigations are focused on laccase-based biooxidation, biotransformation, biosensor, and enzymatic synthesis of organic compounds. By enhancing laccase production using different physiochemical parameters, better understanding of the mechanism for the reactions of interest, and optimizing the catalytic activity of laccase, it can be used in a better way in diverse fields of biotechnology.     

Comparative study of biocatalytic reactions of high and low redox potential fungal and plant laccases in homogeneous and heterogeneous reactions

The homogeneous catalytic and heterogeneous bioelectrocatalytic properties of high redox potential fungal laccases and low redox potential plant laccase have been compared. The fungal and plant laccases exhibit radically different catalytic activities as a function of pH with respect to substrates donating only electrons and substrates donating both hydrogen atoms and electrons, as well as in the bioelectrocatalytic reaction of dioxygen reduction. It is suggested that the difference between the biocatalytic properties of these enzymes correlates with their role in lignin metabolism.     

Direct electron transfer reactions of laccases from different origins on carbon electrodes

Electrochemical studies of laccases from basidiomycetes, i.e., Trametes hirsuta, Trametes ochracea, Coriolopsis fulvocinerea, Cerrena maxima, and Cerrena unicolor, have been performed. Direct (mediatorless) electrochemistry of laccases on graphite electrodes has been investigated with cyclic voltammetry, square wave voltammetry as well as potentiometry. For all mentioned high potential laccases direct electron transfer (DET) has been registered at spectrographic graphite and highly ordered pyrolytic graphite electrodes. The characteristics of DET reactions of the enzymes were analysed under aerobic and anaerobic conditions. It is shown that the T1 site of the laccase is the primary electron acceptor, both in solution (homogenous case) and at surface of the graphite electrode (heterogeneous case). A mechanism of ET for the process of the electro-reduction of oxygen at the laccase-modified graphite electrodes is proposed and the similarity of this heterogeneous process to the laccase catalysed oxygen reduction homogeneous reaction is concluded.     

Native and modified glucose oxidase in reversed micelles

The enzymatic activity of the native and modified glucose oxidase (GOx) from Aspergillus niger in the system of reversed micelles of Aerosol OT in octane was investigated. Two forms of the modified enzyme were studied: a hydrophobized form obtained by the attachment of palmitic chains to lysine amino groups by the reaction with palmitic acid ester of N-hydroxysuccinimide and a glycosylated (hydrophilized) form obtained by the attachment of the cellobiose moieties. The native glucose oxidase and its derivatives, while incorporated into micelles in a surfactant concentration range from 0.05 to 0.3 M, display an enzymatic activity, which is comparable with the activity in aqueous solution. The dependence of the enzymatic activity on hydration degree of surfactant (the molar ratio of water to surfactant, W₀) does not indicate the formation of qualitatively new associated forms of the enzyme subunits inside the micelles. The apparent size of Aerosol OT micelles obtained by dynamic light scattering gradually increases from 10±3 nm at low W₀ up to 25±5 nm at high W₀. Incorporation of the native and hydrophobized glucose oxidase into micelles does not affect their mean size. Kinetic analysis shows that the enzyme specificity is about an order of magnitude greater in the system of reversed micelles as compared with aqueous solution.     

Stabilization of the Cellulase Enzyme Complex as Enzyme Nanoparticle

The native Celluclast BG cellulase enzyme complex consists of different enzymes which can also degrade great substrate molecules as native celluloses. This enzyme complex has been covered by a very thin, a few nanometers thick, polymer layer, in order to improve its stability. It has been proved that the polymer layer around the enzyme molecules does not hinder the digestion as great substrates as crystalline cellulose polymer. The stability of the prepared enzyme nanoparticles (PE) could significantly be increased comparing to that of the native one what was proved by results of the total cellulose activity measured. The pretreated enzyme complex holds its activity often a few magnitudes of orders longer in time than that of the native enzyme complex (enzyme without pretreatment). It retains its activity at least ten times longer than that of the native one, at a temperature range between 20 and 37?°C. The pretreated enzyme complex can have about 50?% of its original activity during 12?h of incubation at even 80?°C, while the native cellulase one totally lost it during 6?h incubation time. The activity of PE has not been significantly reduced even at extreme pH values, namely in the pH range of 1.5 to 12.     

In vitro evolution of a fungal laccase in high concentrations of organic cosolvents

Fungal laccases are remarkable green catalysts that have a broad substrate specificity and many potential applications in bioremediation, lignocellulose processing, organic synthesis, and more. However, most of these transformations must be carried out at high concentrations of organic cosolvents in which laccases undergo unfolding, thereby losing their activity. We have tailored a thermostable laccase that tolerates high concentrations of cosolvents, the genetic product of five rounds of directed evolution expressed in Saccharomyces cerevisiae. This evolved laccase--R2 variant--was capable of resisting a wide array of cosolvents at concentrations as high as 50% (v/v). Intrinsic laccase features such as the redox potential and the geometry of catalytic copper varied slightly during the course of the molecular evolution. Some mutations at the protein surface stabilized the laccase by allowing additional electrostatic and hydrogen bonding to occur.     

Decolorization Potential of Some Reactive Dyes with Crude Laccase and Laccase-Mediated System

In this study, decolorization of dyestuffs, such as Reactive Red 198, Rem Blue RR, Dylon Navy 17, Rem Red RR, and Rem Yellow RR was studied using laccase and laccase-mediated system. The laccases are known to have an important potential for remediation of pollutants. Among these dyestuffs, decolorization of Rem Blue RR and Dylon Navy 17 was performed with crude laccase under optimized conditions. Vanillin was selected as laccase mediator after screening six different compounds with Rem Yellow RR, Reactive Red 198, and Rem Red RR as substrates. However, Rem Yellow RR was not decolorized by either laccase or laccase-mediated system. It is observed that the culture supernatant contained high laccase activity after treatment with catalase that was responsible for the decolorization. Besides, culture supernatant with high laccase activity as enzyme source was treated with catalase; in this way, the hypothesis that laccase was the enzyme responsible for decolorization was supported. The Rem Blue RR was decolorized with 64.84% under the optimum conditions and Dylon Navy 17 with 75.43% with crude laccase. However, using the laccase and vanillin, the decolorization of Reactive Red 198 and Rem Red RR was found to be 62% and 68%, respectively. Our study demonstrated that the decolorization abilities of laccase and/or laccase mediator systems were based on the types of mediator, the dye structure, and the standard experimental conditions. Also, the electrochemical behaviors of some samples were studied. The redox potentials of these samples were determined using cyclic voltammetry on glassy carbon electrode in phosphate buffer (pH 6) solution.     

Banana Peel: A Potential Substrate for Laccase Production by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> VkJ2.4.5 in Solid-State Fermentation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l⁻¹) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l⁻¹) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min⁻¹. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.     

Oxygen effects in aqueous solutions of laccases: Absorption spectra and reactivity

The optical absorption spectra of laccases in aqueous solutions were found to undergo reversible changes in the presence of O₂. It was suggested that dioxygen is coordinated in the active center of the completely oxidized native enzyme. Abnormal behavior of superoxide radical anions upon variation of the laccase concentration was found by pulse radiolysis. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1471–1474, August, 2000.     

Versatile Electrochemical Platform for the Determination of Phenol‐like Compounds Based on Laccases from Different Origins

Simple and fast methods for the monitoring of phenol‐like compounds are relevant in diverse fields ranging from waste management to neurosciences. Laccases are copper‐containing enzymes, which, depending on their origin, are able to oxidize different phenol compounds at different pH conditions. Through adequate laccase immobilization, disposable screen printed electrodes can be used as interphase to build amperometric phenol sensors. In this work three different laccases were studied for the determination of phenol‐like compounds, two of them are isoenzymes from Trametes trogii and the third one from Rhus vernicifera . Their immobilization on screen printed electrodes is presented for the construction of amperometric sensors. The electrode substrate is composed by graphite screen printed electrodes modified with carbon nanotubes and silica microspheres where, depending on the application, one of the three laccases is adsorbed. As each laccase shows an optimum working pH, they were conveniently selected to determine dopamine at physiological pH and catechol at acid pH. Determinations in the micromolar range were possible in both cases. Chronoamperometry shows to be an effective technique for their determinations, simpler than other electrochemical methods already presented in the literature.     

An assessment of the relative contributions of redox and steric issues to laccase specificity towards putative substrates

Laccases catalyze the one-electron oxidation of a broad range of substrates coupled to the 4 electron reduction of O2 to H2O. Phenols are typical substrates, because their redox potentials (ranging from 0.5 to 1.0 V vs. NHE) are low enough to allow electron abstraction by the T1 Cu(II) that, although a relatively modest oxidant (in the 0.4-0.8 V range), is the electron-acceptor in laccases. The present study comparatively investigated the oxidation performances of Trametes villosa and Myceliophthora thermophila laccases, two enzymes markedly differing in redox potential (0.79 and 0.46 V). The oxidation efficiency and kinetic constants of laccase-catalyzed conversion of putative substrates were determined. Hammett plots related to the oxidation of substituted phenols by the two laccases, in combination with the kinetic isotope effect determination, confirmed a rate-determining electron transfer from the substrate to the enzyme. The efficiency of oxidation was found to increase with the decrease in redox potential of the substrates, and the Marcus reorganisation energy for electron transfer to the T1 copper site was determined. Steric hindrance to substrate docking was inferred because some of the phenols and anilines investigated, despite possessing a redox potential compatible with one-electron abstraction, were scarcely oxidised. A threshold value of steric hindrance of the substrate, allowed for fitting into the active site of T. villosa laccase, was extrapolated from structural information provided by X-ray analysis of T. versicolor lac3B, sharing an identity of 99% at the protein level, thus enabling us to assess the relative contribution of steric and redox properties of a substrate in determining its susceptibility to laccase oxidation. The inferred structural threshold is compatible with the distance between two phenylalanine residues that mark the entrance to the active site. Interaction of the substrate with other residues of the active site is commented on.     

Effect of an extracellular laccase ofRigidoporus lignosus onHevea brasiliensis lignin

The abilities of white-rotting fungi to depolymerize lignin and to excrete laccases (p-diphenoloxidases), though brown-rotting fungi do not present these two biological properties, are the main differences between these two types of rotting fungi. Therefore it was assumed that the lignin scission was a result of the laccase reaction. Nevertheless,in vivo, this enzyme may play other major roles such as detoxifying of the medium by oxidation or condensing fungal growth-inhibiting phenolics. AsR. lignosus (causing a white rot on Hevea roots) secretes two laccases, our purpose was to determine whether these enzymes are able to depolymerize the lignin macromolecule or not. This was realized by showing the effect of theR. lignosus purified laccase L1 on the lignin polymerization degree by using Sephadex G100, G50, or G25 gel filtration. The laccase substrate was a lignin extracted from healthy Hevea root tissues by thioglycolic acid. This thioglycolic lignin (TGL) is hydrosoluble and is characterized by a differential ionization spectrum identical to that of a native lignin. This preparation is heterogeneous with regard to molecular weight (mw): 3000 daltons up to very high mw (excluded with the void volume of the G100 column), with a major fraction at nearly 10,000 daltons. The validity of the gel filtration method as for TGL molecular weight determination, was proved by chromatographing two TGL fractions that differ by their mw: a fraction A of high mw and a fraction B of low mw that were isolated by fractionation of crude TGL on a PM 10 Amicon Ultrafilter. The resulting elution patterns show a “normal” distribution for both fraction A and B. Moreover, repeating rechromatographies of three TGL fractions, differing from each other by their mw, demonstrated the high reproducibility of the gel filtration method. After incubation of TGL with the laccase L1, several related events could be observed (incubation in and column elution with phosphate 0.05M pH 6 buffer): o| li]1.|A quick browning of the solution and an increase (up to 50%) of the OD at 280 nm. li]2.|A progressive modification of the differential spectrum: disappearance of the maximum at 300 nm decrease of the maximum at 260 nm increase of the maximum at 365 nm which reflects the decrease of the ionizable phenolic hydroxyl groups and the increase of the number of α-carbonyl groups of the phenylpropane side chain. li]3.|The modification of the elution patterns on G100, G50, and G25, namely: A shift of the major “10,000 dalton peak” to a region where molecules of higher mw (50,000 daltons) are eluted. The appearance of several peaks corresponding to low mw molecules. This is especially clear when the reaction medium is filtrated on G50 or G25. The differential spectrum of one of those fractions shows a maximum at 335 nm, indicating most probably the presence of phenylcoumarone derivatives. These results show that the enzymatic activity of the laccase L1 on TGL results in a modification of the polymerization degree of the macromolecule leading to both condensation and depolymerization. Most probably an equilibrium between those reactions does exist. Nevertheless, it can also be assumed that the chemical bonds that are involved, respectively, in condensing and in splitting reactions, differ from each other.     

Essential Role of the N- and C-terminals of Laccase from Pleurotus florida on the Laccase Activity and Stability

POXA1b is the most thermostable laccase isoenzyme from Pleurotus ostreatus. POXA1b is remarkably stable at alkaline pH (the t_1/2 at pH 10 was 30 days), and its C-terminal affects its catalytic and stability properties. We cloned POXA1c from P. florida, which showed 99 % identity with POXA1b. POXA1c was functionally expressed in Pichia pastoris. The functions of the N and C termini of POXA1c were investigated using site-directed mutagenesis. Compared with POXA1c, the N-terminal R5V site effectively increased the specific activities for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and guaiacol by 2- and 3.5-fold, respectively. A C-terminal truncated mutant, POXA1c△13, also increased the specific activities for ABTS and guaiacol by 2.3- and 3.4-fold, respectively. A double mutant, POXA1cΔ13-R5V, combined the R5V and △13 effects. The specific activity of this double mutant for ABTS was 1,321 U/mg, which indicated a 4-fold increase compared with the wild type. The role of residue V5 on laccase catalytic properties was also observed for laccases from Trametes versicolor and Rigidoporus lignosus. The specific activities of the V5R of the laccases from T. versicolor and R. lignosus were half of that of the wild type. The pH and thermal stability analysis of POXA1c and its mutants showed that the enzymes were remarkably stable because they showed 63 % residual activity after incubation for 108 h at 30 °C over a pH range of 4.5 to 9.0. Similar results were observed for POXA1cΔ13-R5V. POXA1cΔ13-R5V can be widely used in industrial biotechnology because of its excellent catalytic properties.     

Degradation of Polycyclic Aromatic Hydrocarbons by <Emphasis Type="Italic">Rigidoporus lignosus</Emphasis> and its Laccase in the Presence of Redox Mediators

The metabolism of polycyclic aromatic hydrocarbons (PAHs) was studied in vivo and in vitro in systems consisting of Rigidoporus lignosus and its laccase, in the presence of so-called “mediator” compounds. The static culture of the native fungal strain was able to metabolize anthracene and 2-methylanthracene, but not 9-nitroanthracene. The addition of redox mediators 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) or violuric acid (VA) led to a significant increase in the degradation of substrates. The oxidation of PAHs was not significant when purified laccase was used without the addition of mediators. The addition of these compounds increased the oxidation of all substrates by approximately 70–80% after 72 h of incubation. The degradation rate was highest for 2-methylanthracene in the presence of VA.     

A Laccase of <Emphasis Type="Italic">Fomes durissimus</Emphasis> MTCC-1173 and Its Role in the Conversion of Methylbenzene to Benzaldehyde

A laccase has been purified from the liquid culture growth medium containing bagasse particles of Fomes durissimus. The method involved concentration of the culture filtrate by ultrafiltration and anion exchange chromatography on diethyl aminoethyl cellulose. The sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis both gave single protein band indicating that the enzyme preparation was pure. The molecular mass of the purified laccase determined from SDS-PAGE analysis was 75 kDa. Using 2,6-dimethoxyphenol as the substrate, the determined K _m and k _cat values of the laccase are 182 μM and 0.35 s⁻¹, respectively, giving a k _cat/K _m value of 1.92 × 10³ M⁻¹ s⁻¹. The pH and temperature optimum were 4.0 and 35 °C, respectively. The purified laccase has yellow colour and does not show absorption band around 610 nm found in blue laccases. Moreover, it transformed methylbenzene to benzaldehyde in the absence of mediator molecules, property exhibited by yellow laccases.     

Heterologous expression of Trametes versicolor laccase in Pichia pastoris and Aspergillus niger

Convenient expression systems for efficient heterologous production of different laccases are needed for their characterization and application. The laccase cDNAs lcc1 and lcc2 from Trametes versicolor were expressed in Pichia pastoris and Aspergillus niger under control of their respective glyceraldehyde-3-phosphate dehydrogenase promoters and with the native secretion signal directing catalytically active laccase to the medium. P. pastoris batch cultures in shake-flasks gave higher volumetric activity (1.3 U/L) and a better activity to biomass ratio with glucose than with glycerol or maltose as carbon source. Preliminary experiments with fed-batch cultures of P. pastoris in bioreactors yielded higher activity (2.8 U/L) than the shake-flask experiments, although the levels remained moderate and useful primarily for screening purposes. With A. niger, high levels of laccase (2700 U/L) were produced using a minimal medium containing sucrose and yeast extract. Recombinant laccase from A. nigher harboring the lcc2 cDNA was purified to homogeneity and it was found to be a 70-kDa homogeneous enzyme with biochemical and catalytic properties similar to those of native T. versicolor laccase A.     

Effect of the Inducers Veratryl Alcohol, Xylidine, and Ligninosulphonates on Activity and Thermal Stability and Inactivation Kinetics of Laccase from Trametes versicolor

Laccase production from Trametes versicolor was improved in the presence of the inducers ligninosulphonates, veratryl alcohol, and xylidine respectively two-, four-, and eightfold. The thermal inactivation of the produced laccase, after partial purification with ammonium sulfate was kinetically investigated at various temperatures (60?C70?°C) and pH values (3.5, 4.5, and 5.5). The inactivation process followed first-order kinetics for all conditions tested, except for veratryl alcohol, for which a constant activity level was observed at the end of the inactivation, also after first-order decay. Enzyme thermostability was affected by the type of inducer used in the culture medium for the production of laccase and also by the pH of incubation mixture. Generally, laccase stability increased with pH increment, being more stable at pH?5.5, except with xylidine. At pHs?4.5 and 5.5, the three inducers significantly increased laccase thermal stability, with the higher effect being observed for pH?5.5 and ligninosulphonates, where increment of half-life times ranged from 3- to 20-fold, depending on the temperature.     

A Comparative Study of Biocathodes Based on Multiwall Carbon Nanotube Buckypapers Modified with Three Different Multicopper Oxidases

14 Single‐ and multi‐walled carbon nanotubes from different sources were characterized in detail, and the characteristics obtained were carefully analyzed. The carbon material with the highest capacitance, and also other superior properties (“Taunit‐M” from “NanoTechCenter”, Russia), was chosen for further modification and fabrication of buckypaper based electrodes. These electrodes were biomodified with plant and fungal laccases, as well as fungal bilirubin oxidase. The designed biocathodes were investigated in simple buffers and also in a complex physiological fluid (human serum). Biocathodes based on immobilized fungal laccase were bioelectrocatalytically inactive in chloride containing media at neutral pH. In spite of the quite high current densities realized using biodevices based on plant laccase and fungal bilirubin oxidase, the limited thermal stability of the enzymes renders the biocathodes inadequate for practical applications in implanted situations.