Initial Laccase Research Articles

Hierarchically-structured laccase@Ni3(PO4)2 hybrid nanoflowers for antibiotic degradation: Application in real wastewater effluent and toxicity evaluation

Laccase@Ni3(PO4)2 hybrid nanoflowers (HNFs) were prepared by the anisotropic growth of biomineralized nickel phosphate. The immobilization yield was 77.5 ± 3.6 %, and the immobilized enzyme retained 50 % of its initial activity after 18 reusability cycles. The immobilized and free enzymes lost 80 % of their activity after 18 and 6 h incubation in municipal wastewater effluent (MWWE), respectively. The increase in α-helix content (8 %) following immobilization led to a more rigid enzyme structure, potentially contributing to its improved stability. The removal of ciprofloxacin from MWWE by laccase@Ni3(PO4)2·HNFs/p-coumaric acid oxidation system was optimized using a Box-Behnken design. Under the optimized conditions [initial laccase activity (0.05 U mL−1), the concentration of p-coumaric acid (2.9 mM), and treatment time (4.9 h)], the biocatalyst removed 90 % of ciprofloxacin (10 mg L−1) from MWWE. The toxicity of ciprofloxacin against some G+ and G− bacteria was reduced by 35–70 %, depending on their strain. The EC50 of ciprofloxacin for the alga Raphidocelis subcapitata reduced from 3.08 to 1.07 mg L−1 (p-value <0.05) after the bioremoval. Also, the acute and chronic toxicity of identified biodegradation products was lower than ciprofloxacin at three trophic levels, as predicted by ECOSAR software.

Biodegradation and biodetoxification of batik dye wastewater by laccase from Trametes hirsuta EDN 082 immobilised on light expanded clay aggregate.

The online version contains supplementary material available at 10.1007/s13205-021-02806-8.

Green and sustainable synthesis of oligorutin using an enzymatic membrane reactor: Process optimization

The enzymatic polymerization of rutin has been proved as an efficient method for obtaining products with enhanced properties, but toxic organic co-solvents are often involved in this process. In this work, the effect of temperature and enzyme activity were initially studied on 24h batch oligomerization reactions in organic-free reaction media. The optimal conditions, 1000U/L and 25°C, were chosen to operate an ultrafiltration enzymatic membrane reactor (UF-EMR) during three consecutive oligorutin synthesis cycles of 6 and 24h, with the objective of reusing the initial laccase activity. The use of this technology led to the design of a biocompatible and sustainable process with complete rutin conversion and remarkable laccase stability in at least three consecutive 24h cycles with no further enzyme addition. Despite an approximately 80% lower ferric reducing antioxidant power (FRAP), oligorutin displayed up to 1620-fold aqueous solubility and 24-fold iron chelating activity. Additionally, tests involving DNA degradation inhibition in exposure to biologically related radicals showed a better performance of oligorutin when compared to rutin.

Biodegradation of atrazine and ligninolytic enzyme production by basidiomycete strains

BackgroundAtrazine is one of the most widespread chlorinated herbicides, leaving large bulks in soils and groundwater. The biodegradation of atrazine by bacteria is well described, but many aspects of the fungal metabolism of this compound remain unclear. Thus, we investigated the toxicity and degradation of atrazine by 13 rainforest basidiomycete strains.ResultsIn liquid medium, Pluteus cubensis SXS320, Gloelophyllum striatum MCA7, and Agaricales MCA17 removed 30, 37, and 38%, respectively, of initial 25 mg L− 1 of the herbicide within 20 days. Deficiency of nitrogen drove atrazine degradation by Pluteus cubensis SXS320; this strain removed 30% of atrazine within 20 days in a culture medium with 2.5 mM of N, raising three metabolites; in a medium with 25 mM of N, only 21% of initial atrazine were removed after 40 days, and two metabolites appeared in culture extracts. This is the first report of such different outcomes linked to nitrogen availability during the biodegradation of atrazine by basidiomycetes. The herbicide also induced synthesis and secretion of extracellular laccases by Datronia caperata MCA5, Pycnoporus sanguineus MCA16, and Polyporus tenuiculus MCA11. Laccase levels produced by of P. tenuiculus MCA11 were 13.3-fold superior in the contaminated medium than in control; the possible role of this enzyme on atrazine biodegradation was evaluated, considering the strong induction and the removal of 13.9% of the herbicide in vivo. Although 88% of initial laccase activity remained after 6 h, no evidence of in vitro degradation was observed, even though ABTS was present as mediator.ConclusionsThis study revealed a high potential for atrazine biodegradation among tropical basidiomycete strains. Further investigations, focusing on less explored ligninolytic enzymes and cell-bound mechanisms, could enlighten key aspects of the atrazine fungal metabolism and the role of the nitrogen in the process.

Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity.

The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain.

Fabrication and Characterization of a Low-Cost Microfluidic System for the Manufacture of Alginate–Lacasse Microcapsules

The development of microfluidics-based systems in the recent years has provided a rapid and controlled method for the generation of monodisperse microencapsulates for multiple applications. Here, we explore the design, manufacture and characterization of a low-cost microsystem for the encapsulation of the fungal laccase from Pycnoporus sanguineus CS43 in alginate microcapsules. Multiphysics simulations were used to overview the fluid behavior within the device and estimate the resulting capsule size. Polymethylmethacrylate (PMMA) sheets were used for final microsystem manufacture. Different flow rates of the continuous (Qc) and discrete (Qd) phases in the ranges of 83–293 mL/h and 1–5 mL/h, respectively, were evaluated for microcapsule fabrication. Universal Serial Bus (USB) microscope and image analysis was used to measure the final particle size. Laccase encapsulation was evaluated using spectrophotometry and with the aid of fluorescent dyes and confocal microscopy. Results showed microcapsule size was in the range of 203.13–716.00 μm and Qc was found as the dominant parameter to control capsule size. There was an effective enzyme encapsulation of 65.94% with respect to the initial laccase solution.

Effect of Polyethylene Glycol and Triton X-100 on the Enzymatic Treatment of Bisphenol A

The current study includes the screening of indigenous extracellular laccase producing fungus from the soil, identified as Phanerochaete chrysosporiumspecies.The selected strain was applied under the optimized condition for the production of laccase in the solid state fermentation. The effect of polyethylene glycol and Triton X-100 on the enhancement of enzyme activity and biocatalytic removal of bisphenol A using laccase produced by Phanerochaete chrysosporium was investigated at 40°C, initial laccase activity 3U/mL and initial bisphenol A concentration 2 mM after 3 h of treatment. Bisphenol A concentration was evaluated using a colorimetric assay in the presence of 4-AAP. The effect of PEG molecular weight on BPA removal was determined using PEG with a molecular weight between 1500 and 20000.The results showed that the addition of polyethylene glycol and Triton X-100 to the reaction mixture greatly increased the laccase activity and the residual enzyme activity, but no significant changes were found in the removal of bisphenol A. The maximum removal of BPA was obtained at pH 8.0 and 40° C in 90 mg/L of PEG-1500 and 50, 100 mg/L of PEG-6000, while laccase activity at all concentrations of PEG-1500 and 6000 increased.

Laccase Activity as an Essential Factor in the Oligomerization of Rutin

The enzyme-mediated polymerization of bioactive phenolic compounds, such as the flavonoid rutin, has gained interest due to the enhanced physico-chemical and biological properties of the products, which increases their potential application as a nutraceutical. In this work, the influence of enzyme activity on rutin oligomerization was evaluated in reactions with low (1000 U/L) and high (10,000 U/L) initial laccase activities. For both reactions, high molecular weight oligomer fractions showed better properties compared to lower weight oligomers. Products of the reaction with low laccase activity exhibited thermal stability and antioxidant potential similar to control reaction, but led to higher inhibitory activity of xanthine oxidase and apparent aqueous solubility. Oligomers obtained in the reaction with high laccase activity showed better apparent aqueous solubility but decreased biological activities and stability. Their low antioxidant activity was correlated with a decreased phenolic content, which could be attributed to the formation of several bonds between rutin molecules.

Kinetic studies of Bisphenol A in aqueous solutions by enzymatic treatment

Bisphenol A (BPA), which is a major component in the production of various consumer products, is one of the most significant endocrine-disrupting chemicals. In this research, the biocatalytic elimination of Bisphenol A using laccase produced by Phanerochaete chrysosporium was examined and optimized. The response surface methodology was used to study the effect of independent variables such as pH, temperature, initial laccase activity, initial Bisphenol A concentration, and the interactive effects of each variable on the removal of Bisphenol A and residual enzyme activity. Based on response surface plots, the maximum Bisphenol A removal (93%) was detected at a pH value of 8, temperature 40 °C, initial laccase concentration 3 U/ml, and initial Bisphenol A concentration 2 mM after 3 h of treatment. The results showed that pH and temperature are the primary determining factors in Bisphenol A elimination. Maximum reaction rate and Michaelis constant were calculated from the Lineweaver–Burk plots and are 20.12 µM−1 min and 820 µM, respectively. The linearity of the data on the Lineweaver–Burk plot and the excellent correlation coefficient imply that laccase has a propensity to pursue Michaelis–Menten kinetics. In the present study, a high Bisphenol A degradation velocity with a low Km value was attained using the enzyme produced by P. chrysosporium. The degradation of Bisphenol A by the crude extract of laccase using the P. chrysosporium strain would provide a cost effective solution and seems to be an attractive option for the removal of micropollutants, including the recognized and suspected endocrine disruptors.

Enhanced removal of aqueous acetaminophen by a laccase-catalyzed oxidative coupling reaction under a dual-pH optimization strategy

Acetaminophen is one kind of pharmaceutical contaminant that has been detected in municipal water and is hard to digest. A laccase-catalyzed oxidative coupling reaction is a potential method of removing acetaminophen from water. In the present study, the kinetics of radical polymerization combined with precipitation was studied, and the dual-pH optimization strategy (the enzyme solution at pH7.4 being added to the substrate solution at pH4.2) was proposed to enhance the removal efficiency of acetaminophen. The reaction kinetics that consisted of the laccase-catalyzed oxidation, radical polymerization and precipitation were studied by UV in situ, LC-MS and DLS (dynamic light scattering) in situ. The results showed that the laccase-catalyzed oxidation is the rate-limiting step in the whole process. The higher rate of enzyme-catalyzed oxidation under a dual-pH optimization strategy led to much faster formation of the dimer, trimer and tetramer. Similarly, the formation of polymerized products that could precipitate naturally from water was faster. Under the dual-pH optimization strategy, the initial laccase activity was increased approximately 2.9-fold, and the activity remained higher for >250s, during which approximately 63.7% of the total acetaminophen was transformed into biologically inactive polymerized products, and part of these polymerized products precipitated from the water. Laccase belongs to the family of multi-copper oxidases, and the present study provides a universal method to improve the activity of multi-copper oxidases for the high-performance removal of phenol and its derivatives.

Biodegradation Kinetics of Phenol, 2,4,6-Trichlorophenol and Pentachlorophenol with ligninolytic enzymes from Dichomitus Squalens

Biodegradation of phenol, 2,4,6-trichlorophenol and pentachlorophenol was investigated using Dichomitus squalens cultivation media filtrates with initial laccase and manganese peroxidase activities of 1000 U L-1 and 300 U L-1, respectively. Decomposition of all three biocides was studied at T = 28 °C, pH = 4.5 and initial concentrations around 6 mg L-1 and the kinetics evaluated on the basis of the time course of the biocide concentration data. Integral analysis revealed 1st order reaction for all three pollutants and the corresponding reaction rate constants and initial concentrations for phenol, 2,4,6-TCP and PCP were estimated from linearized experimental data. The rate equations obtained allow estimation of the times necessary for a particular biocide conversion. The toxicity of reactants and products to the bacterium Vibrio fischeri before and after 48-hours degradation showed that the products were less toxic than the reactants in all three cases. The initial solutions of the three toxic substances caused 54 - 62 % inhibition of the bacterium Vibrio fischeri, while after 48 hours of degradation, 57 % reduction in the toxicity of phenol, 50 % reduction of 2,4,6-TCP and 29 % reduction of PCP was achieved.

Immobilization of laccase of Pycnoporus sanguineus CS43

Laccase from Pycnoporus sanguineus CS43 was successfully immobilized onto Immobead-150 and Eupergit-C by covalent binding and by entrapment in LentiKats. The highest immobilization was onto Immobead-150 (97.1±1.2%) compared to the other supports, LentiKats (89±1.1%) and Eupergit-C (83.2±1.4%). All three immobilized enzyme systems showed increased thermostability and better mechanical properties than free laccase. Moreover, after 5 cycles of reuse of these systems, 90% of initial laccase activity was retained. Immobead-150 and LentiKats systems exhibited the highest efficiencies in removal of m-cresol under the combined actions of biodegradation and adsorption, while laccase entrapped in LentiKats showed a high ability for degradation of m-cresol within 24h. In addition, the typical Michaelis-Menten enzymatic model effectively described the kinetic profile of m-cresol degradation by the enzyme entrapped in LentiKats. Based on the results obtained in the present study, it can be established that the immobilized biocatalysts developed here possess significant potential for wastewater treatment.

Effective removal of sulfur dyes from water by biosorption and subsequent immobilized laccase degradation on crosslinked chitosan beads

In this study, sulfur dyes were effectively removed from water samples through biosorption and subsequent immobilized laccase degradation on glutaraldehyde-crosslinked chitosan beads (GA-CBs). The Langmuir equation described the biosorption isotherms of the single dyes, sulfur blue 15 (SB15) and sulfur brown GD (SBGD), well. The decreased biosorption of one dye was found in binary mixtures by increasing the concentration of the second dye, and the extended Freundlich equation could reasonably describe such isotherms. Laccase immobilized on GA-CBs showed a high catalytic ability and provided reusability even at low laccase concentrations. In the treatment of single dyes at an initial concentration of 200mgL−1, for example, over 81.6% and 70.8% of SB15 and SBGD, respectively, were removed at a pH of 6.5 after the successive biosorption and enzymatic degradation with a biosorbent dose of 3.6gL−1, an initial laccase activity of 2.24UL−1, and 2.66×10−4mg(gsupport)−1 of immobilized laccase. In the treatment of binary mixtures under the same conditions, the efficiency of each dye decreased. The overall removals of SB15 and SBGD from binary mixtures containing 100mgL−1 of SB15 and 100mgL−1 of SBGD became 78.2% and 52.6%, respectively.

Characterization of combined cross-linked enzyme aggregates from laccase, versatile peroxidase and glucose oxidase, and their utilization for the elimination of pharmaceuticals

In order to transform a wide range of pharmaceutically active compounds (PhACs), the three oxidative enzymes laccase (Lac) from Trametes versicolor, versatile peroxidase (VP) from Bjerkandera adusta and glucose oxidase (GOD) from Aspergillus niger were concomitantly cross-linked after aggregation, thus, making a combined cross-linked enzyme aggregate (combi-CLEA) that was versatile and involved in an enzymatic cascade reaction. From the initial enzymes about 30% of initial laccase activity was recovered along with 40% for each of VP and GOD. The combi-CLEA showed good results in conditions close to those of real wastewater (neutral pH and medium temperature) as well as a good ability to resist to denaturing conditions such as high temperature (60°C) and low pH (3). Batch experiments were realized to test the free enzyme's ability to degrade, a PhACs cocktail, mainly in a synthetic wastewater containing acetaminophen, naproxen, mefenamic acid, indometacin, diclofenac, ketoprofen, caffeine, diazepam, ciprofloxacin, trimethoprim, fenofibrate and bezafibrate, carbamazepine and its by-product 10–11 epoxy-carbamazepine. High removal was achieved (more than 80%) for the five first compounds. Then, the elimination ability of the combi-CLEA with or without hydrogen peroxide, glucose or manganese sulfate was determined. Globally, our results demonstrated that VP has a wider removal spectrum than Lac. These removal features are enhanced under more specific conditions, whereas the combi-CLEA combined advantages of both VP and laccase. Finally, the elimination of PhACs in a municipal wastewater treatment plant effluent using the combi-CLEA was marginally investigated. Concentrations of most of the selected PhACs were below the limit of quantification (lower than 20ng/L) except for acetaminophen. Its combi-CLEA-mediated removal reached up to 25%.

Formulation and characterization of an immobilized laccase biocatalyst and its application to eliminate organic micropollutants in wastewater

Over the past decades, water pollution by trace organic compounds (ng L(-1)) has become one of the key environmental issues for developed countries. To date there is no effective and sustainable remediation strategy available. Laccases from white rot fungi were found particularly attractive for the removal of some micropollutants such as the plasticizer bisphenol A (BPA), the anti-inflammatory drug diclofenac (DF) and the steroidal hormone 17-α-ethinylestradiol (EE2). Laccase immobilization is a prerequisite for their use in continuous water treatment processes. In this study, laccase from Coriolopsis gallica was immobilized on mesoporous silica spheres in a two-step adsorption-crosslinking process. The initial laccase activity, crosslinker (glutaraldehyde) concentration and extra protein (albumin) concentration were varied following a central composite experimental design and optimized with respect to the immobilization yield, activity and thermal stability of the biocatalysts. After a multi-objective optimization of the biocatalyst formulation, a maximum biocatalyst activity of 383 Ug(-1), determined with 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) at pH 4.5, was obtained. Biocatalyst particles were physically characterized by means of scanning electron microscopy, Brunauer-Emmett-Teller surface area and Barrett-Joyner-Halenda pore size analyses revealing few modifications of the surface area and structure during/after the immobilization procedure. The biocatalyst showed a significantly higher thermostability than the free enzyme with a half-life of 31.5 hours and 3.9 hours compared to 6.1 hours and 0.6 hours at 55°C and 75°C respectively. The biocatalyst was able to eliminate in a continuously stirred membrane reactor more than 95% of BPA 10 μM and EE2 10 μM and 70% of DF 10 μM when treated individually and more than 90% when treated as a mixture in aqueous buffered solution (pH 5) for more than 60 reactor volumes. In real wastewater conditions (pH 7.8) the biocatalyst could degrade more than 85% of BPA and EE2 along with 30% of DF when tested in mixture for more than 80 hours, which illustrates the potential of this biocatalyst for the treatment of aquatic micropollutants.

Immobilization of laccase by encapsulation in a sol–gel matrix and its characterization and use for the removal of estrogens

Laccase from Myceliophthora thermophila was immobilized by encapsulation in a sol-gel matrix based on methyltrimethoxysilane and tetramethoxysilane. The amount of laccase used for the preparation of the hydrogel was in the range 2.2-22 mg of protein/mL sol and the corresponding enzymatic activities were in the range 5.5-17.0 U/g biocatalyst. The kinetic parameters of the encapsulated laccase showed that the immobilized enzyme presented lower affinity for the substrate 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). However, the stability of laccase was significantly enhanced after immobilization; thus, both pH and thermal stability improved about 10-30% and tolerance to different inactivating agents (NaN(3) , ZnCl(2) , CoCl(2) , CaCl(2) , methanol, and acetone) was 20-40% higher. The reusability of the immobilized laccase was demonstrated in the oxidation of ABTS for several consecutive cycles, preserving 80% of the initial laccase activity after 10 cycles. The feasibility of the immobilized biocatalyst was tested for the continuous elimination of Acid Green 27 dye as a model compound in a packed-bed reactor (PBR). Removals of 70, 58, 57, and 55% were achieved after four consecutive cycles with limited adsorption on the support: only 10-15%. Finally, both batch stirred tank reactor (BSTR) operated in several cycles and PBR, containing the solid biocatalyst were applied for the treatment of a solution containing the endocrine disrupting chemicals (EDCs): estrone (E1), 17β-estradiol (E2), and 17α-ethinylestradiol (EE2). Eliminations of EDCs in the BSTR were higher than 85% and the reusability of the biocatalyst for the degradation of those estrogens was demonstrated. In the continuous operation of the PBR, E1 was degraded by 55% and E2 and EE2 were removed up to 75 and 60%, at steady-state conditions. In addition, a 63% decrease in estrogenic activity was detected.

Study of the Alkyl Chain Length on Laccase Stability and Enzymatic Kinetic with Imidazolium Ionic Liquids

The activity and stability of laccase and their kinetic mechanisms in water soluble ionic liquids (ILs): 1-butyl-3-methyl imidazolium chloride [C(4)mim][Cl], 1-octyl-3-methyl imidazolium chloride [C(8)mim][Cl], and 1-decyl-3-methyl imidazolium chloride [C(10)mim][Cl] were investigated. The results show that an IL concentration up to 10% is satisfactory for initial laccase activity at pH9.0. The laccase stability was well maintained in [C(4)mim][Cl] IL when compared to the control. The inactivation of laccase increases with the length of the alkyl chain in the IL: [C(10)mim][Cl] > [C(8)mim][Cl] > [C(4)mim][Cl]. The kinetic studies in the presence of ABTS as substrate allowed calculating the Michaelis-Menten parameters. Among the ILs, [C(4)mim][Cl] was the suitable choice attending to laccase activity and stability. Alkyl chains in the ions of ILs have a deactivating effect on laccase, which increases strongly with the length of the alkyl chain.

Immobilization of laccase onto poly(glycidylmethacrylate) brush grafted poly(hydroxyethylmethacrylate) films: Enzymatic oxidation of phenolic compounds

Poly(hydroxyethylmethacrylate), p(HEMA) films were prepared via UV-initiated photo-polymerization. After activation of the hydroxyl groups of p(HEMA) by bromination, surface-initiated atom transfer radical polymerization (ATRP) of glycidylmethacrylate was conducted in dioxane/bipyridine mixture with CuBr as catalyst at 65 °C. The epoxy groups of the poly(glycidylmethacrylate) brushes were converted into amino groups with the reaction of ammonia. The modified p(HEMA-g-GMA)-NH 2 films were used as an ion-exchange support for the immobilization of laccase. The influence of pH and initial laccase concentration on the immobilization capacity of the p(HEMA-g-GMA)-NH 2 films has been investigated. The amount of immobilized laccase on the p(HEMA-g-GMA)-NH 2 films was determined as 139 μg/cm 2 films. The recovered activity of the immobilized laccase on the fibrous polymer grafted films was about 71% compared to free enzyme. The maximum activity ( V max) and Michealis constant ( K m) of laccase immobilized on the films, were found to be 15.4 U/mg and 23 mM, respectively. Finally, the immobilized laccase was operated in a batch system for enzymatic oxidation of phenol, p-chlorophenol and aniline.

Assessment of anthraquinone-dye conversion by free and immobilized crude laccase mixtures

This paper reports on the assessment of two enzyme-based processes for the conversion of synthetic dyes. A crude laccase mixture from Pleurotus ostreatus was immobilised on EUPERGIT C 250L ©, an acrylic resin with epoxy functionalities. This biocatalyst was used in the conversion of the anthraquinonic dye Remazol brilliant blue R. Two different fixed bed reactors were used to assay the solid biocatalyst activity and to characterize the dye conversion kinetics. The immobilisation procedure was optimized providing a maximum immobilisation yield of 7% of the initial laccase activity in solution. The kinetics of dye conversion was in line with a product-inhibited kinetic model. The biocatalyst underwent moderate deactivation along with conversion. The kinetic models were implemented to describe dye conversion in a continuous fixed bed reactor (CFBR), operated with immobilised laccases, and in a stirred tank reactor (STR), operated with free laccases. The CFBR option enables the remediation of a wastewater volume larger than the one processed in the STR under comparable operating conditions. The better performance of CFBR results from the balanced effect of an improved stability of immobilised laccases and of a loss of activity following immobilisation.

Purification and biochemical characterization of a laccase from the aquatic fungus Myrioconium sp. UHH 1-13-18-4 and molecular analysis of the laccase-encoding gene

Myrioconium sp. strain UHH 1-13-18-4 is an ascomycete anamorph isolated from the river Saale, Central Germany. An extracellular, monomeric, and glycosylated laccase with a molecular mass of 72.7 kDa as determined by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and an isoelectric point below 2.8 was purified from CuSO(4) and vanillic acid amended liquid fungal cultures grown in malt extract medium. The catalytic efficiencies (k(cat)/K(m)) for the oxidation of syringaldazine, 2,6-dimethoxyphenol, and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) were 67.3, 46.9, and 28.2 s(-1) mM(-1), respectively, with K(m) values of 4.2, 67.8, and 104.9 microM. After pre-incubation at different pH values and temperatures for 1 h, more than 80% of the initial laccase activity was retained between pH 4 to 6 and 15 degrees C. The laccase-encoding gene was identified and sequenced at both the genomic and complementary DNA (cDNA) level, and corresponding structural characteristics and putative regulatory elements of the promoter region are reported. The identification of two tryptic peptides of the purified enzyme by mass spectrometry confirmed the identity of the functional laccase protein with the translated genomic sequence of the Myrioconium sp. laccase. Myrioconium sp. laccase shows the highest degree of identity with laccases from ascomycetes belonging to the family Sclerotiniaceae, order Helotiales.