Laccase-mediator System Research Articles

Laccase-Mediator Catalyzed Modification of Wood Fibers: Studies on the Reaction Mechanism and Making of Medium-Density Fiberboard

ABSTRACT Owing to the constant increase of prices of the process for petrochemical resin and the possibility of harmful formaldehyde emissions from industrial produced medium-density fiberboards (MDF), enzymatic binder systems are discussed as an environmentally friendly alternative for gluing lignocelluloses such as wood fibers. In this work laccase-mediator systems (LMSs) were used to activate the lignin on wood fiber surfaces. Two different mediators were tested, vanillic acid (VAN) and 4-hydroxybenzoic acid (HBA), of which HBA performed best. Carbon-13 nuclear magnetic resonances (13C-NMR) and electron spin resonances (ESR) of LMS-treated thermomechanical pulp (TMP) fibers were determined for qualitative and quantitative analysis of lignin activation. Analysis outputs were transferred to produce MDF using a dry process. 13C-NMR revealed more structural changes in the wood fibers using LMS with HBA than LMS with VAN. ESR spectroscopy indicated a higher amount of phenoxy radicals after treatment with LMS containing HBA as a mediator. The data correlated well with the quality of MDF. The best mechanical technological properties were achieved by using HBA within the LMS, so that the European Norms could be fulfilled. But VAN also performed well, which showed a high potential to produce ecofriendly MDF by using LMSs in the future.

Banning toxic heavy-metal catalysts from paints: enzymatic cross-linking of alkyd resins

Alkyd resins are polyesters containing unsaturated fatty acids that are used as binding agents in paints and coatings. Chemical drying of these polyesters is based on heavy metal catalyzed cross-linking of the unsaturated fatty acid moieties. Among the heavy-metal catalysts, cobalt complexes are the most effective, yet they have been proven to be carcinogenic. Therefore, strategies to replace the cobalt-based catalyst by environmentally friendlier and less toxic alternatives are under development. Here, we demonstrate for the first time that a laccase–mediator system can effectively replace the heavy-metal catalyst and cross-link alkyd resins. Interestingly, the biocatalytic reaction does not only work in aqueous media, but also in a solid film, where enzyme diffusion is limited. Within the catalytic cycle, the mediator oxidizes the alkyd resin and is regenerated by the laccase, which is uniformly distributed within the drying film as evidenced by confocal laser scanning microscopy. During gradual build-up of molecular weight, there is a concomitant decrease of the oxygen content in the film. A new optical sensor to follow oxygen consumption during the cross-linking reaction was developed and validated with state of the art techniques. A remarkable feature is the low sample amount required, which allows faster screening of new catalysts.

Synthesis of disulfides by laccase-catalyzed oxidative coupling of heterocyclic thiols

A new method employing a laccase–mediator system as the catalyst and aerial oxygen as the oxidant has been developed for the oxidative coupling of heterocyclic thiols to the corresponding disulfides with yields up to 95% under mild reaction conditions.

A new biobleaching sequence for kenaf pulp: Influence of the chemical nature of the mediator and thermogravimetric analysis of the pulp

This paper evaluates five phenolic compounds as mediators for kenaf pulp biobleaching by laccase. The results have been compared with the treatment using a non-phenolic mediator, 1-hydroxybenzotriole and laccase alone. The influence of the nature of the chemical mediators used on various pulp properties is discussed. In addition to oxidizing lignin, the phenolic radicals formed in the process take part in condensation and grafting reactions in enzymatic stage. After biobleaching sequence (LP), syringaldehyde was shown to be the best phenolic mediator, allowing a delignification of 43% and 72% ISO brightness. These results were similar to the use of laccase alone due to the role as mediators of syringyl units resulting from oxidative lignin degradation. As a novelty, the study was supplemented with thermogravimetric analysis, with emphasis on the crystallinity degree of the cellulose surface and the aim of elucidating the action mechanisms of laccase-mediator systems on fiber.

Combinatorial evaluation of laccase-mediator system in the oxidation of veratryl alcohol

Laccases play an important role in the biological break down of lignin and have great potential in the deconstruction of lignocellulosic feedstocks. We examined 16 laccases, both commercially prepared and crude extracts, for their ability to oxidize veratryl alcohol in the presence of various solvents and mediators. Screening revealed complete conversion of veratryl alcohol to veratraldehyde catalyzed by a crude preparation of the laccase from Trametes versicolor ATCC 11235 and the mediator TEMPO in 20% (v/v) tert-butanol.

Prediction model based on decision tree analysis for laccase mediators

A Structure Activity Relationship (SAR) study for laccase mediator systems was performed in order to correctly classify different natural phenolic mediators. Decision tree (DT) classification models with a set of five quantum-chemical calculated molecular descriptors were used. These descriptors included redox potential (ɛ°), ionization energy (Ei), pKa, enthalpy of formation of radical (ΔfH), and OH bond dissociation energy (DO–H). The rationale for selecting these descriptors is derived from the laccase-mediator mechanism. To validate the DT predictions, the kinetic constants of different compounds as laccase substrates, their ability for pesticide transformation as laccase-mediators, and radical stability were experimentally determined using Coriolopsis gallica laccase and the pesticide dichlorophen. The prediction capability of the DT model based on three proposed descriptors showed a complete agreement with the obtained experimental results.

Degradation of some benzodiazepines by a laccase-mediated system in aqueous solution

Purified laccase from the soil ascomycete, Paraconiothyrium variabile was employed in the degradation of 7 benzodiazepine substances in the absence and presence of the enzyme mediators, 1-hydroxybenzotriazole (HBT), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol (DMP), and vanillic acid (VA). In the absence of a laccase mediator, the original concentrations of 10μgmL−1 of nitrazepam, alprazolam, diazepam, and oxazepam decreased by 27.3%, 45.6%, 18.6% and 18.7%, respectively, after 48h treatment using the purified enzyme, whereas the removal percentages for clobazam, chlordiazepoxide, and lorazepam were only 5.6%, 3.6%, and 4.1%, respectively. Among the laccase mediators, HBT was the most efficient compound, increasing the degradation percentages of nitrazepam, alprazolam, diazepam, and oxazepam to 73%, 88.1%, 61.4%, and 71.2%, respectively. The removal percentages of clobazam, chlordiazepoxide, and lorazepam was increased to 8.2%, 4.7%, and 6.5%, respectively, when the laccase-HBT system was used. The data presented suggest that the laccase-mediated system has potential for the elimination of some benzodiazepines in aqueous solution.

EFFECT OF REFINING ON DELIGNIFICATION WITH A LACCASE /XYLANASE TREATMENT

Previous research has demonstrated that a laccase/xylanase system (LXS) from white-rot fungus (Lentinus lepideus) has the same ability to delignify as a laccase/mediator system (LMS). In order to enhance delignification ability of LXS treatment, the effect of refining on delignification with LXS treatment of Masson pine (Pinus massoniana) pulp was investigated by refining the pulp in a PFI mill to different revolutions (14,000/21,000/35,000/42,000/56,000) prior to LXS treatment (enzyme dosage 5/10/15IU/g o.d. pulp). The results indicated that both kappa number and yield of the LXS treated pulp decreased with increasing refining. A substantial delignification without severe yield loss could be achieved by moderate refining prior to LXS treatment. The SEM images suggested that refining increased the accessibility of the material to enzymes and thereby enhanced the delignification ability of LXS. The small cellulase activity detected in the LXS had little effect on the viscosity of the treated pulp even at high enzyme dosage.

Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccases

The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC–MS and 1H NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzyme’s properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C–O coupling type structures being proposed.

Evaluation of the structural and molecular weight changes of lignin during the treatment of hardwood alkaline peroxide mechanical pulp with laccase and a laccase-mediator-system

Alkaline Peroxide Mechanical Pulp (APMP) of triploid of Populus tomentosa was modified by laccase and a Laccase-Mediator-System (LMS). The influence of the following main variables on the pulp physical properties were studied: enzyme dosage, reaction time, treatment temperature, and pH. Under the optimum conditions of laccase treatment – pH 5, temperature 50°C, pulp consistency 4%, and a reaction time of 60 min – the optimum charge of laccase was 2u/g. It was also found that the tensile strength and tear indices of the pulps treated with laccase increased significantly. The two-stage method of enzyme-mild acidic hydrolysis was adopted to isolate lignin from the APMP pulps both before and after enzymatic treatments. The functional groups in all lignin samples were qualitatively and quantitatively analyzed with 31P-NMR spectra. The molecular weight distributions of all the lignin samples were obtained through Gel Permeation Chromatography (GPC) after the lignin samples were benzoylated.

Application of response surface methodology to optimize decolourization of dyes by the laccase-mediator system

Response surface methodology (RSM) was applied to optimize the decolourization of 3 dyes belonging to 3 dye families such as reactive black 5 (diazoic), indigo carmine (indigoid) and aniline blue (anthraquinonic). Crude laccase from Trametes trogii and the laccase-mediator 1-hydroxybenzotriazole (HBT) were used in this study. Box-Behnken design using RSM with six variables namely pH, temperature, enzyme concentration, HBT concentration, dye concentration and incubation time was used in this study to optimize significant correlation between the effects of these variables on the decolourization of reactive black 5 (RB5), indigo carmine (IC) and aniline blue (AB). The optimum of pH, temperature, laccase, HBT, RB5 and reaction time were 4.5, 0.5 U ml(-1), 0.5 mM, 100 mg ml(-1) and 150 min respectively, for a maximum decolourization of RB5 (about 92.92% ± 7.21). Whereas, the optimum decolourization conditions of both IC (99.76% ± 7.75) and AB (98.44% ± 10) were: pH 4.5, temperature of 45 °C, enzyme concentration of 0.1 U ml(-1) and 0.5 U ml(-1), HBT concentration of 0.9 mM and 0.5 mM, dye concentration of 60 mg l(-1) and reaction time of 150 and 90 min, respectively. The experimental values were in good agreement with the predicted ones and the models were highly significant, the correlation coefficient (R(2)) being 0.864, 0.663 and 0.776 for RB5, IC and AB, respectively. In addition, when the kinetic parameters for the three dyes decolourization were calculated according to Hannes-Wolf plot, the following values were obtained: Km of 268.4, 47.94 and 44.64 mg l(-1) then V(max) of 35.58, 10.43 and 9.23 mg l(-1) min for the RB5, IC and AB decolourizations by laccase, respectively.

Laccase-mediated polymerization of 3,4-ethylenedioxythiophene (EDOT)

The oxidative polymerization of 3,4-ethylenedioxythiophene (EDOT) has been carried out using laccase-mediator system. High redox potential laccase isolated from fungi Trametes hirsuta catalyzed the reaction of EDOT oxidation. In this reaction laccase used atmospheric oxygen as the terminal oxidant. Potassium octocyanomolybdate(4+) served as high potential redox mediator. The increase in the rate of EDOT oxidation occurs due to laccase catalysis of octocyanomolybdate(4+) ion to octocyanomolybdate(5+). A template method synthesis of EDOT oligomers with poly(2-acrylamido-2-methyl-1-propanesulfonic) acid (PAMPS) was used. The water dispersible oligoEDOT/PAMPS complex was characterized by UV–vis, Fourier transformed infrared spectra and transmission electron microscopy.

Steam explosion and enzymatic pre-treatments as an approach to improve the enzymatic hydrolysis of Eucalyptus globulus

Abstract Eucalyptus globulus woodchips were subjected to steam explosion under a variety of operational conditions in order to evaluate their effect on a subsequent enzymatic hydrolysis. Steam explosion enhanced the enzymatic hydrolysis of the solid fraction. The application of a laccase-mediator system (LMS) before the enzymatic hydrolysis increased the glucose yield from 24.7 to 27.1%, probably as a result of the detoxification of the substrate. However, when xylanase was used to boost the LMS treatment, the hydrolysis rates did not improve. In order to assess the total amount of sugars obtainable from E. globulus , the liquid fraction obtained from the steam explosion was subjected to hydrolysis too. The enzymatic hydrolysis of the liquid fraction was more efficient than the acid hydrolysis, breaking down 82.7% of the xylooligosaccharide backbone into its monomeric constituent, xylose. The overall yield was 210 g of sugars per kg of raw material.

Surfactant-assisted two phase partitioning bioreactors for laccase-catalyzed degradation of anthracene

The degradation of anthracene by laccase from Trametes versicolor in enzymatic reactors was evaluated. The use of a surfactant (Triton X-100) at concentration above critical micelle concentration (CMC) enhanced anthracene solubility and facilitated its degradation. Moreover, Triton exerted a beneficial effect on the laccase stability and protected it from the oxidative action of the mediator 1-hydroxybenzotriazole (HBT). In a further stage, the combined configuration of a two phase partitioning bioreactor (TPPB) operating with silicone oil as an immiscible solvent and the surfactant achieved the degradation of anthracene at higher conversion rate: 16μmol/LRh. Furthermore, a model for anthracene degradation by laccase-mediator system was developed. The first order kinetic constant (k) and the overall mass transfer coefficient (KLa) were estimated by using the method of least squares. The increased KLa value obtained, 788.1h−1, proved that Triton also improved mass transfer. Anthracene concentration in aqueous phase was close to that corresponding to equilibrium state suggesting that mass transfer mechanism did not limit the global process. The kinetic constant, which is expected to depend on the initial concentration of enzyme, resulted in 52.2h−1. Enzyme inactivation occurred in two stages and could be modeled by using a three parameter biexponential model. The possibility of reusing silicone oil to dissolve more anthracene was proven in three consequent cycles with high percentages of anthracene removal.

STUDIES ON XYLANASE AND LACCASE ENZYMATIC PREBLEACHING TO REDUCE CHLORINE-BASED CHEMICALS DURING CEH AND ECF BLEACHING

The biobleaching efficiency of xylanase and laccase enzymes was studied on kraft pulps from wood and nonwood based raw materials employed in the Indian paper industry. Treatment of these pulps with xylanase enzyme could result in improved properties, showing 2.0% ISO gain in pulp brightness and/or reducing the demand of chlorine-based bleach chemicals by up to 15% with simultaneous reduction of 20 to 25% in AOX generation in bleach effluents. Further, mill-scale trial results revealed that enzymatic prebleaching can be successfully employed with xylanases to reach the same bleach boosting efficacy. Laccase bleaching was also studied on hardwood pulp at a pH around 8.0, where most of the pulp mills in India are operating, in contrast to earlier studies on laccase enzyme bleaching, which were conducted at acidic pHs, i.e. 4.0 to 5.0. In case of laccase bleaching, interesting results were found wherein a bleach-boosting effect was observed even at pH 8.0. Further studies carried out with HOBT as mediator in comparison to the commonly used and expensive ABTS laccase mediator system (LMS) resulted in improvement of the bleaching efficiency with reduction in demand of chlorine dioxide by more than 35%. Potential for further reduction was indicated by the brightness gain, when compared with a control using the DE(p)D bleach sequence.

Effect of steam explosion and enzymatic pre-treatments on pulping and bleaching of Hesperaloe funifera

A non-wood raw material with high potential for pulp and paper applications (Hesperaloe funifera) was subjected to a steam explosion pre-treatment, and the subsequent effect of this pretreatment on biopulping and biobleaching was studied. An increase in the delignification rate, bigger than that reported for autohydrolysis and acid hydrolysis pre-treatments, and a reduction in chemical consumption were found during kraft pulping of the exploded samples. However, biopulping with the laccase-mediator system (LMS) did not lead to a reduction in the kappa number in either non-exploded or exploded unbleached pulps. On the other hand, the steam explosion pretreatment boosted the advantages of the LMS pre-treatment (decrease in kappa number and increase in brightness) favored biobleaching, with a 53.1% delignification rate and a final brightness of 67% ISO. Finally, the steam explosion pre-treatment also improved the color properties of the bleached pulp and reduced the hydrogen peroxide consumption by 24.6%.

Laccase-mediated synthesis of conducting polyaniline

Laccase-mediated system based on potassium octocyanomolybdate (4+) was first used for acceleration of the enzymatic aniline polymerization. The enzymatic reaction yielded oxidized octocyanomolybdate (5+) which can oxidize the aniline monomer to the aniline radical cation. This resulted in the formation of conducting polyaniline with the concomitant regeneration of the redox mediator. The proposed method is environmentally benign, permits a higher degree of control over the kinetics of the reaction and, hence, the synthesis of a conducting polymer with improved physicochemical properties. The optimal conditions for the laccase-mediated synthesis have been found. The redox mediator does not modify the backbone of conducting polyaniline (PANI). The characterization of the polymer obtained by either a laccase-catalyzed method or a laccase-mediator method was carried out using UV–vis, FTIR spectroscopy, TEM investigation and the MALDI-TOF mass spectrometry. The advantage of a laccase-mediated synthesis of PANI compared with the synthesis catalyzed by laccase alone has been shown.

Microbial Enzyme: Applications in Industry and in Bioremediation

Microbial Enzyme: Applications in Industry and in Bioremediation

Modification of old corrugated container pulp with laccase and laccase–mediator system

Modification of the physical properties of old corrugated container (OCC) pulp with laccase or a laccase–mediator (ABTS, HBT, VA) system was investigated under select enzymatic concentrations and reaction times. The optimal conditions for laccase treatment shown to be using a laccase dose of 160U/g o.d. pulp, a treatment time of 20h at 25°C, pH 7 with a pulp consistency of 5%. Results showed that the Lac-HBT treated OCC pulp gave the best strength properties, improving tensile strength by 15.7%. The increase in the carboxyl group content of OCC laccase or Lac-HBT treated pulp led to the increase in the swelling ability and bonding between fibers. Microscope images showed the fiber surface became rougher and more collapsible after Lac-HBT treatment. FT-IR data showed that new carboxylic acid groups were formed during Lac-HBT treatment.

Enzymatic deinking of secondary fibers: cellulases/hemicellulases versus laccase-mediator system

The use of enzymes has been suggested as an environmentally friendly alternative to complement conventional chemical deinking in the recycling of recovered paper. This study compares the use of cellulases/hemicellulases versus the laccase-mediator system for deinking printed fibers from newspapers and magazines. For this purpose, two commercial enzyme preparations with endoglucanase and endoxylanase activities (Viscozyme Wheat from Aspergillus oryzae and Ultraflo L from Humicola insolens, Novozymes) and a commercial laccase (NS51002 from Trametes villosa, Novozymes), the latter in the presence of synthetic or natural (lignin-related) mediators, were evaluated. The enzymatic treatments were studied at the laboratory scale using a standard chemical deinking sequence consisting of a pulping stage; an alkaline stage using NaOH, sodium silicate and fatty acid soap; and a bleaching stage using hydrogen peroxide. The handsheets were then prepared and their brightness, residual ink concentration, and strength properties were measured. Among the different enzymatic treatments assayed, both carbohydrate hydrolases were found to deink the secondary fibers more efficiently. Brightness increased up to 3-4% ISO on newspaper fibers, being Ultraflo 20% more efficient in the ink removal. Up to 2.5% ISO brightness increase was obtained when magazine fibers were used, being Viscozyme 9% more efficient in the ink removal. Regarding the laccase-mediator system, alone or in combination with carbohydrate hydrolases, it was ineffective in deinking both newspaper and magazine fibers, resulting in pulps with worse brightness and residual ink concentration values. However, pulp deinking by the laccase-mediator system was displayed when secondary fibers from printed cardboard were used, obtaining up to 3% ISO brightness increase and lower residual ink concentrations.