Effects of laccase-natural mediator systems on kenaf pulp

This paper reports the first application of laccase-mediator systems (LMS) to kenaf pulp. Five natural phenolic compounds (acetosyringone, syringaldehyde, p-coumaric acid, vanillin and acetovanillone) were used as mediators in combination with laccase in an L stage in order to elucidate their effect on delignification. After LMS treatment, pulp samples were subjected to two alkaline treatments (an E or P stage). The results obtained were compared with those provided by the laccase-1-hydroxybenzotriazole (HBT) system. All natural mediators increased kappa number, decreased brightness and changed optical properties of the pulp after the L stage, suggesting that natural mediators tend to couple to fibers during a laccase-mediator treatment. The greatest delignification and bleaching effects after the P stage were obtained with syringaldehyde and acetosyringone, providing an effective means for delignifying kenaf, whereas those based on the other three could be used to functionalize kenaf with a view to obtaining pulp with novel properties.     

Application of laccase-natural mediator systems to sisal pulp: An effective approach to biobleaching or functionalizing pulp fibres?

The effects of laccase-natural mediator systems (LMS) on sisal pulp and their potential for either biobleaching or functionalizing (via radical-coupling) its fibres were investigated. The enzyme treatment (L stage) was followed by extraction with hydrogen peroxide in order to determine whether observable effects could be enhanced by removing LMS-modified lignin. Four different plant phenols [viz. the p-hydroxycinnamic compounds sinapic acid (SNC), ferulic acid (FRC), coniferyl aldehyde (CLD) and sinapyl aldehyde (SLD)] were used as laccase redox mediators and their effects on pulp and effluents compared with those of the synthetic compound 1-hydroxybenzotriazole (HBT). During the L stage performed with HBT, laccase underwent a loss of 99% and 78% of the initial activity, in the absence and presence of pulp, respectively. With natural mediators inactivation was markedly reduced, being the residual activity between 65% and 100% of the initial one, in the presence of pulp. The pulp was found to protect the enzyme against inactivation: the activity was only reduced by 45% in its presence. Under the operating conditions used the natural mediators proved less efficient than HBT in facilitating pulp bleaching; rather, they tended to bind to pulp fibres. This effect could be used to functionalize fibres in order to improve intrinsic properties of pulp or introducing novel ones (e.g. antimicrobial, antioxidant, optical properties, etc.). This paper shows for the first time the application of laccase-mediator systems to sisal pulp.     

The role of xylanases and laccases on hexenuronic acid and lignin removal

The variation of the contents in hexenuronic acids (HexA) and lignin in Eucalyptus kraft pulp during sequences with the laccase–mediator treatment with or without xylanase pretreatment was studied. The laccase–HBT system (HBT: 1-hydroxybenzotriazole) initially oxidized lignin alone but altered cellulose in the pulp as well after some time. Once all accessible lignin was removed, the system acted on HexA. As a result, the laccase–mediator treatment reduced the HexA content of the pulp, especially if a xylanase pretreatment was applied before. A previously unseen effect was observed here: HexA removal was found to depend on the laccase and HBT doses, but not on the reaction time. In addition, the xylanase pretreatment was found to strongly boost the effects of the laccase–HBT system by facilitating their access to HexA without affecting the lignin content.     

Reactivities of Various Mediators and Laccases with Kraft Pulp and Lignin Model Compounds

Laccase-catalyzed oxygen delignification of kraft pulp offers some potential as a replacement for conventional chemical bleaching and has the advantage of requiring much lower pressure and temperature. However, chemical mediators are required for effective delignification by laccase, and their price is currently too high at the dosages required. To date, most studies have employed laccase from Trametes versicolor. We have found significant differences in reactivity between laccases from different fungi when they are tested for pulp delignification in the presence of the mediators 2,2(prm1)-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1-hydroxybenzotriazole (HBT). A more detailed study of T. versicolor laccase with ABTS and HBT showed that HBT gave the most extensive delignification over 2 h but deactivated the enzyme, and therefore a higher enzyme dosage was required. Other mediators, including 1-nitroso-2-naphthol-3,6-disulfonic acid, 4-hydroxy-3-nitroso-1-naphthalenesulfonic acid, promazine, chlorpromazine, and Remazol brilliant blue, were also tested for their ability to delignify kraft pulp. Studies with dimeric model compounds indicated that the mechanisms of oxidation by ABTS and HBT are different. In addition, oxygen uptake by laccase is much slower with HBT than with ABTS. It is proposed that the dication of ABTS and the 1-oxide radical of HBT, with redox potentials in the 0.8- to 0.9-V range, are required for pulp delignification.     

Supramolecular structure characterization of molecularly thin cellulose I nanoparticles

Unusual fractions of cellulose microfibrils from woody material with dimensions of hundreds of nanometers in length and single digit angstrom thickness were obtained by intensive sonication of TEMPO-oxidized cellulose fibers. These cellulose microfibril fragments, composed of many mono- and bilayer molecular sheets, were analyzed with scattering and spectroscopy techniques to understand the structural changes at the supramolecular level. XRD data indicated that sonication breaks the cellulose microfibrils along its (200) planes, yet some form of the Iβ crystalline structure is still retained with reduced crystallinity. The Raman and FTIR analysis indicated structural changes to the cellulose microfibrils do not occur until after sonication; furthermore, AFM observation indicates that the structural changes began to occur within 5 min of sonication. An altered supramolecular structure is evident after sonication: major features from cellulose I are preserved, although certain spectral features similar to mercerized and ball milled cellulose appeared in its FTIR and Raman spectra. These spectral differences are traced to changes in the methine environment, hydroxymethyl conformations, and skeletal vibrations. By integrating the present findings and previous research, a cellulose molecular sheet delamination scheme is proposed to describe this microfibril fragmentation along its (200) plane.     

Laccase for biobleaching of eucalypt kraft pulp by means of a modified industrial bleaching sequence

Biobleaching of kraft pulp is a possible application of laccase, but it has not been described in detail for complete industrial bleaching sequences yet. Therefore, in this work, the biobleaching of Eucalyptus globulus kraft pulp was performed using a modified industrial totally chlorine‐free sequence. The modification consisted in the substitution of an enzymatic delignification stage, based on the application of laccase from Trametes villosa, for the first alkaline extraction one. The enzymatic stage was performed with several synthetic and natural mediators, namely 1‐hydroxybenzotriazole (HBT), violuric acid (VA), methyl syringate, and syringaldehyde. Several pulp properties were analyzed after each stage of the bleaching process—kappa number, ISO brightness, viscosity, and optical properties of CIEL*a*b* system. The new biobleaching sequence improved the pulp properties, in comparison to the conventional bleaching sequence, if HBT or VA was used as mediators. VA was selected as the best mediator of those tested and the effect of its concentration in the enzymatic stage was subsequently studied. Reducing the initial concentration by 30%, the same pulp quality was obtained, but if the reduction attained 60%, an important decrease in pulp integrity was detected. The modified bleaching sequence could improve the bleached pulp properties (kappa number 10%, ISO brightness 1%, and viscosity 5%) in comparison to the mill sequence. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The effect of the cellulose-binding domain from Clostridium cellulovorans on the supramolecular structure of cellulose fibers

The cellulose-binding domain (CBD) is the second important and the most wide-spread element of cellulase structure involved in cellulose transformation with a great structural diversity and a range of adsorption behavior toward different types of cellulosic materials. The effect of the CBD from Clostridium cellulovorans on the supramolecular structure of three different sources of cellulose (cotton cellulose, spruce dissolving pulp, and cellulose linters) was studied. Fourier-transform infrared spectroscopy (FTIR) was used to record amides I and II absorption bands of cotton cellulose treated with CBD. Structural changes as weakening and splitting of the hydrogen bonds within the cellulose chains after CBD adsorption were observed. The decrease of relative crystallinity index of the treated celluloses was confirmed by FTIR spectroscopy and X-ray diffraction (XRD). X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to confirm the binding of the CBD on the cellulose surface and the changing of the cellulose morphology.     

Laccase-induced HBT-grafting to milled beech wood reduces unspecific protein adsorption

Lignocellulosic biomass is a ubiquitous and renewable feedstock for the production of platform chemicals and biofuels. Typically, this recalcitrant biomass is pretreated by physico-chemical techniques causing disintegration and delignification. An additional treatment with laccase-mediator-systems (LMS) has been found to further improve the subsequent enzymatic cellulose hydrolysis. The aim of this study was to investigate the impact of different LMS on the glucose yield of a subsequent hydrolysis of treated beech wood and to elucidate the underlying effect of LMS treatment. The mediators 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS), 1-hydroxybenzotriazol (HBT) and syringaldehyde were evaluated, but an enhancing effect of LMS treatment on beech wood hydrolysis was only found for HBT. In mass spectrometry analysis of the acid hydrolysate of LMS-treated samples, the mediator HBT could be found in the lignin samples, suggesting a grafting reaction. The fluorescent protein mCherry was used as a reporter for unspecific protein adsorption to biomass samples. LMS treatment with HBT reduced the unspecific adsorption of mCherry to raw beech wood by about 50%, suggesting that the HBT grafting to beech wood lignin decreased the unproductive cellulase binding. In summary, the reduction of unspecific protein adsorption by biomass surface modification with laccase-HBT treatment is proposed to be the underlying mechanism for increased cellulose conversion.     

The properties of enzyme-hydrolyzed cellulose in aqueous sodium hydroxide

Pure natural cellulose (softwood pulp) modified with cellulase is allowed to react with sodium hydroxide in a muller, and changes in structure and properties are investigated by FTIR and DSC. The reactivity of cellulose for some dissolving and derivatization processes is shown to be improved by an enzymatic hydrolysis and admixture with sodium hydroxide. The modified cellulose dissolved at 9% (wt) sodium hydroxide at -10 degrees C at 6% pulp consistency, while the DP of cellulose is >350.     

Microscopy studies reveal delignification and sterol removal from eucalypt kraft pulps by laccase–HBT

Fungal laccases in the presence of mediators are powerful biocatalysts to degrade lignin. Pycnoporus cinnabarinus laccase and 1-hydroxybenzotriazole (HBT) have been successfully used to delignify eucalypt kraft pulp once integrated in a totally chlorine-free bleaching sequence. Real time delignification of kraft pulp by laccase–HBT was verified in situ by monitoring the loss of lignin autofluorescence during the enzymatic treatment using confocal laser scanning microscopy. The highest delignification of pulp fibers occurred over a very short time-span (5 min). Moreover, we demonstrate the removal of sterols, responsible for pitch deposits in hardwood kraft pulps, as an additional effect of laccase-HBT. Spherical structures between pulp fibers localized by low temperature scanning electron microscopy were removed by laccase–HBT. The use of filipin, a specific stain, revealed the sterol nature of many of these structures. At the end of the enzyme-aided bleaching sequence, the fluorescent sterols–filipin signals were almost completely absent.     

Thermogravimetry study of xylanase- and laccase/mediator-treated eucalyptus pulp fibres

Thermogravimetric analyses (TGA) was applied to study the effects of enzymatic bleaching of eucalyptus pulp with xylanase and a laccase-mediator system. The thermal degradation profile of the pulps was sensitive to the enzymatic treatments. Xylanase treatment produced an ordered and clean microfibril, whereas laccase oxidized surface cellulose chains and increased the amorphous (paracrystalline) cellulose content. In this case, pulp viscosity decreased from 972 to 859 mL/g and apparent pulp crystallinity calculated from TGA data decreased almost 50%. Alkaline extraction was necessary to recover pulp crystallinity and to remove oxidized lignin in the laccase-treated samples. TGA data allowed differentiating and quantifying crystalline and amorphous cellulose. This thermogravimetric approach is a simple method in order to monitor superficial changes in cellulosic microfibrils.     

Laccase-mediator biobleaching applied to a direct yellow dyed paper

A laccase-mediator system (LMS) for biobleaching was applied to a bleached chemical pulp dyed with stilbene dye Direct Yellow 11. Of mediators tested, 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) was found to be more effective than either violuric acid (VA) or N-hydroxybenzotriazole (HBT), which had been shown to be superior to ABTS when bleaching virgin chemical pulp. The laccase-ABTS system removed more than 60% of the color.     

Comparing the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp bleaching

Laccase–mediator systems have the disadvantage that the mediator is expensive and potentially toxic. In this work, we used N-hydroxyacetanilide (NHA) in combination with laccase for the first time to bleach eucalypt pulp and found it to be a very promising, advantageous alternative to 1-hydroxybenzotriazole (HBT) as mediator. Thus, NHA is efficiently oxidized by laccase to a radical that absorbs light at 350 nm. Also, NHA is a better substrate for laccase than is HBT. An innovative result is that the enzyme is inactivated to a similar extent by both mediators under the typical treatment conditions of the bleaching step (L). This adverse effect, however, is strongly reduced in the presence of pulp. Moreover, the laccase–NHA system is as efficient as the laccase–HBT system in reducing the kappa number of eucalyptus pulp. Using a xylanase pretreatment or unbleached pulp boosts kappa number reduction and bleaching with the laccase–mediator system. Based on the results of cyclic voltammetry tests, NHA has a slightly lower redox potential than HBT, which further supports use of the former; also, unlike HBT, NHA is oxidized in a reversible, pH-dependent manner. Interestingly, the laccase–NHA system provides more efficient bleaching of eucalyptus pulp at pH 5 than it does at pH 4.     

Quantitative trait locus (QTL) analysis of wood quality traits in <Emphasis Type="Italic">Eucalyptus nitens</Emphasis>

To identify the chromosomal regions affecting wood quality traits, we conducted a genome-wide quantitative trait locus (QTL) analysis of wood quality traits in Eucalyptus nitens. This information is important to exploit the full potential of the impending Eucalyptus genome sequence. A three generational mapping population consisting of 296 progeny trees was used to identify QTL associated with several wood quality traits in E. nitens. Thirty-six QTL positions for cellulose content, pulp yield, lignin content, density, and microfibril angle (MFA) were identified across different linkage groups. On linkage groups (LG)2 and 8, cellulose QTL cluster with pulp yield and extractives QTL while on LG4 and 10 cellulose and pulp yield QTLs cluster together. Similarly, on LG4, 5, and 6 QTL for lignin traits were clustered together. At two positions, QTL for MFA, a physical trait related to wood stiffness, were clustered with QTL for lignin traits. Several cell wall candidate genes were co-located to QTL positions affecting different traits. Comparative QTL analysis with Eucalyptus globulus revealed two common QTL regions for cellulose and pulp yield. The QTL positions identified in this study provide a resource for identifying wood quality genes using the impending Eucalyptus genome sequence. Candidate genes identified in this study through co-location to QTL regions may be useful in association studies.     

Comparative study of the efficiency of synthetic and natural mediators in laccase-assisted bleaching of eucalyptus kraft pulp

The natural phenolic compounds syringaldehyde and vanillin were compared to the synthetic mediators 1-hydroxybenzotriazole, violuric acid and promazine in terms of boosting efficiency in a laccase-assisted biobleaching of eucalyptus kraft pulp. Violuric acid and 1-hydroxybenzotriazole revealed to be the most effective mediators of the bioprocess. Nevertheless, laccase-syringaldehyde system also improved the final pulp properties (28% delignification and 63.5% ISO brightness) compared to the process without mediator (23% and 61.5% respectively), in addition to insignificant denaturation effect over laccase. The efficiency of the biobleaching process was further related to changes in non-conventionally used optical and chromatic parameters of pulp, such as (L( *)), chroma (C( *)) and dye removal index (DRI) showing good correlation. Adverse coupling reactions of the natural phenolic mediators on pulp lignin were predicted by electrochemical studies, demonstrating the complexity of the laccase-mediator reaction on pulp.     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

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.     

On the use of a high-redox potential laccase as an alternative for the transformation of non-steroidal anti-inflammatory drugs (NSAIDs)

The release of pharmaceutical compounds to the environment, such as non-steroidal anti-inflammatory drugs (NSAIDs), implies a great concern because of its negative effects, e.g. potential cytotoxicity to liver and kidney of animal species. In the present study, a high-redox potential laccase is proposed as an alternative system for the biotransformation of selected NSAIDs: naproxen (NPX) and diclofenac (DCF). The effects of pH and the use of synthetic and natural mediators (1-hydroxybenzotriazole (HBT) and syringaldehyde (SA), respectively) were evaluated with the aim of optimizing the removal of NSAIDs by laccase. Results proved high removal yields for NPX (70–94%) after 24 h in the presence of HBT at acidic and neutral pH, while DCF was completely transformed at pH 4 within shorter periods, from 30 min to 4 h, both in the absence or presence of mediators. The operation at a higher pH implied the addition of mediators to remove DCF, with values of 50 and 98% for SA and HBT, respectively. Kinetic parameters of the transformation reactions and laccase inactivation were estimated and compared for the different experimental conditions. Besides, the identification of the major biotransformation products of DCF was attempted: decarboxylated compounds were detected by gas chromatography-mass spectrometry and corresponding reaction pathways were proposed. Moreover, laccase-catalyzed treatment was demonstrated to significantly improve the aerobic biodegradability of the medium containing DCF in comparison with untreated solution, suggesting that DCF transformation products are less toxic than the parent compound.     

Production and Stability Evaluation of Modified-Release Microparticles for the Delivery of Drug Combinations

Production and evaluation of novel formulations of tizanidine and tramadol microparticles was the chief purpose of this project. Microparticles of both drugs were prepared separately via temperature change method. To extend the release of formulations, ethyl cellulose was employed. Higuchi, zero-order, first-order, and Korsmeyer–Peppas kinetic models were applied to appraise the mechanism and mode of drugs release. Higuichi model was found to be best for all release profiles. Stability of microparticles at 40°C/75% RH over a 3-month duration was determined by Fourier transform infrared (FTIR), X-ray diffractometry (XRD), and drugs assay. Microparticles were compatible and stable as no significant differences were observed when subjected to drug assay, FTIR, and XRD during accelerated stability studies.     

Enhanced delignification of mixed wood pulp by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> laccase mediator system

An environmentally sound biobleaching to get high quality paper pulp from mixed wood pulp was attempted employing laccase from Aspergillus fumigatus VkJ2.4.5 for lignin removal. Laccase treatment was performed in the presence of a mediator N-hydroxybenzotriazole (HBT, 1.5% w/w), resulting into notably higher level of delignification of the pulp. Enzyme at 10 Ug⁻¹ of pulp at 50°C, pH 6.0, for 2 h with a pulp consistency of 10% was found suitable for enabling maximum decrease in the kappa number. The kappa number and yellowness decreased by 14 and 4% whereas ISO brightness improved by 7%. The presence of a characteristic peak at 280 nm indicated the presence of lignin in the effluent during biobleaching. Analysis of FTIR spectra of residual lignin revealed characteristic modifications following enzymatic bleaching by laccase mediator system (LMS). Variations in morphology and crystallinity of pulp were evaluated by scanning electron microscopy and X-ray diffraction analysis.