Chemical and structural transformations of lignin in sesame seed hull during roasting.

To investigate the mechanism of lignin degradation during sesame roasting, structural transformations of milled wood lignin (MWL) from sesame seed hull samples roasted at 190-250 °C for 30 min were investigated. The findings revealed that, with increasing temperature, the degradation extent of carbohydrates from lignin carbohydrate complex in the fractions deepened, which reduced total sugar content (from 8.59 % to 0.45 %). Compared to that of the original sesame seed hull lignin (LSSH), the molecular weight of MWL fractions showed a tendency to decline (Mw 4377-2235 Da) with the rise of roasting temperature (210-250 °C). During roasting, lignins in the sesame seed hull underwent degradation and condensation. Due to demethoxylation, the H-type lignin proportion increased from 2.7 % to 26.1 %. Compared to G- and C-type lignin, S-type lignin was more stable. The ß-O-4 linkages decreased from 5.8 to 1.2/100 Ar due to CO bond breaking, and ß-ß linkages from 26.3 to 9.6/100 Ar decreased due to condensation of CC. As the roasting temperature increased, more chemical bonds between lignin structural units were broken, resulting in the generation of more phenolic hydroxyl groups (1.80-2.53 mmol/g). This study helps to elucidate the contribution of lignin degradation during roasting to the oxidative stability of sesame oil.

Revealing the structural changes of lignin in Chinese quince (Chaenomeles sinensis) fruit as it matures.

Chinese quince fruits (Chaenomeles sinensis) contain substantial amounts of lignin; however, the exact structure of lignin remains to be investigated. In this study, milled wood lignins (Milled wood lignin (MWL)-1, MWL-2, MWL-3, MWL-4, MWL-5, and MWL-6) were extracted from fruits harvested once a month from May to October 2019 to investigate their structural evolution during fruit growth. The samples were characterized via High-performance anion exchange chromatography (HPAEC), Fourier transform-infrared spectroscopy (FT-IR), gel permeation chromatography (GPC), thermogravimetric (TGA), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) and NMR (2D-heteronuclear single quantum coherence (HSQC) and 31P). The MWL samples in all fruit growth stages were GS-type lignin and lignin core undergoing minimal alterations during fruit development. The predominant linkage in the lignin structure was ß-O-4', followed by ß-ß' and ß-5'. Galactose and glucose were the main monosaccharides associated with MWL. In MWL-6, the lignin exhibited the highest homogeneity and thermal stability. As the fruit matured, a gradual increase in the ß-O-4' proportion and the ratio of S/G was observed. The results provide comprehensive characterization of the cell wall lignin of quince fruit as it matures. This study could inspire innovative applications of quince fruit lignin and provide the optimal harvest time for lignin utilization.

Selecting Suitable Near-Native Lignins for Research.

There are several methods to isolate near-native lignins, including milled-wood lignin, enzymatic lignin, cellulolytic enzyme lignin, and enzymatic mild-acidolysis lignin. Which one is the most representative of the native lignin? Herein, near-native lignins were isolated from different plant groups and structurally analyzed to determine how well these lignins represented their native lignin counterparts. Analytical methods were applied to understand the molecular weight, monomer composition, and distribution of interunit linkages in the structure of the lignins. The results indicated that either enzymatic lignin or cellulolytic enzyme lignin may be used to represent native lignin in softwoods and hardwoods. None of the lignins, however, appeared to represent native lignins in grasses (monocot plants) because of substantial syringyl/guaiacyl differences. Complicating the understanding of grass lignin structure, large amounts of hydroxycinnamates acylate their polysaccharides and, when released, are often conflated with actual lignin monomers.

Evaluation of a dye-decolorizing peroxidase from Comamonas serinivorans for lignin valorization potentials.

Although dye-decolourising peroxidases (DyPs) are well-known for lignin degradation, a comprehensive understanding of their mechanism remains unclear. Therefore, studying the mechanism of lignin degradation by DyPs is necessary for industrial applications and enzyme engineering. In this study, a dye-decolourising peroxidase (CsDyP) gene from C. serinivorans was heterologously expressed and studied for its lignin degradation potential. Molecular docking analysis predicted the binding of 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), veratryl alcohol (VA), 2, 6-dimethylphenol (2, 6- DMP), guaiacol (GUA), and lignin to the substrate-binding pocket of CsDyP. Evaluation of the enzymatic properties showed that CsDyP requires pH 4.0 and 30 °C for optimal activity and has a high affinity for ABTS. In addition, CsDyP is stable over a wide range of temperatures and pH and can tolerate 5.0 mM organic solvents. Low NaCl concentrations promoted CsDyP activity. Further, CsDyP significantly reduced the chemical oxygen demand decolourised alkali lignin (AL) and milled wood lignin (MWL). CsDyP targets the ß-O-4, CO, and CC bonds linking lignin's G, S, and H units to depolymerize and produce aromatic compounds. Overall, this study delivers valuable insights into the lignin degradation mechanism of CsDyP, which can benefit its industrial applications and lignin valorization.

Structural Characterization of the Milled-Wood Lignin Isolated from Sweet Orange Tree (Citrus sinensis) Pruning Residue.

The pruning of sweet orange trees (Citrus sinensis) generates large amounts of lignocellulosic residue. Orange tree pruning (OTP) residue presents a significant lignin content (21.2%). However, there are no previous studies describing the structure of the native lignin in OTPs. In the present work, the "milled-wood lignin" (MWL) was extracted from OTPs and examined in detail via gel permeation chromatography (GPC), pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), and two-dimensional nuclear magnetic resonance (2D-NMR). The results indicated that the OTP-MWL was mainly composed of guaiacyl (G) units, followed by syringyl (S) units and minor amounts of p-hydroxyphenyl (H) units (H:G:S composition of 1:62:37). The predominance of G-units had a strong influence on the abundance of the different linkages; therefore, although the most abundant linkages were ß-O-4' alkyl-aryl ethers (70% of total lignin linkages), the lignin also contained significant amounts of phenylcoumarans (15%) and resinols (9%), as well as other condensed linkages such as dibenzodioxocins (3%) and spirodienones (3%). The significant content of condensed linkages will make this lignocellulosic residue more recalcitrant to delignification than other hardwoods with lower content of these linkages.

Exploring Alkyl-O-Alkyl Ether Structures in Softwood Milled Wood Lignins.

Recent studies have suggested that there are significant amounts of various alkyl ether (Alk-O-Alk; Alk = alkyl) moieties in a spruce native lignin preparation, milled wood lignin (SMWL). However, the comprehensive NMR assignment to these moieties has not been addressed yet. This study focused on investigating different types of Alk-O-Alk structures at the α- and γ-positions of the lignin side chain in an heteronuclear single-quantum coherence (HSQC) spectrum of SMWL using experimental NMR data of lignin and synthesized model compounds. Ambiguous structural features were predicted by computer simulation of 1H and 13C NMR spectra to complement the experimental NMR data. As a result, specific regions in the HSQC spectrum were attributed to different Alk-O-Alk moieties of Alk-O-Alk/ß-O-4 and Alk-O-Alk/ß-ß' structures. However, the differences between the specific regions were rather subtle; they were not well separated from each other and some major lignin moieties. Furthermore, SMWL contained a large variety of Alk-O-Alk moieties but in minute individual amounts, resulting in rather broad, superimposing resonances. Thus, evaluation did not allow assigning individual types of Alk-O-Alk moieties from the HSQC spectra; instead, they were quantified as total (α- and γ-linked) Alk-O-Alk based on the balance of structural units in the 13C NMR spectra. At last, potential formation mechanisms of various Alk-O-Alk ether structures in lignin biosynthesis, lignin aging, and during ball milling of wood were hypothesized and discussed.

Structural and Thermal Characterization of Milled Wood Lignin from Bamboo (Phyllostachys pubescens) Grown in Korea.

The structural and thermal characterization of milled wood lignin (MWL) prepared from bamboo (Phyllostachys pubescens) grown in Korea was investigated, and the results were compared with bamboo MWLs from other studies. The C9 formula of the bamboo MWL was C9H7.76O3.23N0.02 (OCH3)1.41. The Mw and Mn of MWL were 13,000 and 4400 Da, respectively, which resulted in a polydispersity index (PDI) of 3.0. The PDI of the prepared MWL was higher than other bamboo MWLs (1.3-2.2), suggesting a broader molecular weight distribution. The structural features of MWL were elucidated using FT-IR spectroscopy and NMR techniques (1H, 13C, HSQC, 31P NMR), which indicate that MWL is of the HGS-type lignin. The major lignin linkages (ß-O-4, ß-ß, ß-5) were not different from other bamboo MWLs. The syringyl/guaiacyl ratio, determined from 1H NMR, was calculated as 0.89. 31P NMR revealed variations in hydroxyl content, with a higher aliphatic hydroxyl content in MWL compared to other bamboo MWLs. Thermal properties were investigated through TGA, DSC, and pyrolysis-GC/MS spectrometry (Py-GC/MS). The DTGmax of MWL under inert conditions was 287 °C, and the Tg of MWL was 159 °C. Py-GC/MS at 675 °C revealed a syringyl, guaiacyl, p-hydroxyphenyl composition of 17:37:47.

Fast pyrolysis of guaiacyl-syringyl (GS) type milled wood lignin: Product characteristics and CH4 formation mechanism study.

It is anticipated that the insight into the demethylation and mechanism of CH4 formation from natural lignin using in-situ diffuse reflectance infrared Fourier transform spectroscopy (in-situ FTIR) combined with two-dimensional perturbation correlation infrared spectroscopy (2D-PCIS) and density functional theory (DFT) calculation analysis would contribute to a deeper insight of bond cleavage mechanism of lignin pyrolysis. Herein, GS-type lignin (poplar MWL) was characterized by Fourier transform infrared spectroscopy (FTIR) and heteronuclear Single-Quantum Correlation Nuclear Magnetic Resonance (HSQC), and its pyrolysis at different temperatures was performed in a lab-scale fixed-bed reactor. The biochar, gaseous and liquid products were qualitative, and quantitative analysis of gases and bio-oil is demonstrated using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The key of CH4 formation is the homolytic cleavage of the methoxyl functional group generating methyl radical and further verified via in-situ FTIR combined with 2D-PCIS and DFT calculation. The study established a new methodology based on multiple factor analysis to evaluate the CH4 formation mechanism in GS-type milled wood lignin at the molecular level, which is of positive significance for increasing lignin valorization and improving the environment.

The Effect of Ball Milling Time on the Isolation of Lignin in the Cell Wall of Different Biomass.

Ball milling technology is the classical technology to isolate representative lignin in the cell wall of biomass for further investigation. In this work, different ball milling times were carried out on hardwood (poplar sawdust), softwood (larch sawdust), and gramineous material (bamboo residues) to understand the optimum condition to isolate the representative milled wood lignin (MWL) in these different biomass species. Results showed that prolonging ball milling time from 3 to 7 h obviously increased the isolation yields of MWL in bamboo residues (from 39.2% to 53.9%) and poplar sawdust (from 15.5% to 35.6%), while only a slight increase was found for the MWL yield of larch sawdust (from 23.4% to 25.8%). Importantly, the lignin substructure of ß-O-4 in the MWL samples from different biomasses can be a little degraded with the increasing ball milling time, resulting in the prepared MWL with lower molecular weight and higher content of hydroxyl groups. Based on the isolation yield and structure features, milling time with 3 and 7 h were sufficient to isolate the representative lignin (with yield over 30%) in the cell wall of bamboo residues and poplar sawdust, respectively, while more than 7 h should be carried out to isolate the representative lignin in larch sawdust.

Chemical Characterization of Kraft Lignin Prepared from Mixed Hardwoods.

Chemical characterization of kraft lignin (KL) from mixed hardwoods (Acacia spp. from Vietnam and mixed hardwoods (mainly Quercus spp.) from Korea) was conducted for its future applications. To compare the structural changes that occurred in KL, two milled wood lignins (MWLs) were prepared from the same hardwood samples used in the production of KL. Elemental analysis showed that the MWL from acacia (MWL-aca) and mixed hardwood (MWL-mhw) had almost similar carbon content, methoxyl content, and C9 formula. KL had high carbon content but low oxygen and methoxyl contents compared to MWLs. The C9 formula of KL was determined to be C9H7.29O2.26N0.07S0.12(OCH3)1.24. The Mw of KL and MWLs was about 3000 Da and 12,000-13,000 Da, respectively. The structural features of KL and MWLs were investigated by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance spectrometry (1H, 13C NMR). The analyses indicated that KL underwent severe structural modifications, such as γ-carbon cleavage, demethylation, and polycondensation reactions during kraft pulping, which resulted in increased aromatic content and decreased aliphatic content. The main linkages in lignin, ß-O-4 moieties, were hardly detected in the analysis as these linkages were extensively cleaved by nucleophilic attack of SH- and OH- during pulping.

How Pseudo-lignin Is Generated during Dilute Sulfuric Acid Pretreatment.

Pseudo-lignin is generated from lignocellulose biomass during pretreatment with dilute sulfuric acid and has a significant inhibitory effect on cellulase. However, the mechanism of pseudo-lignin generation remains unclear. The following main points have been addressed to help elucidate the pseudo-lignin generation pathway. Cellulose and xylan were pretreated with sulfuric acid at different concentrations; aliquots were periodically collected; and the changes in the byproducts of the prehydrolysate were quantified. Milled wood lignin (MWL) mixed with cellulose and xylan was pretreated to evaluate the impact of lignin on pseudo-lignin generation. Furfural, 5-hydroxymethylfurfural, and MWL were pretreated as model compounds to investigate pseudo-lignin generation. The result indicated that the increasing acid concentration significantly promoted the generation of pseudo-lignin. When the acid concentration was increased from 0 to 1.00 wt %, pseudo-lignin was increased from 1.36 to 4.05 g. In addition, lignin promoted the pseudo-lignin generation through the condensation between lignin and the generated intermediates.

New strategy to elucidate the positive effects of extractable lignin on enzymatic hydrolysis by quartz crystal microbalance with dissipation.

BACKGROUND: The presence of lignin normally affects enzymatic saccharification of lignocellulose detrimentally. However, positive effects of lignin on enzymatic hydrolysis have been recently reported. Enzyme-lignin interactions could be the key to reveal the underlying mechanism of their discrepant behaviors. In this study, to elucidate the positive effects of extractable lignin (EL) on enzymatic hydrolysis of ethanol organosolv-pretreated wood sawdust, two lignin fractions, EL and milled wood lignin (MWL), were isolated sequentially from pretreated substrates. Quartz crystal microbalance with dissipation (QCM-D) was then used to investigate the lignin aggregation effects on enzyme adsorption. RESULTS: We found that both EL and MWL had a narrow molecular weight distribution. However, MWL had an obviously higher molecular weight than EL. This indicated that EL and MWL likely represent two distinct lignin fractions from ethanol organosolv-pretreated substrates. HSQC NMR analysis revealed that less ß-O-4, ß-ß, and ß-5 linkages and a higher S/G ratio was present in EL, as compared to MWL. QCM-D analysis showed that the enzyme adsorption on lignin was highly relevant to these lignin structural characteristics. An obviously lower maximum enzyme adsorption capacity was observed on EL films (152.63-168.09 ng/cm2) compared to MWL films (196.71-224.73 ng/cm2). Furthermore, enzyme desorption on lignin films was determined. A significantly lower irreversible enzyme adsorption was observed on EL (75.40 ng/cm2) compared to MWL (137.35 ng/cm2). More importantly, two reconstructed lignin films were designed to investigate lignin assembly on enzyme adsorption. The results indicated that the presence of EL reduced irreversible enzyme adsorption on the reconstructed lignin films by 39.2-45.0%. CONCLUSIONS: Lignin structure determined the interaction between enzyme and lignins. A positive correlation was observed between molecular weight, the content of ß-5 linkages, and enzyme adsorption on lignin. EL, which was more depolymerized and less condensed, had the lower enzyme adsorption of the two preparations tested. Additionally, the presence of EL reduced enzyme adsorption on reconstructed lignin films, perhaps through a mechanism involving the blocking of non-productive enzyme binding sites on the MWL. This could be the mechanism for the positive effects of EL on enzymatic hydrolysis.

Lignin for white natural sunscreens.

Long-time exposure to the sun's ultraviolet (UV) radiation is harmful and causes various skin problems. Natural sun blockers have been drawing considerable attention recently. Even though lignin, an abundant aromatic polymer from plants, is a natural UV screening agent, its unfavorable dark color hinders its high value-added applications in sunscreens and cosmetics. In this study, we separate lignin under mild conditions (at room temperature with neutral solvents) in order to prevent darkening occurring during delignification and apply the resultant lignin as a natural sunscreen ingredient for the first time. Lignins isolated from Miscanthus sacchariflorus (MWL-M) and from Pinus densiflora (MWL-P) are compared with organosolv lignin (OL), which showed the best sunscreen performance, in color and UV protection. MWLs separated under mild conditions were light in color unlike conventional lignins extracted under harsh conditions. UV absorption of light-colored MWL-M was revealed to be as high as dark-colored OL. MWLs also showed synergistic effects with a commercial sunscreen; exposure of the MWL-added sunscreen to UVA radiation greatly enhanced the sun protection factor (SPF) value of the sunscreen.

Lignin-carbohydrate complexes: properties, applications, analyses, and methods of extraction: a review.

The complexity of lignin and hemicellulose segmentation has been known since the middle of the ninetieth century. Studies confirmed that all lignin units in coniferous species and 47-66% of lignin moieties in deciduous species are bound to hemicelluloses or cellulose molecules in lignin-carbohydrate complexes (LCC). Different types and proportions of lignin and polysaccharides present in biomass lead to the formation of LCC with a great variety of compositions and structures. The nature and amount of LCC linkages and lignin substructures affect the efficiency of pulping, hydrolysis, and digestibility of biomass. This review paper discusses the structures, compositions, and properties of LCC present in biomass and in the products obtained via pretreating biomass. Methods for extracting, fractionating, and analyzing LCC of biomass, pulp, and spent pulping liquors are critically reviewed. The main perspectives and challenges associated with these technologies are extensively discussed. LCC could be extracted from biomass following varied methods, among which dimethyl sulfoxide or dioxane (Björkman's) and acetic acid (LCC-AcOH) processes are the most widely applied. The oxidation and methylation treatments of LCC materials elucidate the locations and frequency of binding sites of hemicelluloses to lignin. The two-dimensional nuclear magnetic resonance analysis allows the identification of the structure and the quantity of lignin-carbohydrate bonds involved in LCC. LCC application seems promising in medicine due to its high anti-HIV, anti-herpes, and anti-microbial activity. In addition, LCC was successfully employed as a precursor for the preparation of spherical biocarriers.

Ball Milling's Effect on Pine Milled Wood Lignin's Structure and Molar Mass.

The effect of ball milling expressed as the yield of milled wood lignin (MWL) on the structure and molar mass of crude milled wood lignin (MWLc) preparation is studied to better understand the process' fundamentals and find optimal conditions for MWL isolation (i.e., to obtain the most representative sample with minimal degradation). Softwood (loblolly pine) MWLc preparations with yields of 20⁻75% have been isolated and characterized based on their molar mass distribution (by Size Exclusion Chromatography (SEC)), hydroxyl groups of different types (31P NMR), methoxyl groups (HS-ID GC-MS), and sugar composition (based on methanolysis). Classical MWL purification is not used to access the whole extracted lignin. The results indicate that lignin degradation during ball milling occurs predominantly in the high molar mass fraction and is less pronounced in the low molar mass fraction. This results in a significant decrease in the Mz and Mw of the extracted MWLc with an increase in the yield of MWLc, but has only a very subtle effect on the lignin structure if the yield of MWLc is kept below about 55%. Therefore, no tedious optimization of process variables is necessary to achieve the required MWLc yield in this range for structural studies of softwood MWL. The sugar composition shows higher amounts of pectin components in MWLs of low yields and higher amounts of glucan and mannan in high-yield MWLs, confirming that lignin extraction starts from the middle lamella in the earlier stages of MWL isolation, followed by lignin extraction from the secondary wall region.

Structural Characterization of Lignin in Fruits and Stalks of Chinese Quince.

Chinese quince (Chaenomeles sinensis) is used in food and pharmaceutical products, but it is seldom eaten as a raw fruit due to its astringent, woody flesh. The structural characterization of lignin fractions from Chinese quince was very important to investigate the structure-activity relationships of lignin. In this investigation, to characterize the structure of lignin in Chinese quince fruits, the milled wood lignin sample was isolated from the fruits (FMWL) and the chemical structure of FMWL was investigated by sugar analysis, FT-IR, GPC, pyrolysis-GC/MS analysis, UV spectra analysis, thermogravimetric analysis (TGA), and advanced NMR spectroscopic techniques. In addition, the lignin fraction from the stalk of Chinese quince (SMWL) was also prepared for comparison to obtained more information of lignin structure in the fruits. The results showed that the two lignin fractions isolated from fruit and stalk of Chinese quince exhibited different structural features. The two MWL samples were mainly composed of ß-O-4 ether bonds, ß-5 and ß-ß' carbon-carbon linkages in the lignin structural units. Compared to the SMWL, the FMWL fraction had the higher S/G ratio and more carbohydrates linkages. The predominant carbohydrates associated with FMWL and SMWL fractions were glucans-type hemicelluloses and xylan-type hemicelluloses, respectively. Understanding the structure of lignin could give insight into the properties of the lignin and enable the food processing industry to separate lignin more efficiently.

Integrated hot-compressed water and laccase-mediator treatments of Eucalyptus grandis fibers: structural changes of fiber and lignin.

Eucalyptus grandis fibers were treated with hot-compressed water (HCW) and laccase mediator to enhance the fiber characteristics and to produce an active lignin substrate for binderless fiberboard production. The composition, morphology, and crystallinity index (CrI) analysis of fibers showed that the HCW treatment increased the CrI and lignin content of the treated fibers through partial removal of hemicelluloses. Simultaneously, the HCW treatment produced some granules and holes on the surface of the fibers, which possibly facilitated the accessibility of the laccase mediator. Milled wood lignins and enzymatic hydrolysis lignins isolated from the control and treated fibers were comparatively characterized. A reduction of molecular weight was observed, which indicated that a preferential degradation of lignin occurred after exposure to the laccase mediator. Quantitative (13)C, 2D-HSQC and (31)P NMR characterization revealed that the integrated treatment resulted in the cleavage of ß-O-4' linkages, removal of G' (oxidized α-ketone) substructures, and an increase in the S/G ratio and free phenolic hydroxyls.

Effects of lignin modification on wheat straw cell wall deconstruction by Phanerochaete chrysosporium.

BACKGROUND: A key focus in sustainable biofuel research is to develop cost-effective and energy-saving approaches to increase saccharification of lignocellulosic biomass. Numerous efforts have been made to identify critical issues in cellulose hydrolysis. Aerobic fungal species are an integral part of the carbon cycle, equip the hydrolytic enzyme consortium, and provide a gateway for understanding the systematic degradation of lignin, hemicelluloses, and cellulose. This study attempts to reveal the complex biological degradation process of lignocellulosic biomass by Phanerochaete chrysosporium in order to provide new knowledge for the development of energy-efficient biorefineries. RESULTS: In this study, we evaluated the performance of a fungal biodegradation model, Phanerochaete chrysosporium, in wheat straw through comprehensive analysis. We isolated milled straw lignin and cellulase enzyme-treated lignin from fungal-spent wheat straw to determine structural integrity and cellulase absorption isotherms. The results indicated that P. chrysosporium increased the total lignin content in residual biomass and also increased the cellulase adsorption kinetics in the resulting lignin. The binding strength increased from 117.4 mL/g to 208.7 mL/g in milled wood lignin and from 65.3 mL/g to 102.4 mL/g in cellulase enzyme lignin. A detailed structural dissection showed a reduction in the syringyl lignin/guaiacyl lignin ratio and the hydroxycinnamate/lignin ratio as predominant changes in fungi-spent lignin by heteronuclear single quantum coherence spectroscopy. CONCLUSION: P. chrysosporium shows a preference for degradation of phenolic terminals without significantly destroying other lignin components to unzip carbohydrate polymers. This is an important step in fungal growth on wheat straw. The phenolics presumably locate at the terminal region of the lignin moiety and/or link with hemicellulose to form the lignin-carbohydrate complex. Findings may inform the development of a biomass hydrolytic enzyme combination to enhance lignocellulosic biomass hydrolysis and modify the targets in plant cell walls.

Recent developments and applications of immobilized laccase.

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.