Laccase multi-point covalent immobilization: characterization, kinetics, and its hydrophobicity applications.

Laccase from Myceliophthora thermophila was immobilized using one-point and multi-point covalent attachment on both a native and a modified new commercial epoxy carrier (Immobead 150P). After 10 cycles of operation at pH 3.0 and temperature 70 °C, the multi-point covalently immobilized laccase on the modified Immobead 150P performed best in terms of immobilization characteristics, retaining 95% of its initial activity. Thermodynamic parameters of thermal inactivation emphasized the positive impact of the immobilization procedure. At 50 °C, the immobilized and free enzyme activity levels dropped by 27 and 73%, respectively, after 48 h of incubation. The immobilized enzyme enhanced its stability in alkaline conditions, resuming 95% of its original activity after 3 h at pH 9.0. Immobilization reduced substrate affinity because the free laccase's Km value was lower than that of the immobilized laccase. Finally, the application of immobilized laccase in an innovative wood treatment process was tested by grafting lauryl gallate (LG) in order to provide hydrophobic properties to the wood. The results showed a relative water contact angle of 85.7% for treated wood, whereas the control showed only 26.6%, after 4 min of contact between water and beechwood surface. KEY POINTS: • Multi-point covalent immobilization of a commercial laccase on a commercial support. • Enzymatic parameters generally improved by immobilization process. • New application of immobilized laccase: enzymatic-assisted wood hydrophobization.

Enzymatic Functionalization of Wood as an Antifouling Strategy against the Marine Bacterium Cobetia marina.

The protection of wood in marine environments is a major challenge due to the high sensitivity of wood to both water and marine microorganisms. Besides, the environmental regulations are pushing the industry to develop novel effective and environmentally friendly treatments to protect wood in marine environments. The present study focused on the development of a new green methodology based on the laccase-assisted grafting of lauryl gallate (LG) onto wood to improve its marine antifouling properties. Initially, the enzymatic treatment conditions (laccase dose, time of reaction, LG concentration) and the effect of the wood specie (beech, pine, and eucalyptus) were assessed by water contact angle (WCA) measurements. The surface properties of the enzymatically modified wood veneers were assessed by X-ray photoelectron spectroscopy (XPS), Fourier transform-infrared spectroscopy (FTIR). Antifouling properties of the functionalized wood veneers against marine bacterium Cobetia marina were studied by scanning electron microscopy (SEM) and protein measurements. XPS and FTIR analysis suggested the stable grafting of LG onto the surface of wood veneers after laccase-assisted treatment. WCA measurements showed that the hydrophobicity of the wood veneers significantly increased after the enzymatic treatment. Protein measurements and SEM pictures showed that enzymatically-hydrophobized wood veneers modified the pattern of bacterial attachment and remarkably reduced the bacterium colonization. Thus, the results observed in the present study confirmed the potential efficiency of laccase-assisted treatments to improve the marine antifouling properties of wood.

Kinetic and thermodynamic study of laccase cross-linked onto glyoxyl Immobead 150P carrier: Characterization and application for beechwood biografting.

In this study, we cross-linked aminated Thermothelomyces thermophilus laccase onto Immobead 150P epoxy carrier, and achieved an immobilization yield of 99.84 %. The optimum temperature and pH values for the oxidation of ABTS by laccase were determined to be 70 °C and pH 3.0. After 6 h at 50 °C, laccase activity was diminished by about 13 % in the free form and 28 %, in the immobilized form. Km values for both free and cross-linked laccase were 0.051 and 0.567 mM, whereas Vmax values were 2.027 and 0.854 µmol. min-1, respectively. The immobilized laccase was able to preserve its full activity for 6 weeks, retaining approximately 95 % and 78 % of its initial activity after 8 and 20 weeks, respectively. The contact angles were two-fold higher when the laccase enzyme was occupied in the biografting reaction, revealing that the hydrophobic compound bonded stably onto beechwood samples.

3D Printable Filaments Made of Biobased Polyethylene Biocomposites.

Two different series of biobased polyethylene (BioPE) were used for the manufacturing of biocomposites, complemented with thermomechanical pulp (TMP) fibers. The intrinsic hydrophilic character of the TMP fibers was previously modified by grafting hydrophobic compounds (octyl gallate and lauryl gallate) by means of an enzymatic-assisted treatment. BioPE with low melt flow index (MFI) yielded filaments with low void fraction and relatively low thickness variation. The water absorption of the biocomposites was remarkably improved when the enzymatically-hydrophobized TMP fibers were used. Importantly, the 3D printing of BioPE was improved by adding 10% and 20% TMP fibers to the composition. Thus, 3D printable biocomposites with low water uptake can be manufactured by using fully biobased materials and environmentally-friendly processes.

Green Binder Based on Enzymatically Polymerized Eucalypt Kraft Lignin for Fiberboard Manufacturing: A Preliminary Study.

The capability of laccase from Myceliophthora thermophila to drive oxidative polymerization of Eucalyptus globulus Kraft lignin (KL) was studied as a previous step before applying this biotechnological approach for the manufacturing of medium-density fiberboards (MDF) at a pilot scale. This method, which improves the self-bonding capacity of wood fibers by lignin enzymatic cross-linking, mimics the natural process of lignification in living plants and trees. An interesting pathway to promote these interactions could be the addition of lignin to the system. The characterization of E. globulus KL after enzymatic treatment showed a decrease of phenolic groups as well as the aromatic protons without loss of aromaticity. There was also an extensive oxidative polymerization of the biomolecule. In the manufacture of self-bonded MDF, the synergy generated by the added lignin and laccase provided promising results. Thus, whenever laccase was present in the treatment, MDF showed an increase in mechanical and dimensional stability for increasing amounts of lignin. In a pilot scale, this method produced MDF that meets the requirements of the European standards for both thickness swell (TS) and internal bonding (IB) for indoor applications.

Stability and kinetic behavior of immobilized laccase from Myceliophthora thermophila in the presence of the ionic liquid 1-ethyl-3-methylimidazolium ethylsulfate.

The use of ionic liquids (ILs) as reaction media for enzymatic reactions has increased their potential because they can improve enzyme activity and stability. Kinetic and stability properties of immobilized commercial laccase from Myceliophthora thermophila in the water-soluble IL 1-ethyl-3-methylimidazolium ethylsulfate ([emim][EtSO4 ]) have been studied and compared with free laccase. Laccase immobilization was carried out by covalent binding on glyoxyl-agarose beads. The immobilization yield was 100%, and the activity was totally recovered. The Michaelis-Menten model fitted well to the kinetic data of enzymatic oxidation of a model substrate in the presence of the IL [emim][EtSO4 ]. When concentration of the IL was augmented, the values of Vmax for free and immobilized laccases showed an increase and slight decrease, respectively. The laccase-glyoxyl-agarose derivative improved the laccase stability in comparison with the free laccase regarding the enzymatic inactivation in [emim][EtSO4 ]. The stability of both free and immobilized laccase was slightly affected by small amounts of IL (<50%). A high concentration of the IL (75%) produced a large inactivation of free laccase. However, immobilization prevented deactivation beyond 50%. Free and immobilized laccase showed a first-order thermal inactivation profile between 55 and 70°C in the presence of the IL [emim][EtSO4 ]. Finally, thermal stability was scarcely affected by the presence of the IL.

Immobilization of laccase on modified silica: stabilization, thermal inactivation and kinetic behaviour in 1-ethyl-3-methylimidazolium ethylsulfate ionic liquid.

Laccase was immobilized on modified silica carrier. The immobilization conditions, pH and enzyme concentration were optimized. Operational stability of 10 reaction cycles showed that immobilized laccase in buffer was stable, presenting an activity loss <30%. Nevertheless, a high decrease >80% was obtained in ionic liquid (IL) solution. Activity of immobilized laccase was maintained when incubated in IL. After 7days of incubation, immobilized laccase lost 30-50% of its initial activity. Immobilization also improved thermal stability of laccase in the presence of IL. Enzyme kinetics was modelled with Michaelis-Menten model. The Km value for free laccase increases significantly with the IL concentration. Slight differences were found in Vm for free enzyme. Unusual kinetic behaviour was obtained for immobilized laccase in IL: Both Vm and Km increased with IL concentration, resulting in increased catalytic efficiency of the immobilized enzyme in presence of IL.

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.

Role of laccase and low molecular weight metabolites from Trametes versicolor in dye decolorization.

The studies regarding decolorization of dyes by laccase may not only inform about the possible application of this enzyme for environmental purposes, but also may provide important information about its reaction mechanism and the influence of several factors that could be involved. In this paper, decolorization of crystal violet and phenol red was carried out with different fractions of extracellular liquids from Trametes versicolor cultures, in order to describe the role of laccase in this reaction. Moreover, the possible role of the low molecular weight metabolites (LMWMs) also produced by the fungus was evaluated. The results confirm the existence of a nonenzymatic decolorization factor, since the nonprotein fraction of the extracellular liquids from cultures of T. versicolor has shown decolorization capability. Several experiments were performed in order to identify the main compounds related to this ability, which are probably low molecular weight peroxide compounds.

Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes.

The white-rot fungus Trametes versicolor growing in submerged culture on a basal medium, with barley bran as a carbon source, produced two laccase isoenzymes LacI and LacII. The addition of metal ions to the culture medium was performed to improve the total laccase activity and to determine the effect on the production of laccase isoenzymes. From all the tested metals, only Cu2+ increased laccase activity (up to 12-fold with respect to control cultures) and T. versicolor in presence of all metals produced the two isoenzymes in different proportion with ratios of activity (LacI/LacII) varying between 0.11 and 0.51. This factor played an important role in the decolourisation of the textile dye Indigo Carmine.