Laccase Loading Research Articles

Highly permeable biocatalytic membrane prepared by 3D modification: Metal-organic frameworks ameliorate its stability for micropollutants removal

Aquatic micropollutants seriously affect human health. Biocatalytic membranes with immobilized laccase show a great potential for micropollutants removal. However, highly efficient and stable biocatalytic membranes are still under development. Herein, the separation and support layers of polyacrylonitrile (PAN) ultrafiltration membrane are sequentially modified by polyethyleneimine (PEI), metal–organic frameworks (MOFs), laccase and polydopamine (PDA), termed as “three dimensional (3D) modification”. The PEI polymers inserted in the selective separation layer result in charge reversal, thus immobilizing laccase mainly by electrostatic adsorption. The water-stable MOFs particles adhered on the support layer fibers can enhance the membrane adsorption capacity to laccase and micropollutants (called dual adsorption effect). PEI and MOFs dominate laccase loading and distribution in the membrane, thus greatly affecting micropollutants removals. The LacPAN-MIL-101-L biocatalytic membrane shows high permeability with desirable micropollutants removal, wide pH operating range and good reusability. Theoretically, this “3D modification” strategy can enable all the composite membranes to be biocatalytic membrane prepared by simple reverse filtration and subsequent coating.

Interactive effect of enzymes and surfactant on the cellulose digestibility of un-washed and washed dilute ammonia pretreated energy cane bagasse

The interaction effect of cellulase (Cellic® CTec2), xylanase (Cellic® HTec2) and laccase along with a non-ionic surfactant (Tween® 80) on cellulose digestibility of unwashed and washed liquid ammonia pretreated energy cane bagasse was investigated. A polynomial quadratic model was fitted and solved for the optimum value of each variable for the desired response using response surface methodology (RSM). Statistical analysis of the results showed that the interactive effect of all variables with cellulose were significant (p < .05) and resulted in 75.85% and 12.74% improvement in cellulose digestibility of unwashed and washed biomass, respectively. Highest cellulose digestibilities observed were 84.30% and 97.10% for values set within the design range for the unwashed and washed biomass, respectively. Optimum enzymatic hydrolysis conditions for unwashed substrate were 19.39% (mass ratio of glucan) CTec2, 12.04% (mass ratio of glucan) HTec2, a laccase loading on dry biomass of 46.32 IU g−1, and 10.15% (mass ratio of biomass) Tween® 80. Optimum enzymatic hydrolysis conditions for washed substrate were 16.90% (mass ratio of glucan) CTec2, 14.17% (mass ratio of glucan) HTec2 (w/w), a laccase loading on dry biomass of 34.64 IU g−1, and 14.86% (mass ratio of biomass) Tween® 80.

Chitosan based metal-chelated copolymer nanoparticles: Laccase immobilization and phenol degradation studies

Immobilization improves the stability and reusability of enzymes. In this study, laccase, which is a green biocatalyst, was immobilized onto Cu (II)-chelated chitosan nanoparticles via adsorption and successfully applied to remove phenol from aqueous solution. Cu (II)-chelated chitosan-graft-poly (glycidyl methacrylate) nanoparticles were prepared using poly (ethylene imine), PEI, which is employed as both a spacer arm and metal chelator and used to immobilize laccase by coordination. Properties of chitosan based nanoparticles were characterized using FTIR, TGA, SEM and zeta-sizer analysis. The maximum laccase loading capacity of Cu (II)-chelated chitosan based nanoparticles (CHT-PGMA-PEI-Cu (II) NPs) was calculated as 65.75 ± 2.51 mg/g. This immobilized system exhibited broad pH and temperature profiles, and better stability and reusability than free enzyme; after eight cycles of continuous use, the activity of the immobilized enzyme remained above 50 ± 0.62%. The Km and Vmax values of free and immobilized laccase were 0.055 mM, 0.070 mM, and 0.19 U/mg, 0.14 U/mg, respectively. The catalytic efficiencies (Vmax/KM) of the free and immobilized laccase were 3.45 and 2.0, respectively. Feasibility of the laccase immobilized system in the removal of phenol was investigated in a batch system. The results showed that the CHT-PGMA-PEI-Cu (II) NPs have great potential for industrial applications. More than 96% of phenol was removed with laccase immobilized metal chelated NPs in the presence of mediator, ABTS, indicating that the metal-chelated chitosan based NPs is a promising support for both laccase immobilization and further applications in the removal of phenolic compounds.

Laccase – methacrylyol functionalized magnetic particles: Highly immobilized, reusable, and efficacious for methyl red decolourization

Magnetically retrievable formulations of laccase potentially promising for biomedical and environmental applications were constructed by immobilization of the enzyme on the surface of magnetic nanoparticles functionalized by siloxane layers with active amino and methacrylyol moieties. The functional magnetic particles were prepared using a hydrolytic polycondensation reaction of tetraethoxysilane with 3-methacryloxypropyltrimethoxysilane (MPS). The results indicated that the amounts of laccase loading on the methacrylyol functional magnetic particles were approximately 150mg/g under the optimum conditions. The stability of the immobilized laccase was highly enhanced against thermal treatments and provided better long-term storage. Compared to free laccase, the immobilized laccase exhibited significantly higher decolourisation efficiency in the decolorization of a representative dye, methyl red, which resulted from the magnetic particles adsorption and enzymatic catalysis. These results show its great potential for industrial applications.

Laccase Activity in CTAB-Based Water-in-Oil Microemulsions.

The aim of this study was to develop a microemulsion system as a medium for laccase-catalyzed reactions. Phase behavior studies were conducted by constructing partial pseudo-ternary phase diagrams for systems comprising of cetyltrimethylammonium bromide (CTAB), various organic solvents as the oil phase (i.e., hexane, cyclohexane, heptane, octane, isooctane, toluene, isopropyl myristate), two co-surfactants (i.e., 1-butanol and 1-hexanol) and citrate buffer solution, at various surfactant/co-surfactant weight ratios (Rsm). A monophasic, transparent, non-birefringent area (designated as microemulsion domain) was seen to occur in some phase diagrams along the surfactant/organic solvent axis, the extent of which was dependent mainly upon the nature of co-surfactant and Rsm. On each phase diagram, three different water-in-oil (w/o) microemulsion systems with less than 50 wt% surfactant mixture and less than 20 wt% of aqueous phase were selected for laccase loading and activity measurements. Results revealed that the catalytic activity of laccase in CTAB-based w/o microemulsions decreased considerably, compared with its activity in the buffer solution, the extent of which depended upon the type of component and their compositions in the microemulsions. It was suggested that the conformational changes due to the electrostatic interactions between the cationic head group of CTAB and the negative enzyme might be the reason for the reduction of laccase activity, once entrapped in the microemulsion.

Effect of synthesis conditions on physical properties, laccase catalytic activity and stability of sol–gel laccase

The physical properties, laccase catalytic activity and stability of sol–gel laccases prepared at various synthesis conditions were investigated. The sol–gel laccases were characterized using the scanning electron microscope (SEM), Fourier transform infrared spectrophotometer and nitrogen adsorption/desorption analyzer. The laccase catalytic activity of the synthesized sol–gel laccases was determined by using 2,6-dimethoxyphenol as a standard substrate. The SEM results confirmed that the sol–gel laccases have a spherical agglomerated particle morphology having surface area and pore properties varied with synthesis conditions. It was found that the laccase catalytic activity and stability of sol–gel laccases were enhanced depending on synthesis conditions used. There was no significant laccase leaching observed, suggesting that most of the laccase molecules were sterically confined in the silica matrices. The highest laccase catalytic activity was observed for the SOLAC04 (480 AU) which also had the highest surface area (445 m2/g), pore size (30.7 nm) and pore volume (1.14 cm3/g). The optimal conditions for the laccase loading were 10 mg/mL, pH for both free laccase and SOLAC04 was 6, and temperature for the free laccase and SOLAC04 was 50 and 40 °C, respectively. The laccase catalytic activity of SOLAC04 could retain almost 90 % of its original activity after 34-day storage duration at 27 °C. These results demonstrated the advantages offered by the sol–gel laccases.

Selective laccase-catalyzed dimerization of phenolic compounds derived from lignin: Towards original symmetrical bio-based (bis) aromatic monomers

A laccase-catalyzed process was developed to prepare, selectively, in high yield, dimers of lignin-based phenolic compounds without any purification. The influence of experimental parameters such as laccase loading, nature of solvent and the presence of oxygen on the conversion of vanillin was investigated. After the dimerization, the product obtained as a precipitate is filtered off and the solution containing the enzyme can be re-used several times, which improves the process economics. A phenolic-substrate screening reveals that such process enables to dimerize regioselectively, six ortho-methoxy-para-substituted phenols (vanillin, 4-hydroxy-3-methoxybenzonitrile, acetovanillon, methyl vanillate, 2-methoxy-4-methylphenol, and eugenol) with yields ranging from 87% to 96% and one ortho-disubstituted phenol (2,6-dimethoxyphenol) with 80% yield.

Enhancing catalytic performance of laccase via immobilization on chitosan/CeO2 microspheres

In this study, laccase from Trametes versicolor was immobilized onto chitosan/CeO2 microspheres (CCMS) by adsorption or covalent binding after activating the amine groups of chitosan with glutaraldehyde (GA). The results indicated that the laccase loading on chitosan/CeO2 microspheres was approximately 73mg/g under the optimum conditions (pH 5.4, 6h), and the activity recovery was 66.9%. In comparison with free laccase, the thermal and operational stabilities of the immobilized laccase were significantly improved. The catalytic activity of the immobilized laccase was also demonstrated by the decolorization of two reactive dyes (methyl red and orange II). The laccase immobilized on CCMS was very effective for the removal of textile dyes from an aqueous solution. The removal rates of methyl red and orange II by the immobilized laccase were 83.3% and 92.6%, respectively, which are much higher than that of free laccase (i.e., 49.0% and 67.1%, respectively).

A Sequential Combination of Laccase Pretreatment and Enzymatic Hydrolysis for Glucose Production from Furfural Residues

Furfural residues (FRs) were pretreated with laccase or a laccase-mediator (1-hydroxybenzotriazole, HBT) system to produce fermentable sugar for bioethanol production. Compared to laccase-only pretreatment, laccase-mediator pretreatment dissolved more lignin. Approximately 10.5% of the initially present lignin was removed when FRs were treated with a laccase loading of 100 U/g of dry substrate in 1% (w/w) HBT at 48 °C for 24 h in an acetate buffer (pH 4.8). The enzymatic saccharification process was carried out by a combined laccase or laccase-mediator pretreatment without washing of the treated solids. The results showed that active laccase had a negative effect on the rate and yield of enzymatic hydrolysis. Laccase-oxidized HBT seriously reduced glucose yield. However, non-oxidized HBT increased glucose yield when laccase was deactivated at 121 °C for 20 min prior to enzymatic hydrolysis. The highest glucose yield, 80.9%, was obtained from the substrate pretreated with 100 U/g of dry substrate laccase and 1% (w/w) HBT at 48 °C for 24 h in an acetate buffer (pH 4.8). Furthermore, the structures of FRs before and after laccase-mediator pretreatment were characterized by scanning electron microscopy (SEM) and Fourier Transform Infrared spectroscopy (FT-IR).

Laccase immobilization on chitosan/poly(vinyl alcohol) composite nanofibrous membranes for 2,4-dichlorophenol removal

A novel method of laccase immobilization on electrospun chitosan/poly(vinyl alcohol) composite nanofibrous membranes was developed. Laccase from Trametes versicolor was immobilized on the membranes by glutaraldehyde crosslinking. Its physical, chemical, and biochemical properties, as well as its application in 2,4-dichlorophenol removal were investigated. Scanning electron microscopy and transmission electron microscopy images showed that the fiber diameters were 50–200nm, and that the membranes were porous and continuous. The laccase loading on the membranes was about 853mg/g under the optimum conditions (pH 4, 10h). Compared with free laccase, immobilized laccase was less sensitive to changes in pH, temperature, and oscillation, thereby exhibiting higher stability and reusability. The catalytic performances of free and immobilized laccases were evaluated by the degradation of 2,4-dichlorophenol. The removal efficiency of 2,4-dichlorophenol by immobilized laccase was about 87.6% under the optimum conditions (pH 6, 50°C) after 6h, which was much higher than that of free laccase (82.7%).

Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment

Laccase, capable of selectively degrading lignin while keeping cellulose intact, has been widely applied for the modification and bio-bleaching of pulp. In this study Sclerotium sp. laccase (MSLac) was employed in combination with steam explosion to evaluate the effect of this treatment on cellulose hydrolysis. Combined steam explosion with laccase pretreatment enhanced the cellulose conversion rate of wheat straw no matter in the case of successive (MSLac-Cel) and simultaneous (MSLac+Cel) MSLac and cellulase hydrolysis. The highest cellulose conversion rate of 84.23% was obtained when steam-exploded wheat straw (SEWS) (1.3MPa, 5min) was treated by MSLac+Cel at a laccase loading of 0.55Ug−1 substrate. FT-IR and SEM analyses indicated that MSLac oxidized the phenol and changed electron configuration of the ring, which contributed to loosening the compact wrap of lignin–carbohydrate complex and consequently enhancing the enzymatic hydrolysis efficiency of cellulose. This article provided a promising method for lignocellulose bio-pretreatment.

Effects of laccase on lignin depolymerization and enzymatic hydrolysis of ensiled corn stover

The aim of this study was to explore the synergies of laccase, a ligninolytic enzyme, with cellulose and hemicellulase amendments on ensiled corn stover. Molecular signals of lignin decomposition were observed by tetramethylammonium hydroxide thermochemolysis and gas chromatography–mass spectroscopy (TMAH–GC–MS) analysis. The significant findings suggest that ensilage might provide a platform for biological pretreatment. By partially hydrolyzing cellulose and hemicellulose into soluble sugars, ensilage facilitates laccase penetration into the lignocellulose complex to enhance lignin degradation. Downstream cellulose hydrolysis was improved 7% with increasing laccase loading rate. These results demonstrate the potential of enzymes, either directly amended or expressed by microbes during ensilage, to maximize utilization of corn stover for cellulosic biofuels and other downstream fermentations.

The Catalytic Activity of Laccase Immobilized in Sol-Gel Silica

The removal potentially harmful chemicals such as substituted phenols and aromatic amines by laccase oxidative biocatalyst has been attracting interest, particularly in view of certain unsolved problems of oxidation reactions in conventional chemical process. These environmental pollutants mostly do not dissolve in an aqueous solution due to their high hydrophobicity, hence organic solvents are required to dissolve and concentrate them. The biodegradation of these pollutants using enzymatic process has been hindered by instability of enzyme in organic solvents. In order to increase enzyme stability, several methods of stabilization through immobilization and encapsulation have been reported successfully for various enzymes. Enzyme immobilization by physical entrapment has a wide benefit and may provide relatively small perturbation of the enzyme native structure and function. Therefore, in afford to enhance the laccase activity and stability; encapsulation of laccase by sol-gel silica method was studied. The catalytic activity of laccase immobilized in sol-gel silica was demonstrated by an enzymatic assay using 2, 6-dimethoxyphenol as a substrate. After immobilization, the laccase was proven to be active in a wider pH range and the laccase loading in sol-gel silica has a significant effect on laccase activity.