Immobilization Of Laccase Research Articles

Preparation and Characterization of Chitosan-Coated Manganese-Ferrite Nanoparticles Conjugated with Laccase for Environmental Bioremediation.

Bioremediation with immobilized enzymes has several advantages, such as the enhancement of selectivity, activity, and stability of biocatalysts, as well as enzyme reusability. Laccase has proven to be a good candidate for the removal of a wide range of contaminants. In this study, naked or modified MnFe2O4 magnetic nanoparticles (MNPs) were used as supports for the immobilization of laccase from Trametes versicolor. To increase enzyme loading and stability, MNPs were coated with chitosan both after the MNP synthesis (MNPs-CS) and during their formation (MNPs-CSin situ). SEM analysis showed different sizes for the two coated systems, 20 nm and 10 nm for MNPs-CS and MNPs-CSin situ, respectively. After covalent immobilization of laccase by glutaraldehyde, the MNPs-CSin situ-lac and MNPs-CS-lac systems showed a good resistance to temperature denaturation and storage stability. The most promising system for use in repeated batches was MNPs-CSin situ-lac, which degraded about 80% of diclofenac compared to 70% of the free enzyme. The obtained results demonstrated that the MnFe2O4-CSin situ system could be an excellent candidate for the removal of contaminants.

Immobilization of yeast cell walls with surface displayed laccase from Streptomyces cyaneus within dopamine-alginate beads for dye decolorization

High amounts of toxic textile dyes are released into the environment due to coloring and wastewaters treatment processes' inefficiency. To remove dyes from the environment and wastewaters, researchers focused on applying immobilized enzymes due to mild reaction conditions and enzyme nontoxicity. Laccases are oxidases with wide substrate specificity, capable of degradation of many different dye types. Laccase from Streptomyces cyaneus was expressed on the surface of Saccharomyces cerevisiae EBY100 cells. The specific activity of surface-displayed laccase was increased by toluene-induced lysis to 3.1 U/g of cell walls. For cell wall laccase immobilization within hydrogel beads, alginate was modified by dopamine using periodate oxidation and reductive amination and characterized by UV–Vis, FTIR, and NMR spectroscopy. Cell wall laccase was immobilized within alginate and dopamine-alginate beads additionally cross-linked by oxygen and laccase. The immobilized enzyme's specific activity was two times higher using dopamine-alginate compared to native alginate beads, and immobilization yield increased 16 times. Cell wall laccase immobilized within dopamine-alginate beads decolorized Amido Black 10B, Reactive Black 5, Evans Blue, and Remazol Brilliant Blue with 100% efficiency and after ten rounds of multiple-use retained decolorization efficiency of 90% with Evans Blue and 61% with Amido Black.

Fabrication of hollow covalent-organic framework microspheres via emulsion-interfacial strategy to enhance laccase immobilization for tetracycline degradation

• Hollow microspheres H-COF-OMe were prepared by emulsion interfacial polymerization. • Interfacial-defects in H-COF-OMe were formed by confined growth on emulsion droplets. • Lac@H-COF-OMe showed both high laccase loading and activity recovery. • Lac@H-COF-OMe displayed excellent structure stability and recyclability. • Lac@H-COF-OMe exhibited better adsorption uptake and faster degradation rate for tetracycline. Hollow covalent organic framework microsphere (H-COF-OMe) using TAPB and DMTP with enriched interfacial defects was fabricated via emulsion interfacial polymerization and in turn applied as a novel host for high laccase loading and tetracycline (TC) degradation. Benefited from the space-confined growth in the multiphase solvent interface, H-COF-OMe exhibited hollow spherical microstructure, high surface area and unique defects-rich interface. Attributed to these intriguing aspects, H-COF-OMe achieved maximum loading capacity of 567 mg/g and activity recovery of 85% for laccase. H-COF-OMe efficiently stabilized the active conformation of laccase from structural distortion via multiple binding sites, which endowed Lac@H-COF-OMe significantly higher pH, thermal, and storage stabilities, and reusability than free laccase and Lac@COF-OMe. Interestingly, the hollow morphology and defective interface of Lac@H-COF-OMe accelerated the diffusion of TC and shortened the reaction pathway, which endowed it with markedly enhanced TC degradation and recycling performance than many state-of-the-art catalysts. Significantly, Lac@H-COF-OMe (20 mg) could achieve 99% degradation of 50 mg/L tetracycline (50 mL) within 100 min. Monitoring of the intermediate products indicated that Lac@H-COF-OMe showed outstanding detoxification performance of the degradation products. This work suggested a novel COF synthesis strategy as laccase immobilization supporters for high TC degradation, which makes it as a promising candidate for degradation of organic pollutants.

Immobilization of laccases onto cellulose nanocrystals derived from waste newspaper: relationship between immobilized laccase activity and dialdehyde content

The purpose of this work is to evaluate the possibility of using cellulose nanocrystals (CNC) derived from waste newspaper as a carrier for immobilizing laccases. Firstly, the cellulose nanocrystals extracted from waste newspaper were oxidized with sodium periodate, and the hydroxyl groups on the second and third carbons (C2 and C3) of the CNC molecules were converted to dialdehyde groups. Then, the laccase was successfully immobilized on the dialdehyde-modified cellulose nanocrystals (DMC) through covalent bonding. The DMC was characterized using zeta potential, Fourier-transform infrared spectroscopy, X-ray diffraction, scanning electron microscopy, and transmission electron microscopy, and the relationship between the activity of the immobilized laccase and the dialdehyde group content of the DMC was thoroughly investigated. It was found that the yield of the laccase immobilization is 64.94%, and the highest activity of the immobilized laccase (1.108 U/mg) is obtained at an aldehyde content of the DMC equal to 50.64%, a mass ratio of the carrier to the laccase equal to 45:3, a pH of 4.5, and a reaction time of 2 h. Compared to the free laccase, the immobilized laccase exhibits excellent stability in a wider range of pH values and temperatures. Furthermore, the immobilized laccase showed excellent reusability as evidenced by its retained relative activity of 67% after 6 cycles.

Sustainable liquid supports for laccase immobilization and reuse: Degradation of dyes in aqueous biphasic systems.

Novel liquid supports for enzyme immobilization and reuse based on aqueous biphasic systems (ABS) constituted by cholinium-based ionic liquids (ILs) and polymers for the degradation of dyes are here proposed. The biocatalytic reaction for dye decolorization using laccase occured in the biphasic medium, with the enzyme being "supported" in the IL-rich phase and the dye and degradation products being enriched in the polymer-rich phase. An initial screening of the laccase activity in aqueous solutions of ABS constituents, namely cholinium dihydrogen citrate ([Ch][DHC]), cholinium dihydrogen phosphate ([Ch][DHP]), cholinium acetate ([Ch][Acet]), polypropylene glycol 400 (PPG 400), polyethylene glycol 400 (PEG 400) and K2 HPO4 was carried out. Compared to the buffered control, a relative laccase activity of up to 170%, 257%, and 530% was observed with PEG 400, [Ch][DHP], and [Ch][DHC], respectively. These ABS constituents were then investigated for the in situ enzymatic biodegradation of the Remazol Brilliant Blue R (RBBR) dye. At the optimized conditions, the ABS constituted by PPG 400 at 46 wt% and [Ch][DHC] at 16 wt% leads to the complete degradation of the RBBR dye, further maintaining the enzyme activity. This ABS also allows an easy immobilization, recovery, and reuse of the biocatalyst for six consecutive reaction cycles, achieving a degradation yield of the dye of 96% in the last cycle. In summary, if properly designed, high enzymatic activities and reaction yields are obtained with ABS as liquid supports, while simultaneously overcoming the safety and environmental concerns of conventional organic solvents used in liquid-liquid heterogeneous reactions, thus representing more sustainable biocatalytic processes.

Preparation of Functionalized Mesoporous Silica as a Novel Carrier and Immobilization of Laccase.

Amino-modified mesoporous silica (SBA-15-NH2) was prepared by hydrothermal method, which is a kind of excellent carrier for enzyme immobilization. The structure of SBA-15 was characterized by SEM and FTIR, which proved that amino group was successfully attached to the surface of SBA-15. The carrier had good mesoporous structure proved by nitrogen adsorption and desorption test. Using SBA-15-NH2 as the carrier, the optimal conditions of laccase immobilization by two different cross-linking methods were explored. At the same time, the properties of the immobilized enzyme and free enzyme were compared. The results showed that the activity of immobilized laccase by two-step method (2977.5 U/g) was much higher than that by one-step method (239.5 U/g). The optimal conditions were as follows: free laccase (35°C, pH=4.5), two-step immobilized laccase (40°C, pH=4.0), one-step immobilized laccase (35°C, pH=4.0). The two-step method was more adaptable to temperature. The pH adaptation range of the immobilized enzyme is wider, and the thermal stability is greatly enhanced. After five cycles of repeated reaction, the residual enzyme activity of two-step and one-step methods was 56% and 43% of the original. The treatment of simulated wastewater containing 2,4-dichlorophenol (2,4-DCP) by immobilized laccase was also studied. Under optimum conditions (40°C, pH=5.0, 20 mg/L), the removal of 2,4-DCP reached 89.06%. The immobilized laccase is really effective for treatment of 2,4-DCP-containing wastewater.

Modeling and experimental validation of covalent immobilization of Trametes maxima laccase on glyoxyl and MANA-Sepharose CL 4B supports, for the use in bioconversion of residual colorants.

Our novel strategy for the rational design of immobilized derivatives (RDID) is directed to predict the behavior of the protein immobilized derivative before its synthesis, by the usage of mathematic algorithms and bioinformatics tools. However, this approach needs to be validated for each target enzyme. The objective of this work was to validate the RDID strategy for covalent immobilization of the enzyme laccase from Trametes maxima MUCL 44155 on glyoxyl- and monoaminoethyl-N-aminoethyl (MANA)-Sepharose CL 4B supports. Protein surface clusters, more probable configurations of the protein-supports systems at immobilization pHs, immobilized enzyme activity, and protein load were predicted by RDID1.0 software. Afterward, immobilization was performed and predictions were experimentally confirmed. As a result, the laccase-MANA-Sepharose CL 4B immobilized derivative is better than laccase-glyoxyl-Sepharose CL 4B in predicted immobilized derivative activity (63.6% vs. 29.5%). Activity prediction was confirmed by an experimentally expressed enzymatic activity of 68%, using 2,6-dimethoxyphenol as substrate. Experimental maximum protein load matches the estimated value (11.2±1.3 vs. 12.1protein mg/support mL). The laccase-MANA-Sepharose CL 4B biocatalyst has a high specificity for the acid blue 62 colorant. The results obtained in this work suggest the possibility of using this biocatalyst for wastewater treatment.

Immobilization of laccase from Bacillus subtilisDS on chitosan beads and standardization of process for biodegradation of textile dyes

AbstractBACKGROUNDThe textile industry generates a huge amount of effluents containing different hazardous dyes. Indigo carmine and malachite green are the most commonly used dyes in textile and dye industries. Pollution caused by these dyes affects aquatic life.RESULTSBacillus subtilis DS produces an alkali‐thermostable laccase. This enzyme was immobilized on chitosan beads and conditions for immobilization were optimized. After immobilization, the maximum activity of 26 IU g–1 beads was found under optimized conditions of 1% glutaraldehyde, 3 h cross‐linking time and 5 h immobilization time. The immobilized enzyme retained a temperature and pH profile similar to that of the free enzyme. Significant enzyme activity was retained after ten reaction cycles with >60% stability after 15 days, allowed reuse of enzyme over multiple cycles. Immobilized laccase was used to degrade textile dyes and the conditions were standardized statistically for its optimal degradation. Under optimized conditions a high degradation rate was achieved for indigo carmine (95.24%) and malachite green (90%), respectively.CONCLUSIONImmobilized laccase is a highly suitable candidate for bioremediation of textile industry dyes because of its stability and reusability. © 2021 Society of Chemical Industry

Evaluation of a Trametes pubescens laccase concentrated extract on allura red AC decolorization without the addition of synthetic mediators

Synthetic dye bioremediation is a topic of great importance since these pollutants possess toxic effects, and huge quantities of them are being discharged into water bodies. Ligninolytic enzyme treatment stands out for being a cost-effective methodology, capable of obtaining high decolorization levels. In this work, a laccase enzyme treatment was evaluated to effectively perform a cycle of dye bioremediation. Furthermore, a dye decolorization improvement was also assessed through laccase immobilization. Particularly, a Trametes pubescens enzyme extract was concentrated, immobilized onto calcium alginate beads, and characterized to assess its dye decolorization potential. Ammonium sulfate precipitation and vacuum evaporation were evaluated to concentrate the crude extract and to decolorize allura red AC. Both treatments reached a high enzyme yield recovery (>90%), but only the vacuum-evaporated extract achieved a high allura red AC decolorization level after 16 h of contact time. This suggested that essential compounds for allura red AC decolorization were present in the crude extract, implying that neither a complete laccase purification process nor an addition of synthetic mediators are necessary. Under optimized immobilization conditions, 94.6% immobilization efficiency and 49.8% activity recovery were obtained with 0:1 alginate:enzyme (v/v), 100 mM CaCl2, and 5.0% w/v sodium alginate. Furthermore, by immobilizing the laccase concentrated extract, both the pH and temperature stabilities were improved. The decolorization of allura red AC by free and immobilized laccase was 68.4% and 4.6%, respectively, showing that although the enzyme stability was improved, dye decolorization was negatively affected. Thus, an efficient allura red AC decolorization was obtained with concentrated-free laccase by a feasible and low-cost methodology.

α-Cellulose Fibers of Paper-Waste Origin Surface-Modified with Fe3O4 and Thiolated-Chitosan for Efficacious Immobilization of Laccase.

The utilization of waste-paper-biomass for extraction of important α-cellulose biopolymer, and modification of extracted α-cellulose for application in enzyme immobilization can be extremely vital for green circular bio-economy. Thus, in this study, α-cellulose fibers were super-magnetized (Fe3O4), grafted with chitosan (CTNs), and thiol (-SH) modified for laccase immobilization. The developed material was characterized by high-resolution transmission electron microscopy (HR-TEM), HR-TEM energy dispersive X-ray spectroscopy (HR-TEM-EDS), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FT-IR) analyses. Laccase immobilized on α-Cellulose-Fe3O4-CTNs (α-Cellulose-Fe3O4-CTNs-Laccase) gave significant activity recovery (99.16%) and laccase loading potential (169.36 mg/g). The α-Cellulose-Fe3O4-CTNs-Laccase displayed excellent stabilities for temperature, pH, and storage time. The α-Cellulose-Fe3O4-CTNs-Laccase applied in repeated cycles shown remarkable consistency of activity retention for 10 cycles. After the 10th cycle, α-Cellulose-Fe3O4-CTNs possessed 80.65% relative activity. Furthermore, α-Cellulose-Fe3O4-CTNs-Laccase shown excellent degradation of pharmaceutical contaminant sulfamethoxazole (SMX). The SMX degradation by α-Cellulose-Fe3O4-CTNs-Laccase was found optimum at incubation time (20 h), pH (3), temperatures (30 °C), and shaking conditions (200 rpm). Finally, α-Cellulose-Fe3O4-CTNs-Laccase gave repeated degradation of SMX. Thus, this study presents a novel, waste-derived, highly capable, and super-magnetic nanocomposite for enzyme immobilization applications.

Rapid and Oriented Immobilization of Laccases on Electrodes via a Methionine-Rich Peptide

Different from other laccases, copper efflux oxidase (CueO) from Escherichia coli possesses an additional methionine-rich segment (MetRich), which is generally considered to be detrimental to its o...

Fiber-optic biosensor based on the laccase immobilization on silica-functionalized fluorescent carbon dots for the detection of dopamine and multi-color imaging applications in neuroblastoma cells

An efficient and cost-effective biosensor is of the great demand for the detection of the biologically significant neurotransmitter dopamine. In this context, enzymatic biosensors show excellent sensitivity and selectivity. In this study, we developed a laccase immobilized fiber-optic biosensor based on the fluorescence principle for the detection of dopamine. To design this biosensor, we used microwave irradiation to synthesize carbon dots (CDs) using curcumin and dimethylformamide, and the resulting CDs were called CDD-CDs. These were functionalized with a silicon precursor, 3-(aminopropyl)-triethoxysilane, and were referred to as APT-CDs. Furthermore, laccase was covalently immobilized to the APT-CDs to construct a novel bioprobe. The CDD-CDs, APT-CDs, and bioprobe showed orange (λem = 586 nm) green (λem = 533 nm), and blue-colored emissions (λem = 476 nm) at 430, 380, and 360 nm excitation wavelengths, respectively. The CDD-CDs and bioprobe showed quantum yields of 14.8% and 10.2%, respectively. The CDD-CDs displayed solvatochromism in various solvents. Bioprobe showed a significant fluorescence quenching for dopamine in the linear range of 0–30 μM with a detection limit of 41.2 nM. Bioprobe was immobilized on the tapered optical fiber using ethyl cellulose by a simple dip-coating method and investigated for multi-color imaging applications. The resulting tapered optical fiber achieved a satisfactory detection limit of 46.4 nM in the dopamine concentration range of 0–10 μM. The bioprobe demonstrated high biocompatibility, long-lasting photostability, and thermal stability, and had sufficient cytotoxicity in human neuroblastoma cells (SH-SY5Y) with excellent multi-color imaging potential. The practicality of the bioprobe was investigated in human serum and cerebrospinal fluid.

Carbon nanowalls functionalization for efficient O2 reduction catalyzed by laccase using design of experiment

An enzymatic biofuel cell cathode was elaborated by nano-structuration and functionalization of graphite using two consecutive plasma treatments before immobilization of laccase as a biocatalyst to perform oxygen reduction reaction (ORR) by direct electron transfer ((DET). Nano-structuration of graphite surfaces by synthesis of vertical graphen-like carbon nanowalls (CNWs) was performed by microwave excited plasma enhanced chemical vapor deposition (PECVD). Atmospheric pressure plasma jet (APPJ) system was then used to functionalize chemically the CNWs surface via a rapid and dry route. A partial fractional design evidenced two significant parameters of the APPJ treatment in terms of ORR current density: the treatment time and the inter-distance between the plasma jet and the surface of the carbon material. With the optimized APPJ treatment parameters, a four-fold enhancement of the ORR current density (-440 ± 70 μA/cm2) could be obtained on CNWs, compared to graphite electrodes. The best current density obtained was about −1 mA/cm2 with oxidized laccase covalently immobilized on the electrode without any agitation of the solution, confirming the main importance of enzyme orientation on the electrode surface for efficient DET transfer, the relevance of electrode nanostructuration and the advantage of covalent linking of the protein on the electrode surface.

Study on adsorption-degradation of 2,4-dichlorophenol by modified biochar immobilized laccase

In this paper, biochar was modified by cetyltrimethylammonium bromide (CTAB) and KOH solution, and it was characterized by TGA, FT-IR, XRD, BET, SEM. Then, the modified biochar (MB) was used to immobilize free laccase (FL) by adsorption method to prepare modified biochar immobilize laccase (MBL). The adsorption and degradation performance for 2,4-DCP via MB, FL, MBL were studied. The results showed that under the optimal conditions, the adsorption rate of MB on 2,4-DCP was up to 42.5%, adsorption capacity was 85.13 mg/g, and the adsorption of 2,4-DCP by MB was proved to conform to the pseudo-second-order model and the Langmuir adsorption isotherm model; the maximum degradation rate of FL on 2,4-DCP was 97.7%, and the degradation of 2,4-DCP by FL was proved to conform to the first-order kinetic model; under the optimal conditions, the removal rate of MBL on 2,4-DCP can reach almost 100%. MBL combines physical–chemical adsorption and biodegradation, and the contribution of biodegradation is greater than adsorption. Finally, the possible degradation mechanism and pathways of 2,4-DCP were given via high-performance liquid chromatography (HPLC) analysis and gas chromatography-mass spectrometry (GC–MS) analysis.

Preparation of uniform polyurea microspheres at high yield by precipitation polymerization and their use for laccase immobilization

Uniform polyurea (PU) microspheres were prepared using isophorone diisocyanate as the only monomer either in binary solvents of water/acetone (H2O/AT) or water/acetonitrile (H2O/AN), or in ternary solvent of H2O/AN/DMF, by precipitation polymerization without any shaking or stirring. Through careful examination, highly uniform microspheres were obtained at monomer concentration of 28.0 wt% at 65 °C in H2O/AN at 20/80 by mass, a value much higher than the previously (13.0 wt%) achieved in H2O/AT/DMF. The microspheres were analyzed by FTIR and NMR to confirm their PU structure and used as support for covalent immobilization of laccase via glutaraldehyde coupling. The immobilized laccase showed higher storage and thermal stability than the free laccase, and exhibited higher catalytic activity and better reusability in biodegradation of Congo Red and RBBR, compared with reported studies. In addition to enhancing the productivity of uniform microspheres, this study provides a new pathway to supported catalyst for enzymatic remediation of dyes-polluted water.

A novel process for the covalent immobilization of laccases on silica gel and its application for the elimination of pharmaceutical micropollutants.

In the present work, pharmaceutical micropollutant degradation by laccase immobilized on silica through an innovative process is proposed. The influence of different parameters on the immobilization conditions was evaluated by a 23 full factorial design, and parameters leading to the highest activity were identified. Under these conditions, laccase activity reached 14 ± 2 U g-1 of silica with a protein immobilization yield of 35%. The biocatalyst characterization did not show any change in pH and thermal stabilities but enhanced the long-term storage of laccases. Immobilized T. versicolor laccases were then tested to remove four pharmaceutical micropollutants (amoxicillin, ciprofloxacin, carbamazepine, and sulfamethoxazole) in the presence of redox mediators (syringaldehyde, p-coumaric acid, and ABTS). High removal yields (50-100% according to the pollutant) were obtained within 4 h of treatment due to the synergistic effect of laccase-mediator biotransformation and adsorption on the support. Overall, the pharmaceuticals' removal efficiency was highly influenced by their physicochemical properties; however, the presence of redox mediators impacted not only the oxidation mechanism but also the interactions between the biocatalyst and micropollutants. Finally, the reusability of the biocatalyst was proved during 7 degradation cycles.

Laccase Immobilization Strategies for Application as a Cathode Catalyst in Microbial Fuel Cells for Azo Dye Decolourization.

Enzymatic biocathodes have the potential to replace platinum as an expensive catalyst for the oxygen reduction reaction in microbial fuel cells (MFCs). However, enzymes are fragile and prone to loss of activity with time. This could be circumvented by using suitable immobilization techniques to maintain the activity and increase longevity of the enzyme. In the present study, laccase from Trametes versicolor was immobilized using three different approaches, i.e., crosslinking with electropolymerized polyaniline (PANI), entrapment in copper alginate beads (Cu-Alg), and encapsulation in Nafion micelles (Nafion), in the absence of redox mediators. These laccase systems were employed in cathode chambers of MFCs for decolourization of Acid orange 7 (AO7) dye. The biocatalyst in the anode chamber was Shewanella oneidensis MR-1 in each case. The enzyme in the immobilized states was compared with freely suspended enzyme with respect to dye decolourization at the cathode, enzyme activity retention, power production, and reusability. PANI laccase showed the highest stability and activity, producing a power density of 38 ± 1.7 mW m−2 compared to 25.6 ± 2.1 mW m−2 for Nafion laccase, 14.7 ± 1.04 mW m−2 for Cu-Alg laccase, and 28 ± 0.98 mW m−2 for the freely suspended enzyme. There was 81% enzyme activity retained after 1 cycle (5 days) for PANI laccase compared to 69% for Nafion and 61.5% activity for Cu-alginate laccase and 23.8% activity retention for the freely suspended laccase compared to initial activity. The dye decolourization was highest for freely suspended enzyme with over 85% decolourization whereas for PANI it was 75.6%, Nafion 73%, and 81% Cu-alginate systems, respectively. All the immobilized laccase systems were reusable for two more cycles. The current study explores the potential of laccase immobilized biocathode for dye decolourization in a microbial fuel cell.

Application of Immobilized Laccase on Polyurethane Foam for Ex-Situ Polycyclic Aromatic Hydrocarbons Bioremediation

There is growing concern about developing treatment technologies for the hazardous Polycyclic Aromatic Hydrocarbons (PAHs), because the rising levels of these compounds in the environment by human activities. The application of laccases has been evaluated as one of the most promising treatments. Thus, laccase immobilization on polyurethane foam (PUF)—low-cost material—was evaluated for bioremediation in batch mode of simulated groundwater using a combination of 16 polycyclic aromatic hydrocarbons as model pollutants. Conditions closer to a real contaminated site (non-optimal) were considered on our experimental design, leading to the formation of new degradation intermediaries, even more degraded than the usual ones. The bioremediation of PAH (mg L−1) using immobilized laccase on PUF reached 92.35% of removal for anthracene (Ant) and 97% for benzo(a)pyrene (BaP). After the treatment, the biodegradation products were identified as diisooctyl phthalate and tetradecane. The biodegradation mechanism was proposed, where PAHs oxidation processes and aromatic ring fission led to quinone and diethyl phthalate formation. Then, through the latter processes besides, polymerization and methylation, lead to the identified biodegradation product formation. The immobilized enzyme improvement in the removal yield of 8 of the other 14 PAHs tested in μg L−1, compared to the free counterpart. Laccase immobilized on PUF achieved final anthracene concentration of 0.95 mg L−1, up to 38 μg L−1 of chrysene (77% removal) and 98 μg L−1 of pyrene (32% removal), under the intervention limits of environmental protection policies. Thus, laccase immobilized on PUF for use in bioreactors can be considered a potential approach for PAHs bioremediation for an ex-situ treatment.

Modification of Electrospun Regenerate Cellulose Nanofiber Membrane via Atom Transfer Radical Polymerization (ATRP) Approach as Advanced Carrier for Laccase Immobilization.

This study aimed to modify an electrospun regenerated cellulose (RC) nanofiber membrane by surface grafting 2-(dimethylamino) ethyl methacrylate (DMAEMA) as a monomer via atom transfer radical polymerization (ATRP), as well as investigate the effects of ATRP conditions (i.e., initiation and polymerization) on enzyme immobilization. Various characterizations including XPS, FTIR spectra, and SEM images of nanofiber membranes before and after monomer grafting verified that poly (DMAEMA) chains/brushes were successfully grafted onto the RC nanofiber membrane. The effect of different ATRP conditions on laccase immobilization was investigated, and the results indicated that the optimal initiation and monomer grafting times were 1 and 2 h, respectively. The highest immobilization amount was obtained from the RC-Br-1h-poly (DMAEMA)-2h membrane (95.04 ± 4.35 mg), which increased by approximately 3.3 times compared to the initial RC membrane (28.57 ± 3.95 mg). All the results suggested that the optimization of initiation and polymerization conditions is a key factor that affects the enzyme immobilization amount, and the surface modification of the RC membrane by ATRP is a promising approach to develop an advanced enzyme carrier with a high enzyme loading capacity.

Immobilization of laccase via cross-linked enzyme aggregates prepared using genipin as a natural cross-linker

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.