Laccase Loading Research Articles

Removal of aflatoxin M1 in milk using magnetic laccase/MoS2/chitosan nanocomposite as an efficient sorbent

The current study tries to find the impact of the integration of laccase enzyme (Lac) onto magnetized chitosan (Cs) nanoparticles composed of molybdenum disulfide (MoS2 NPs) (Fe3O4/Cs/MoS2/Lac NPs) on the removal of AFM1 in milk samples. The Fe3O4/Cs/MoS2/Lac NPs were characterized by FT-IR, XRD, BET, TEM, FESEM, EDS, PSA, and VSM analysis. The cytotoxic activity of the synthesized nanoparticles in different concentrations was evaluated using the MTT method. The results show that the synthesized nanoparticles don't have cytotoxic activity at concentrations less than 20 mg/l. The ability of the prepared nanoparticles to remove AFM1 was compared by bare laccase enzyme, MoS2, and Fe3O4/Cs/MoS2 composite, indicating that the Fe3O4/Cs/MoS2/Lac NPs the highest adsorption efficiency toward AFM1. Besides, the immobilization efficiency of laccase with a concentration range of 0.5–2.0 was investigated, indicating that the highest activity recovery of 96.8% was obtained using 2 mg/ml laccase loading capacity. The highest removal percentage of AFM1 (68.5%) in the milk samples was obtained by the Fe3O4/Cs/MoS2/Lac NPs at a contact time of 1 h. As a result, Fe3O4/MoS2/Cs/Lac NPs can potentially be utilized as an effective sorbent with high capacity and selectivity to remove AFM1 from milk samples.

Remediation effect and mechanism of immobilized laccase on actual trichloromethane-contaminated soil samples

Trichloromethane (TCM) in soil causes adverse effects on the soil ecosystem and human health. Therefore, it is urgent to develop an efficient and low-cost soil TCM removal technology. In this study, we prepared sodium alginate-biochar-laccase immobilized pellets (SA-BC-LC) using composite immobilization technology, exploring the fitting impact of artificial neural network model (ANN) and general linear model (GLM) models on laccase loading and the correlation properties of SA-BC-LC. The SA-BC-LC exhibited a porous multi-layer network structure internally. Compared to free laccase, the relative activity of SA-BC-LC remained above 50 % after 5 reuses and still maintained at 48 % after 50 days of storage. Moreover, SA-BC-LC demonstrated significant capacity for adsorption and degradation of TCM in soil with ratios approximately at 31.3 % and 68.7 %, respectively. During the remediation process of TCM-contaminated soil from Taizhou Chemical plant, there was a rapid decrease in TCM concentration within the first hour. Under IM (Immobilization) treatment (added with 5 % SA-BC-LC), the removal rate of TCM reached up to 88.9 % in the first hour. The adsorption of TCM by SA-BC-LC was mainly chemical adsorption, which induced the improvement of the relative abundance of the co-metabolic dechlorinated microbial Pseudomonas. Our research presents a promising material for effectively remediating soils contaminated with chlorinated hydrocarbons (CHs), with excellent removal efficiency.

Degradation of 2,4-DCP by immobilized laccase on modified biochar carrier.

Rape straw was used as the raw material for the biochar in this study, which was then changed using acid, alkali, and magnetic techniques. The laccase was attached using the adsorptions-crosslinking process, and the three modified biochars served as the carriers. The ideal circumstances for laccase immobilization were explored, and both biochar and immobilized laccase's characteristics were examined. The removal of 2,4-dichlorophenol (2,4-DCP) by immobilized laccase from modified biochar and its degradation products were researched. The main conclusions are as follows: the optimal concentration of glutaraldehyde (GLU) was 4%, and the pH was four, and the enzyme dosage was 1.75mg/mL for the immobilized laccase of acid-modified biochar (SBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 2mg/mL for immobilized laccase from alkali-modified biochar (JBC@LAC). The optimal concentration of GLU was 5%; the pH was four, and the enzyme dosage was 1.75mg/mL for immobilized laccase from magnetically modified biochar (CBC@LAC). SEM images could show the changes in the surface morphology of biochar caused by three modification methods. The BET results demonstrated that acid and magnetic modification increased the specific surface area of biochar, and alkali modification mainly expanded the pore size of biochar. FT-IR and XRD showed that modification and laccase loading had little effect on the structure of biochar. The stability of immobilized laccase was better than that of free laccase in acid-base, heat, and storage. Among the three modified biochar immobilized laccases, JBC@LAC showed the best acid-base stability and thermal stability, and the relative enzyme activity changed the least when pH and temperature conditions changed. The storage stability of SBC@LAC is the best. After 30days of storage, the relative enzyme activity is still 83%. The removal rates of 2,4-DCP were 57, 99, and 63%, respectively, by SBC@LAC, JBC@LAC, and CBC@LAC. After five reuses, the removal rates of 2,4-DCP by SBC@LAC, JBC@LAC and CBC@LAC were 26, 42, and 27%, respectively. The intermediate products of 2,4-DCP degradation by immobilized laccase were p-phenol, p-benzoquinone and maleic acid.

Facile co-immobilization of laccase and mediator by polyethyleneimine-induced biomimetic mineralization for dye removal

Laccase, a green biocatalyst, has been widely used in the field of biodegradation with the assistance of electron mediators. Herein, a novel strategy, polyethyleneimine (PEI)-induced biomimetic mineralization, was explored and applied to co-immobilize laccase and 2,2′-azino bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS). With the PEI doping, calcium phosphate (CaP) could facilely mineralize within 20 min to form PEI@CaP; ABTS could be co-mineralized with PEI to form (PEI + ABTS)@CaP. Laccase, a negatively charged protein, was then immobilized via electrostatic adsorption onto the embedded PEI in the carriers. Both of the immobilized laccase preparations, denoted as Lac-PEI@CaP and Lac-(PEI + ABTS)@CaP, showed high activity recoveries (96.4 % and 88.0 %, respectively), and increased pH tolerance and storage stability as compared to free laccase. ABTS and laccase loading densities of Lac-(PEI + ABTS)@CaP reached 0.83 mmol g−1 and 100 mg g−1, respectively. Lac-(PEI + ABTS)@CaP presented higher malachite green (MG) degradation capability than free laccase and Lac-PEI@CaP with free ABTS, possibly due to the proximity effect of the co-immobilized laccase and ABTS. Moreover, Lac-(PEI + ABTS)@CaP exhibited reusability and kept 83.2 % of MG degradation activity after three recycles and 37.9 % in the sixth. This work provided a new strategy for the co-immobilization of laccase and electron mediator, and demonstrated the potential of the biocatalyst for organic pollutant removal.

Construction of a biomimetic core-shell PDA@Lac bioreactor from intracellular laccase as a nano-confined biocatalyst for decolorization

Enzymes immobilized on the surface of the carriers are difficult to maintain their conformation and high activity due to the influence of the external harsh environments. A biomimetic core-shell PDA@Lac bioreactor was constructed by depositing polydopamine (PDA) on the surface of the recombinant Escherichia coli with CotA laccase gene, and releasing intracellular laccase into the PDA shell using ultrasound to break the cell wall of the bacteria. The bioreactor provided a nano-confined environment for the laccase and accelerated the mass and electron transfer in the volume-confined space, with a 2.77-fold increase in Km compared with the free laccase. Since there was no barrier of the cell wall, the crystal violet dye can enter the bioreactor to participate in the enzymatic reaction. As a result, PDA@Lac achieved excellent decolorization performance even without ABTS as an electron mediator. Moreover, the cytoplasmic solution retained in the PDA shell promoted the enzyme's tolerance to pH, temperature and harsh environments. In addition to PDA encapsulation, carbonyl and –NH2 groups of PDA were bound covalently with –NH2 and –COOH on the laccase in the PDA@Lac, resulting in an extremely high laccase loading of 817.59 mg/g. Also, the relative activity of the bioreactor maintained approximately 75% after 10 cycles of reuse. In addition, the protection of the PDA shell increased the resistance of laccase to UV irradiation. This work provides a novel method of laccase immobilization for application in wastewater treatment.

Degradation of phenolic inhibitors by laccase immobilized on tannic acid/polyethylenimine modified magnetic nanoparticles

Magnetic Fe3O4 nanoparticles were successfully functionalized with the low-cost mussel-inspired tannic acid (TA)/polyethylenimine (PEI) under mild conditions and then activated with glutaraldehyde (GA) to covalently immobilize laccase for degradation of four model phenolic inhibitors commonly found in lignocellulosic hydrolysate. The immobilized laccase showed the highest relative enzyme activity with the laccase loading of 39.9 mg/g and the enzyme activity recovery of 53.1%. Compared with free laccase, immobilized laccase possessed better relative enzyme activity at acidic pH and wide temperature range, as well as exhibited excellent storage stability and satisfactory reusability. Moreover, it was found that the phenolic degradation products by immobilized laccase did not exhibit obvious inhibition on subsequent enzymatic cellulose hydrolysis. Thus, the low-cost TA/PEI coating for modification of magnetic nanoparticles will provide a promising support for laccase immobilization and the immobilized laccase has great potential in removal of phenolic inhibitors present in lignocellulosic hydrolysate.

Biodecolorization and Ecotoxicity Abatement of Disperse Dye-Production Wastewater Treatment with Pycnoporus Laccase.

The biological treatment efficiency of dye wastewater using activated sludge (AS) is largely limited to the chromaticity and ecotoxicity of dyestuff. To alleviate this limitation, eleven industrial-grade disperse dyes were obtained from a fiber-dyeing factory, and for the first time, we studied the decolorization and detoxification effects of using the Pycnoporus laccase enzyme. Efficient decolorization was achieved with the following conditions: dye concentration 50 mg/L, 1-hydroxybenzotriazole (HBT) 0.15 mM, temperature 65 °C, pH 4, and laccase 0.33 U/mL. The decolorization rate of disperse dyes, ranging from 51 to 96% in this investigation, was highly dependent on the dye type, concentration, laccase loading, and HBT. The ecotoxicity of dyes was evaluated by studying the germination/growth of wheat seed as well as the respiratory rate of aerobic AS. Laccase treatment mitigated the phytotoxicity of dyes because of the higher wheat germination (e.g., increase of 38% for Black ECT 200%) and growth rate (e.g., increase of 91% for Blue 2BLN 200%). The reduced ecotoxicity of decolorized dye solution towards microorganisms was also confirmed by the finding that the oxygen uptake by aerobic AS was increased relative to that of the untreated samples (e.g., increase of 14 folds for Blue HGL 200%). In addition, the chemical oxygen demand (COD) of decolorized dye solution was slightly lower than that without decolorization during the respiratory test. The experimental results suggest that enzymatic decolorization and detoxification can be potentially used as a pretreatment method for disperse dye wastewater followed by AS treatment.

Eco-friendly laccase and cellulase enzymes pretreatment for optimized production of high content lignin-cellulose nanofibrils

Lignin-cellulose nanofibrils (LCNF) are of attracting an increasing interest due to the benefits of maintaining the lignin in the nanomaterial composition. The production of LCNF requires considerable energy consumption, which has been suppressed employing pretreatment of biomass, in which it highlights those that employ enzymes that have the advantage of being more environmentally friendly. Some negative aspects of the presence of lignin in the fiber to obtain cellulose nanofibrils is that it can hinder the delamination of the cell wall and act as a physical barrier to the action of cellulase enzymes. This study aimed to evaluate the impact of a combined enzymatic pretreatment of laccase and endoglucanase for high content lignin LCNF production. The morphological and chemical properties, visual aspect and stability, crystallinity, mechanical properties, rheology, barrier properties and quality index were used to characterize the LCNF. The laccase loading used was efficient in modifying the lignin to facilitate the action of the endoglucanase on cellulose without causing the removal of this macromolecule. This pretreatment improved the quality of LCNF (61 ± 3 to 71 ± 2 points) with an energy saving of 42% and, therefore, this pretreatment could be suitable for industrial production for a variety of applications.

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.

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.

Thiolation of Chitosan Loaded over Super-Magnetic Halloysite Nanotubes for Enhanced Laccase Immobilization.

This study focuses on the development of a nanosupport based on halloysite nanotubes (HNTs), Fe3O4 nanoparticles (NPs), and thiolated chitosan (CTs) for laccase immobilization. First, HNTs were modified with Fe3O4 NPs (HNTs-Fe3O4) by the coprecipitation method. Then, the HNTs-Fe3O4 surface was tuned with the CTs (HNTs-Fe3O4-CTs) by a simple refluxing method. Finally, the HNTs- Fe3O4-CTs surface was thiolated (-SH) (denoted as; HNTs- Fe3O4-CTs-SH) by using the reactive NHS-ester reaction. The thiol-modified HNTs (HNTs- Fe3O4-CTs-SH) were characterized by FE-SEM, HR-TEM, XPS, XRD, FT-IR, and VSM analyses. The HNTs-Fe3O4-CTs-SH was applied for the laccase immobilization. It gave excellent immobilization of laccase with 100% activity recovery and 144 mg/g laccase loading capacity. The immobilized laccase on HNTs-Fe3O4-CTs-SH (HNTs-Fe3O4-CTs-S-S-Laccase) exhibited enhanced biocatalytic performance with improved thermal, storage, and pH stabilities. HNTs-Fe3O4-CTs-S-S-Laccase gave outstanding repeated cycle capability, at the end of the 15th cycle, it kept 61% of the laccase activity. Furthermore, HNTs-Fe3O4-CTs-S-S-Laccase was applied for redox-mediated removal of textile dye DR80 and pharmaceutical compound ampicillin. The obtained result marked the potential of the HNTs-Fe3O4-CTs-S-S-Laccase for the removal of hazardous pollutants. This nanosupport is based on clay mineral HNTs, made from low-cost biopolymer CTs, super-magnetic in nature, and can be applied in laccase-based decontamination of environmental pollutants. This study also gave excellent material HNTs-Fe3O4-CTs-SH for other enzyme immobilization processes.

Eggshell membrane as feedstock in enzyme immobilization

Eggshell membrane, an eco-compatible, safe and cheap by-product was employed as carrier for the laccase from Trametes versicolor immobilization. In order to evaluate the best protocol to apply for the syringic acid degradation, two different types of laccase loading on eggshell membrane were used by incubation in solution or by enzyme-dropping. Chemicals (covalent) and physicals (adsorption) immobilizations were tested for both procedure using native or periodate-oxidized laccase. It is shown that immobilization of periodate-oxidized laccase on NiCl2-pretreated eggshell membrane was the best method for the first procedure (immobilized activity 1300 U/Kg, a residual activity of 30 % for 6 reuse). For the enzyme-dropping protocol a covalent method with the bifunctional cross linker (glutaraldehyde) was the best method (immobilized activity 3500 U/Kg, a residual activity of 45 % for 6 reuse).

Chitosan-Grafted Halloysite Nanotubes-Fe3O4 Composite for Laccase-Immobilization and Sulfamethoxazole-Degradation.

A surface-engineered nano-support for enzyme laccase-immobilization was designed by grafting the surface of halloysite nanotubes (HNTs) with Fe3O4 nanoparticles and chitosan. Herein, HNTs were magnetized (HNTs-M) by a cost-effective reduction-precipitation method. The synthesized HNTs-M were grafted with 0.25%, 0.5%, 1%, and 2% chitosan (HNTs-M-chitosan), respectively. Synthesized HNTs-M-chitosan (0.25%), HNTs-M-chitosan (0.5%), HNTs-M-chitosan (1%) and HNTs-M-chitosan (2%) were linked with glutaraldehyde (GTA) for laccase immobilization. Among these formulations, HNTs-M-chitosan (1%) exhibited the highest laccase immobilization with 95.13% activity recovery and 100.12 mg/g of laccase loading. The optimized material was characterized thoroughly by scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HR-TEM), X-ray powder diffraction (XRD), thermal gravimetric analysis (TGA), and vibrating sample magnetometer (VSM) analysis. The immobilized laccase (HNTs-M-chitosan (1%)-GTA-Laccase) exhibited higher pH, temperature, and storage stabilities. The HNTs-M-chitosan (1%)-GTA-Laccase possesses excellent reusability capabilities. At the end of 10 cycles of the reusability experiment, HNTs-M-chitosan (1%)-GTA-Laccase retained 59.88% of its initial activity. The immobilized laccase was utilized for redox-mediated degradation of sulfamethoxazole (SMX), resulting in 41%, 59%, and 62% degradation of SMX in the presence of 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), guaiacol (GUA), and syringaldehyde (SA), respectively. Repeated SMX degradation (57.10% after the sixth cycle) confirmed the potential of HNTs-M-chitosan (1%)-GTA-Laccase for environmental pollutant degradation. Thus, we successfully designed chitosan-based, rapidly separable super-magnetic nanotubes for efficacious enhancement of laccase biocatalysis, which can be applied as nano-supports for other enzymes.

Functionalized hierarchical wrinkled-silica spheres for laccases immobilization

Functionalized mesoporous SiO2 are common supports for some enzymes of industrial interest, such as laccases. However, the incorporation of specific functionalities and the loading of enzymes with dimensions close to the diameters of the pores obstructs the porous system. For biotechnological applications, tailored porous supports are still needed to enhance the laccase loading. The hierarchical meso/macroporous system in wrinkled-SiO2 spheres (w-SiO2) is a suitable option to overcome this issue. Herein, (3-aminopropyl) triethoxysilane and glutaraldehyde were use as functionalizing agents for the immobilization of laccase on w-SiO2. The functionalization occurs in the mesopores of the wrinkled walls and the preservation of the macroporous entries facilitates the diffusion of the laccase inside the particle. The enzyme performance was evaluated by means of the crystal violet bleaching. The enzyme is stabilized through the imine groups provided by glutaraldehyde, allowing the retention of the activity after several reaction cycles. The bleaching can be boosted by acetosyringone, highlighting the possibility of using redox mediators to expand the range of oxidizable substrates. Understanding the effect of w-SiO2 functionalization on laccases loading and performance could be extrapolated to other enzymes with biotechnological interest that requires this type of hierarchical porous silica.

Construction and characterization of sandwich-type laccase electrode based on functionalized conducting polymers

Laccase (Lac) is a multi-active-center glycoprotein. Its heterogeneous electron transfer efficiency depends on the distance between the substrate binding site and the electrode surface. Laccase encapsulation by conducting polymers should be favorable for the direct electron transfer (DET) process, thus improving the electrocatalytic efficiency of the laccase electrode. Here, a sandwich-type laccase electrode based on functionalized conducting polymers was developed. First, porous poly (3-aminophenylboronic acid) (PAPBA) conductive film was electrodeposited on a glassy carbon electrode (GCE), and on which laccase was then immobilized via the specific binding of the boronic acid group from PAPBA and the glycosyl group on laccase. In addition to higher laccase loading, the present Lac/PAPBA/GCE electrode is more robust than that prepared by physical adsorption of laccase on conducting polymer. For laccase to be wired better, a poly (3,4-ethylenedioxythiophene) (PEDOT) conductive film with polystyrenesulfonate anion (PSS−) as dopant was electrochemically covered on the Lac/PAPBA/GCE. In addition to the compatibility among the stability of the cyclic boronic acid ester, the biological activity of the laccase, and the polymer conductivity, the sandwich-type laccase electrode was demonstrated to be more efficient than the counterpart without the second layer for the electroreduction of O2 probably due to a so-called “cage effect”. The "cage effect" of the sandwich-type conducting polymers reduces the requirement for laccase orientation and greatly improves the electrocatalytic performance of the laccase electrode.

Layer-by-layer assembly based low pressure biocatalytic nanofiltration membranes for micropollutants removal

Biocatalytic nanofiltration (NF) membranes incorporated with enzymes show high capacity for micropollutants (MPs) removal. However, there remains significant challenges such as the lack of molecular-level tailoring for skin layer design and effective strategy for enzyme immobilization. In this work, layer-by-layer (LBL) assembly based biocatalytic NF membranes were fabricated for bisphenol (BPA) removal by immobilizing laccase into the skin layer during the LBL polyelectrolytes assembly with controlled crosslinking and immobilization. This strategy enables simultaneous enzyme immobilization and NF skin layer formation. Three laccase immobilization strategies (i.e., post immobilization, post crosslinking, and post crosslinking and immobilization) on skin layer were explored to prepare NF membrane for evaluating BPA removal efficiency. The post immobilization was identified as the optimal strategy, which endowed the biocatalytic NF membrane with a pure water permeability of 10.9 ± 0.4 LMH/bar and MgCl2 rejection of 97.2 ± 0.3% under 2 bar pressure, alongside competitive laccase loading (238.8 ± 3.5 μg/cm2) and laccase activity (0.6 U/cm2). The optimal biocatalytic NF membrane exhibited an improvement in BPA removal of 79.5% under an incubation mode and 92.5% under a full recycling mode. The removal efficiencies were ~240% higher than that of the unmodified LBL membrane and clearly comparable to other reported biocatalytic membranes. This performance was attributed to the synergistic effect of membrane rejection, adsorption and laccase catalysis. The optimal biocatalytic NF membrane was found to be robust after six cycles within 14 days, while maintaining a relatively high BPA removal efficiency and salt rejection. Overall, our results open up a new avenue for enzyme immobilization into the skin layer of membranes for designing high-efficient biocatalytic NF membranes for MPs removal.

Biodegradation of Cibacron Blue 3GA by insolubilized laccase and identification of enzymatic byproduct using MALDI-ToF-MS: Toxicity assessment studies by Daphnia magna and Chlorella vulgaris

The presented paper describes a detailed study on the use of immobilized laccase for effective degradation of Cibacron Blue 3GA dye. The amount of laccase loading on the cyclic carbonate groups containing poly(hydroxyethyl methacrylate-co-vinylene carbonate), p(HEMA-co-VC), microbeads was 27.8 mg g−1, and the retained immobilized enzyme activity was 73% compared to free enzyme. The toxicity of the dye and its byproducts were studied using Daphnia magna as test organism. The micro-algal growth inhibition was also studied using a green micro algae "Chlorella vulgaris". MALDI-ToF-MS was used to verify dye degradation byproducts. After 60 min of incubation period, Cibacron Blue 3GA (CB3GA) and its byproducts disappeared from the medium. After 60-min enzymatic treatment, the non-toxic nature of medium was confirmed by toxicity studies. On the other hand, the initial byproducts of the dye seemed to be more toxic than the later formed dye products. It should be noted that the information obtained from this study can be beneficial for understanding the initial degradation byproducts toxicities of the enzymatically treated dyes to provide information about environmental protection.

Biomimetic dynamic membrane for aquatic dye removal

This study utilized physical adsorption and filtration of carbon nanotubes (CNTs) and laccases to fabricate biomimetic dynamic membrane (BDM) for the advanced treatment of dye wastewater. In BDM, the adsorption, enzymatic degradation and membrane separation demonstrated a synergism effect on pollutant removal. At first, the fabrication methods of BDM were investigated, and the mixed filtration for laccases and CNTs showed a better performance than the stepwise filtration. Furthermore, the operation parameters of BDM, including CNTs and laccase loading amounts, dye concentration, agitation speed and transmembrane pressure (TMP), were studied. Suitable CNTs and laccase amounts could reduce filtration resistance and increase catalysis efficiency, while moderate TMP and agitation speed were in favor of boosting the BDM structure for catalysis and permeability. Optimized operation parameters (CNT loading amount = 20 g m−2, laccase loading amount = 74.6 g m−2, agitation speed = 100 rpm, and TMP = 1.0 bar) sustained a high removal rate, and the flux was over 120 L m−2 h−1, even for 7 operation cycle’ tests. BDM exhibited an excellent dye removal rate, stable flux and great antifouling capacity, on the ground that adsorption saturation and foulant may be alleviated “online and in-situ” by the enzymatic degradation. Afterwards, the bionic layer on BDM, after absorption saturation and catalyst deactivation, could be eliminated rapidly by carrying out a simple backwash cleaning operation, then a new one could be fabricated immediately. Therefore, BDM is a good candidate for functional membrane materials in future water treatment.

Covalently immobilized laccase onto graphene oxide nanosheets: Preparation, characterization, and biodegradation of azo dyes in colored wastewater

In this study, graphene oxide (GO) was synthesized via modified Hummer's method and exploited as an ideal enzyme immobilization support due to its exclusive chemical and structural features. Then, laccase from genetically modified Aspergillus was covalently immobilized onto GO (nanobiocatalyst). Enzymatic characterization of the nanobiocatalyst exhibited promising results: laccase loading of 156.5 mg g−1 and immobilization yield of 64.6% at laccase concentration of 0.9 mg/ mL. Further employment of various structural characterization techniques including Fourier Transform Infrared Spectroscopy (FTIR), X-ray Powder Diffraction (XRD), Scanning Electron Microscopy (SEM), Thermo-Gravimetric Analysis (TGA), and Transmission Electron Microscopy (TEM) comprehensively confirmed the morphological properties of the prepared nanomaterials. Furthermore, bioconversion of two anionic dyes (i.e. Direct Red 23 (DR23) and Acid Blue 92 (AB92)) using nanobiocatalyst, was investigated and optimized. Also, the average decolorization effectiveness of the nanobiocatalyst was more than 75% for both of dyes after six cycles, implies its excellent operational stability and good reusability.

A L-proline/O2 biofuel cell using L-proline dehydrogenase (LPDH) from Aeropyrum pernix.

To utilize amino acids from food waste as an energy source, L-proline/O2 biofuel cell was constructed using a stable enzyme from hyperthermophilic archaeon for long-term operation. On the anode, the electrocatalytic oxidation of L-proline by L-proline dehydrogenase from Aeropyrum pernix was observed in the presence of ferrocenecarboxylic acid as mediator. On the cathode, electrocatalytic oxygen reduction was detected. Ketjenblack modification of carbon cloth substrate increased the current density due to increased laccase loading and enhanced electron transfer reaction. The biofuel cell using these electrodes achieved a current density of 6.00µA/cm2. We successfully constructed the first biofuel cell that generates power from L-proline.