Covalent 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.

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.

Covalent immobilization of laccase on Fe3O4–graphene oxide nanocomposite for biodegradation of phenolic compounds

Covalent immobilization of laccase on Fe3O4–graphene oxide nanocomposite for biodegradation of phenolic compounds

Novel fabrication of a laccase biosensor to detect phenolic compounds using a carboxylated multiwalled carbon nanotube on the electropolymerized support

Biosensors research is one of the fastest growing fields in which tens of thousands of papers have been published over the years; even more, numerous biosensors have been developed for the detection of phenolic compounds, such as catechol which reacts with an appropriate enzymatic bioreceptor like laccase. A biosensing electrode for catechol detection was investigated by covalent immobilization of laccase on a glassy carbon electrode modified by conducting polymers built of poly(3,4-ethylenedioxythiophene), gold nanoparticles and carboxylated multiwalled carbon nanotubes. The fabrication process of the sensing surface was investigated by Fourier transform infrared spectroscopy, scanning electron microscopy and electrochemical procedures. The electrochemical results demonstrate that the enzyme was immobilized covalently onto the modified glassy carbon electrode by the interaction between carboxyl groups of the carboxylated multiwalled carbon nanotubes and laccase. The biosensor demonstrates a direct electron transfer between the electrode and immobilized laccase. Under optimum conditions, it presented two linear responses in the range of 0.1–0.5 and 11.99–$$94.11\,\upmu \hbox {M}$$. The limits of detection were found to be 0.11 and $$12.26\,\upmu \hbox {M}$$.

Covalent immobilization of laccase by one pot three component reaction and its application in the decolorization of textile dyes

In present study, laccase from Myceliophthora thermophila, was immobilized on epoxy-functionalized silica via one-pot three component reaction as a novel and efficient method for immobilization. The results revealed immobilization of 50 mg of M. thermophila laccase on 1 g of the supports in presence of cyclohexyl isocyanide after 12 h of incubation. The immobilized enzyme exhibited notable activity (50 U/g) with improved stability toward pH, temperature and organic solvents. Laccase derivative was used for decolorization of five textile dyes (acid orange 156, acid red 52, coomassie brilliant blue, methyl violet, malachite green) with or without the redox mediators such as 1-hydroxybenzotriazole (HBT), catechol, syringaldehyde and N-hydroxyphthalimide (HPT). The results showed that laccase/mediator systems were effective biocatalysts for the treatment of textile dyes.

Covalent immobilization of laccase on citric acid functionalized micro-biochars derived from different feedstock and removal of diclofenac

Immobilization of enzymes on the solid supports can improve the stability as well as catalytic properties of enzymes. In this study, biochar derived from various feedstocks were used as immobilization support considering biochars carbon negative as well as sustainable properties. Partially purified (concentrated) crude laccase was covalently immobilized onto pine wood (BC-PW), pig manure (BC-PM) and almond shell (BC-AS) micro-biochars using optimized 5% w/v glutaraldehyde. Moreover, citric acid pretreatment improved the laccase binding capacity of all the micro-biochars and the highest laccase binding of 40.2 ± 1.8 U g−1 was observed with BC-PM in comparison with raw BC-PM (34.1 ± 1.1 U g−1). The enhanced binding of laccase on BC-PM over wood derived biochars was attributed to the higher surface area (46.1 m2 g−1) of BC-PM. Moreover, feedstock selection as well as a method of production influenced the biochar physicochemical properties such as surface area and consequently, different biochars showed various laccase immobilization efficiencies. BC-PW showed better pH, thermal, storage, and operational stability, compared with BC-AS and BC-PM. While applying the laccase bound micro-biochar, complete removal was observed in 2 h under batch mode with 0.5 g of laccase bound BC-PM at an environmentally relevant concentration of 500 µg L−1 in wastewater effluent. About 40% of the laccase activity was retained with all the laccase-bound micro-biochars after 5 cycles of diclofenac treatment.

Development of a Superparamagnetic Laccase Nanobiocatalyst for the Enzymatic Biotransformation of Xenobiotics

The development of nanotechnology has provided a range of diverse nanoscale carriers that can be potentially applied for enzyme immobilization. Among the different types of support, magnetic nanoparticles (mNPs) have received increasing attention because they can be easily recovered from the reaction medium by applying an external magnetic field. In the present study, silica-coated magnetic nanoparticles (Fe3O4@SiO2) were prepared, characterized, and used for covalent immobilization of laccase from Trametes versicolor. Different concentrations of the functionalization agent (3-aminopropyltriethoxysilane), cross-linker (glutaraldehyde), and laccase allowed a maximum enzyme loading of 2.68±0.08 U mg−1 mNP. Several factors, including pH, T, presence of inactivating compounds, enzyme stability, and reusability of the support, were evaluated. The oxidative action of the enzyme toward xenobiotics was proven in the formation of the chromogenic radical of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and the biotransformation of the endocrine-disrupting compound Bisphenol A (BPA). The enhancement of catalytic activity and stability of the nanobiocatalyst was evidenced by the increase of ABTS oxidation. Moreover, the superparamagnetic characteristic of the support allowed simple and fast recovery of the nanobiocatalyst. With the development of nanobiocatalyst-driven applications in mind, the development of magnetic nanoparticle synthesis at large scale as well as the design of bioreactors that assure the retention and recovery of the nanobiocatalyst need to addressed.

Covalent Immobilization of Laccase onto Nanofibrous Membrane for Degradation of Pharmaceutical Residues in Water

Enzymatic degradation with ligninolytic enzyme, e.g., laccase, is a potential green solution for removal of pharmaceutical compounds that are released into the environment through wastewater effluent. However, the deficiencies of using the enzyme in its free forms, such as reusability and stability, should be addressed before industrial applications. In this study, laccase was immobilized onto tailor-made polyacrylonitrile–biochar composite nanofibrous membrane through covalent bonding, and the parameters of immobilization were optimized. The obtained biocatalyst was utilized for removal of chlortetracycline (CTC), carbamazepine (CBZ), and diclofenac (DCF) at an environmentally relevant concentration in batch mode. These pharmaceutical compounds represented three main categories of pharmaceutical compounds, i.e., antibiotics, antidepressant, and anti-inflammatory. The results showed that the immobilized laccase has improved storage, temperature, and pH stability compared to free laccase. Also, it maintain...

Laccase-immobilized bacterial cellulose/TiO2 functionalized composite membranes: Evaluation for photo- and bio-catalytic dye degradation

Bacterial cellulose (BC) was prepared by the fermentation of Komagataeibacter xylinus. Subsequently, through site-directed surface oxidation chemistry, the hydroxyl groups of BC were successfully oxidized into aldehyde groups that served as anchors for covalent immobilization of laccase (Lac) to the newly developed oxidized BC (OBC) membrane. TiO2 was additionally co-immobilized to OBC to produce a novel material in which dye degradation was carried out under specific conditions. Atomic Force Microscopy (AFM) and Scanning Electron Microscope (SEM) confirmed the installation of both TiO2 and laccase on the surface of OBC nanofiber membrane. The optimum pH, temperature, thermal stability, operational stability of the OBC/Lac and OBC/TiO2-Lac membrane were also studied in detail. In addition, the effect of the temperature and pH on dye degradation was also investigated. The results showed that the oxidation process successfully introduced aldehyde groups onto the BC (FT-IR), and also improved the stability of the immobilized laccase. Compared with free laccase, the optimum pH of immobilized laccase shifted to lower pH, while the optimum temperature decreased from 55°C to 50°C. The dye degradation experiments showed that the optimum pH for dye degradation was 5.0–6.0, while the optimum temperature was ~40°C. Under UV illumination, the dye degradation efficiency was significantly improved. Therefore, functionalized composite bacterial cellulose nanofiber membranes with a combined bio- and photo- catalytic property are a potentially valid approach for industrial textile dye degradation.

Laccase-Functionalized Graphene Oxide Assemblies as Efficient Nanobiocatalysts for Oxidation Reactions.

Multi-layer graphene oxide-enzyme nanoassemblies were prepared through the multi-point covalent immobilization of laccase from Trametes versicolor (TvL) on functionalized graphene oxide (fGO). The catalytic properties of the fGO-TvL nanoassemblies were found to depend on the number of the graphene oxide-enzyme layers present in the nanostructure. The fGO-TvL nanoassemblies exhibit an enhanced thermal stability at 60 °C, as demonstrated by a 4.7-fold higher activity as compared to the free enzyme. The multi-layer graphene oxide-enzyme nanoassemblies can efficiently catalyze the oxidation of anthracene, as well as the decolorization of an industrial dye, pinacyanol chloride. These materials retained almost completely their decolorization activity after five reaction cycles, proving their potential as efficient nano- biocatalysts for various applications.

Reduced graphene oxide-Sb2O5 hybrid nanomaterial for the design of a laccase-based amperometric biosensor for estriol

A novel reduced graphene oxide/Sb2O5 hybrid nanomaterial was prepared by a one-pot reaction process. The nanomaterial was characterized by different spectroscopic, microscopic and electrochemical techniques, demonstrating that the graphene sheets were doped with a Sb2O5 thin film. A glassy carbon electrode coated with the hybrid material was further employed as support for the covalent immobilization of laccase to develop an electrochemical biosensor for estriol. The enzyme biosensor showed high sensitivity (275mA/M) and fast analytical response (4s) for the hormone, with a limit of detection of 11nM in the range of 25nM to 1.03μM. The biosensor showed high selectivity for the analytical detection of estriol.

Laccase immobilization on the electrode surface to design a biosensor for the detection of phenolic compound such as catechol

Biosensors based on the coupling of a biological entity with a suitable transducer offer an effective route to detect phenolic compounds. Phenol and phenolic compounds are among the most toxic environmental pollutants. Laccases are multi-copper oxidases that can oxide phenol and phenolic compounds. A method is described for construction of an electrochemical biosensor to detect phenolic compounds based on covalent immobilization of laccase (Lac) onto polyaniline (PANI) electrodeposited onto a glassy carbon (GC) electrode via glutaraldehyde coupling. The modified electrode was characterized by voltammetry, Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM) techniques. The results indicated that laccase was immobilized onto modified GC electrode by the covalent interaction between laccase and terminal functional groups of the glutaraldehyde. The laccase immobilized modified electrode showed a direct electron transfer reaction between laccase and the electrode. Linear range, sensitivity, and detection limit for this biosensor were 3.2×10−6 to 19.6×10−6M, 706.7mALmol−1, 2.07×10−6M, respectively.

Covalent immobilization of laccase in green coconut fiber and use in clarification of apple juice

Waste materials from agroindustry constituted from lignocellulose have been used in the immobilization process of technological interest enzymes to reduce costs, increasing the value of such products. In the present study, green coconut husk was used to obtain fibers (CF) that were treated through thermal decompression in combination with either acid or alkaline medium. A solid support for enzyme immobilization was prepared using the pretreated CF activated with glyoxyl or glutaraldehyde and was used to immobilize the laccase enzyme (EC 1.10.3.2) produced by Trametes versicolor. Immobilized enzyme retained up to 59±1% of the initial activity and showed maximum immobilization profile of 98±1%. The thermal stability was higher when laccase was immobilized on alkaline pretreated support with increments of 6.8-fold (laccase-glutaraldehyde-FC) up to 16.5-fold (laccase-glyoxyl-FC) of the soluble enzyme. The laccase-glutaraldehyde-CF achieved excellent results in the clarification of apple juice, reducing 61±1% of the original juice color and 29±1% of its turbidity, retaining up to 100% of the initial activity after a 10-times reuse assay. This study is pioneer in the utilization of low-cost support for laccase immobilization with application in the juice fruits clarification.

Reversible covalent immobilization of Trametes villosa laccase onto thiolsulfinate-agarose: An insoluble biocatalyst with potential for decoloring recalcitrant dyes.

The development of a solid-phase biocatalyst based on the reversible covalent immobilization of laccase onto thiol-reactive supports (thiolsulfinate-agarose [TSI-agarose]) was performed. To achieve this goal, laccase-producing strains isolated from Eucalyptus globulus were screened and white rot fungus Trametes villosa was selected as the best strain for enzyme production. Reduction of disulfide bonds and introduction of "de novo" thiol groups in partially purified laccase were assessed to perform its reversible covalent immobilization onto thiol-reactive supports (TSI-agarose). Only the thiolation process dramatically improved the immobilization yield, from 0% for the native and reduced enzyme to 60% for the thiolated enzyme. Mild conditions for the immobilization process (pH 7.5 and 4°C) allowed the achievement of nearly 100% of coupling efficiency when low loads were applied. The kinetic parameters, pH, and thermal stabilities for the immobilized biocatalyst were similar to those for the native enzyme. After the first use and three consecutives reuses, the insoluble derivative kept more than 80% of its initial capacity for decolorizing Remazol Brilliant Blue R, showing its suitability for color removal from textile industrial effluents. The possibility of reusing the support was demonstrated by the reversibility of enzyme-support binding.

Development of a nanocomposite system and its application in biosensors construction

Abstract The present work describes the development of a nanocomposite system and its application in construction of a new amperometric biosensor applied in the determination of total polyphenolic content from propolis extracts. The nanocomposite system was based on covalent immobilization of laccase on functionalized indium tin oxide nanoparticles and it was morphologically and structural characterized. The casting of the developed nanocomposite system on the surface of a screen-printed electrode was used for biosensor fabrication. The analytical performance characteristics of the settled biosensor were determined for rosmarinic acid, caffeic acid and catechol (as laccase specific substrate). The linearity was obtained in the range of 1.06×10−6 − 1.50×10−5 mol L−1 for rosmarinic acid, 1.90×10−7 − 2.80×10−6 mol L−1 for caffeic acid and 1.66×10−6 − 7.00×10−6 mol L−1 for catechol. A good sensitivity of amperometric biosensor 141.15 nA µmol−1 L−1 and fair detection limit 7.08×10−8 mol L−1 were obtained for caffeic acid. The results obtained for polyphenolic content of propolis extracts were compared with the chromatographic data obtained by liquid-chromatography with diode array detection.

Direct electron transfer reaction of laccase on a glassy carbon electrode modified with 1-aminopyrene functionalized reduced graphene oxide

A biosensor was developed based on the covalent immobilization of laccase (Lac) on 1-aminopyrene (1-AP)-functionalized reduced graphene oxide (rGO)-modified glassy carbon electrode (GCE) by encapsulation with chitosan. X-ray photoelectron spectroscopy and cyclic voltammetry were used to optimize the mass ratio between 1-AP and rGO (rAP/rGO) to be 1/8. Direct electron transfer behavior of Lac immobilized on 1-AP-functionalized rGO (AP-rGO) was investigated by cyclic voltammogram. Results indicate that the AP-rGO is a friendly platform for the immobilization of enzyme, which facilitates the electron exchange between Lac and the modified electrode surface. On AP-rGO/GCE, Lac displayed rapid electron transfer with one participating proton. Furthermore, it was also confirmed that Lac immobilized on the modified electrode exhibited the electrocatalytic response to an established unspecific Lac substrate, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate)diammonium salt (ABTS). Without the aid of an electron mediator between Lac and modified electrode, the enzyme-based biosensor shows good reproducibility and high sensitivity to ABTS with a detection limit of 0.1 μM (S/N = 3). Within the concentration of 2–1000 μM, a linear relationship was achieved between the current intensity of the electro-catalytic oxidation of ABTS and the concentration of ABTS. The proposed biosensor achieved 95% of steady-state reduction currents within 5 s.

An amperometric biosensor based on laccase immobilized onto nickel nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode for determination of phenolic content in fruit juices

A method is described for construction of an enzyme electrode for detection of phenolic compounds based on covalent immobilization of laccase onto nickel nanoparticles (NiNPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto gold (Au) electrode. The modified electrode was characterized at different stages of its construction by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammograms and electrochemical impedance spectroscopy (EIS). An amperometric biosensor for phenolic compounds was fabricated by connecting enzyme electrode (Lac/NiNPs/cMWCNTs/PANI/AuE) as working electrode, with Ag/AgCl as reference electrode and Pt wire as auxiliary electrode through potentiostat. The biosensor showed optimum response at pH 5.5 (0.1M acetate buffer) and 35°C, when operated at a scan rate of 20mVs−1. Linear range, response time, detection limit and sensitivity of biosensor were 0.1–10μM (lower concentration range) and 10–500μM (higher concentration range), 8s, 0.05μM and 0.694μAμM−1cm−2 respectively. The biosensor measured total phenolic content in fruit juices. The enzyme electrode was used 200 times over a period of four months, when stored at 4°C.

An amperometric biosensor based on laccase immobilized onto Fe3O4NPs/cMWCNT/PANI/Au electrode for determination of phenolic content in tea leaves extract

A method is described for the construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase onto iron oxide nanoparticles (Fe3O4NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto a gold (Au) electrode. The modified electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response within 3s at pH 6.0 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, detection limit were 0.1–10μM (lower concentration range) and 10–500μM (higher concentration range), and 0.03μM respectively. The sensor measured total phenolic content in tea leaves extract. The enzyme electrode lost 25% of its initial activity after its 150 uses over a period of 4 months, when stored at 4°C.

An amperometric biosensor based on laccase immobilized onto MnO2NPs/cMWCNT/PANI modified Au electrode

A method is described for construction of an amperometric biosensor for detection of phenolic compounds based on covalent immobilization of laccase (Lac) onto manganese dioxide nanoparticles (MnO2NPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/PANI composite electrodeposited onto a gold (Au) electrode through N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry. The modified electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The biosensor showed optimum response at pH 5.5 (0.1M sodium acetate buffer) and 35°C, when operated at 0.3V vs. Ag/AgCl. Linear range, response time, detection limit were 0.1–10μM (lower concentration range) and 10–500μM (higher concentration range), 4s and 0.04μM, respectively. Biosensor measured total phenolic content in tea leaves extract. The enzyme electrode was used 150 times over a period of 5months.

Amine functional monodisperse microbeads via precipitation polymerization of N-vinyl formamide: Immobilized laccase for benzidine based dyes degradation

Densely cross-linked poly(vinylamine) microbeads (∼2 μm) were prepared by precipitation copolymerization of N-vinyl formamide and ethylene glycoldimethacrylate in acetonitrile. The formamido groups of the microbeads were hydrolyzed into amino groups. Then, amino-functionalized microbeads were used for covalent immobilization of laccase via glutaraldehyde coupling. The average amount of immobilized enzyme was 18.7 mg/g microbeads. Kinetic parameters, V max and K m values were determined as 20.7 U/mg protein and 2.76 × 10 −2 mmol/L for free enzyme and 15.8 U/mg protein and 4.65 mmol/L for the immobilized laccase, respectively. The immobilized laccase was operated in a batch reactor for the degradation of two different benzidine based dyes (i.e., Direct Blue 1 and Direct Red 128). The laccase immobilized on the microbeads was very effective for removal of these dyes which interfere with the hormonal system.