Free Laccase Research Articles

A pilot study for enhanced transformation of a metabolite 3,5-dichloroaniline derived from dicarboximide fungicides through immobilized laccase mediator system.

This pilot investigation aimed to evaluate the removal efficiency and the underlying biocatalytic pathways of immobilized fungal laccase during the oxidative biotransformation of a non-phenolic metabolite, 3,5-dichloroaniline (3,5-DCA) derived from dicarboximide fungicides. The maximum loading of laccase on the microporous support surfaces could reach 36.4mg/g. The immobilized laccase on the microporous support surfaces exhibited excellent thermal stability, pH adaptability, storage stability, and reusability compared to free laccase. The ILMS assay indicated that the immobilized laccase efficiently removed studied 3,5-DCA (99-100%) in the aqueous medium, within 72h in the presence of catechol. In this study, we identified three coupling reaction products during the removal of 3,5-DCA through an ILMS assay. Based on the identified coupling reaction products, we proposed the reaction pathway for the biotransformation of 3,5-DCA by immobilized laccase, which was shown to be potentially useful in the sustainable environmental remediation of aniline metabolite (i.e., 3,5-DCA) derived from dicarboximide fungicides.

Preparation optimization and stability comparison study of alkali-modified biochar immobilized laccase under multi-immobilization methods

Alkali modified biochar has attracted many interests as a potential enzyme immobilization carrier. In this paper, the preparation of alkali-modified immobilized laccases under multi-immobilization methods (adsorption: A/lac@A-MB; adsorption-crosslinking: A-C/lac@A-MB; covalent bonding: CB/lac@A-MB) were optimized via Design-Expert for the first time, and the stability of alkali-modified immobilized laccases was compared with other literatures. In addition, SEM, BET, FT-IR and XRD analyses were used to characterize the prepared immobilized enzyme samples. The results showed that the three kinds of immobilized laccases have improved the stability to different degrees relative to free laccase. In the pH range of 3–7, temperature range of 25–55 °C, and storage for more than 30 days, after 5 reaction cycles, the relative activity of the laccase immobilized on three different supports can still be greater than 50%. In addition, CB/lac@A-MB obtained the highest Vmax (0.94 U/mg), specific activity (0.66 U/mg) and activity recovery (66.20%); A-C/lac@A-MB obtained the highest immobilization yield (67.40%) and enzyme loading (180.81 mg/g); and A/lac@A-MB obtained the lowest Km of 140 μM. The above results are comparable to other reports, aiming to provide a reference for practical application of different methods of immobilized laccase in the industrial field.

Fast anisotropic growth of the biomineralized zinc phosphate nanocrystals for a facile and instant construction of laccase@Zn3(PO4)2 hybrid nanoflowers

Organic-inorganic hybrid nanoflowers (HNFs) of laccase@Zn3(PO4)2 were fabricated through a facile, simple, and rapid one-step strategy. In this process, laccase was involved in nucleation and fast anisotropic growth reactions with Zn (II) and phosphate ions. The average pore size of the prepared HNFs was 54.5 nm, and its BET-specific surface area was 59.5 m2 g−1. In comparison with the free laccase, the entrapped enzyme activity in the constructed HNFs was 86.4%. In addition, the hybrid biocatalyst displayed a maximum rate of reaction (Vmax) of 1640.2 ± 3.6 μmol min−1 with respect to the native enzyme. The constructed HNFs maintained 45.1% and 60% of the original laccase activity after 12 successive reusability cycles and 30 days of storage at 4 °C, respectively. The as-obtained HNFs demonstrated a high bioremoval percentage of Direct blue-71 (94.1%) within a 10-h-treatment at 40 °C and 15 mg l−1 of the dye concentration. The pseudo-first order and second order were the best-fitted kinetic models for the dye removal using Zn3(PO4)2 nanoflakes and the fabricated HNFs, respectively. Besides, liquid chromatography-mass spectrometry (LC-MS) revealed biotransformation of the dye into less toxic metabolites as verified by testing on some bacterial strains.

Insolubilization of Tramates versicolor laccase as cross-linked enzyme aggregates for the remediation of trace organic contaminants from municipal wastewater

The novelty of this study deals with the biocatalytic treatment of trace organic contaminants (TrOCs) from municipal wastewater by insolubilized laccase. Laccase from Trametes versicolor was aggregated by three-phase partitioning technique followed by cross-linking with glutaraldehyde to produce insolubilized laccase as cross-linked enzyme aggregates (CLEAs). The optimal conditions for CLEAs preparation include ammonium sulphate concentration of 83% (w/v), crude to t-butanol ratio of 1.00: 1.05 (v/v), pH 5.3, and glutaraldehyde concentration of 20 mM obtained via statistical design. The efficiency of insolubilization of the CLEAs laccase based on the kcat/km ratio was approximately 4.8-fold greater than that of free laccase. The developed CLEAs showed greater resistance to product inhibition mediated by ABTS than the free enzyme and exhibited excellent catalytic activity even after the tenth successive cycle. Further, free laccase and the synthesized CLEAs laccase were utilized to treat five analgesics, two NSAIDS, three antibiotics, two antilipemics, and three pesticides in the municipal wastewater. Under the batch process with operating conditions of pH 7.0 and 20 °C, 1000 U/L of CLEAs, laccase removed 11 TrOCs in the range of about 20–99%. However, the inactivated CLEAs only adsorbed 2–25% of TrOCs. It was observed that acetaminophen, mefenamic acid, trimethoprim, and metolachlor depicted almost complete removal with CLEAs laccase. The performance of CLEAs laccase in a perfusion basket reactor was tested for the removal of TrOCs from municipal wastewater.

Optimized degradation of bisphenol A by immobilized laccase from Trametes versicolor using Box-Behnken design (BBD) and artificial neural network (ANN)

This study represents one of the first attempts towards bisphenol A (BPA) degradation by Trametes versicolor laccase immobilized on Ba-alginate beads. The effect of input variables including temperature (20–40 °C), BPA concentration (2–10 mg/L) and time (10–90 min) were studied using Box-Behnken design (BBD) and artificial neural network (ANN). The maximum BPA degradation of 84.34% was obtained when the temperature was 40 °C, BPA concentration 2 mg/L and time 50 min, which was accurately predicted by BBD (83.48%) and ANN (84.33%), proving the accuracy of prediction for both models. The values of R2 and MSE for BBD were found to be 0.98, 9.88, while for ANN were 0.97, 38.25, respectively. Based on higher R2 value and lower MSE, BBD was slightly better than ANN. The immobilized laccase showed higher storage stability than free laccase by retaining 68.64% and 44.62% of their activity at the same experimental conditions. Furthermore, BPA transformation was confirmed by Fourier-transform infrared spectroscopy (FTIR) analysis. GC-MS had detected the oxidative degradation products from BPA. Results showed that Ba-alginate immobilized laccase could be a promising biocatalyst in treating organic pollutants.

Printing toner used as carrier for immobilization of laccase

The search for seeking novel immobilized enzyme carriers is a hot topic in the field of enzyme engineering . As a kind of common office consumable, magnetic printing toner has been terribly wasted, leading to potential harm to the environment. We try to use printing toner as carrier for enzyme immobilization to realize waste reuse. In this paper, the laccase extracted from Agaricus bisporus was successfully immobilized on chitosan coated magnetic printing toner by glutaraldehyde linking. The use of printing toner is proved to be feasible. The results show that the optimal temperature of immobilized laccase is 40 °C, and the optimal pH is 3.0. Compared with free laccase, the optimal temperature and optimal pH have been obviously changed after immobilization. And the pH tolerance has been significantly improved, which can remain 53% activity after 5 cycles. Because of the use of magnetic printing toner, it is more convenient to recycle the immobilized enzyme. In this study, printing toner was used as carrier for the immobilization of enzyme and the discarded printing toner recycling was realized. • The immobilized enzyme carrier with printing toner as the main body was successfully constructed. • Laccase was successfully immobilized with the new carrier. • The new carrier uses discarded print toner to reduce environmental pollution.

A comparative study of free laccase and laccase immobilized in copper alginate

Enzyme immobilization has gained considerable attention due to the incredible properties exhibited by the enzymes in immobilized condition. Therefore, in the present study, a comparative analysis of free and immobilized laccase is reported using various substrates. The ideal substrate for immobilization was found to be copper alginate with an immobilization yield of 91.078%. The optimum pH was 4 and 5, respectively, for free and immobilized enzymes while the optimum temperature was found to be 50 and 60C, respectively. The kinetic parameters Vmax and Km were found from the Line weaver-Burk plot and were 48.076 U/mL and 0.480 mM for free enzyme while 55.55 U/mL and 1.277 mM for the immobilized enzyme, respectively. The catalytic efficiency Kcat was found as 100.01s1 for free enzyme and 43.5s1 for its immobilized counterpart. Out of the various metal ions used, Co2+ was found to enhance the activity of an immobilized enzymes. The storage stability of the enzyme was studied and was found that only 32.44% of initial activity was retained by free enzyme whereas, 70.21% of activity was retained by immobilized enzyme upon storage for four weeks at 4C. The thermal stability studies shows that the immobilized enzyme retained 32.60% of its initial activity and the free enzyme retained 1.14% of initial activity on exposure to 60C for 3 h. Finally, the reusability of immobilized laccase beads was evaluated by decolorization of methyl orange for five repeated cycles and a percentage decolorization of 32.04% could be retained at the fifth cycle. This study therefore, suggests copper alginate-immobilized beads to be an effective option for various applications

Recent Developments in the Immobilization of Laccase on Carbonaceous Supports for Environmental Applications - A Critical Review.

Τhe ligninolytic enzyme laccase has proved its potential for environmental applications. However, there is no documented industrial application of free laccase due to low stability, poor reusability, and high costs. Immobilization has been considered as a powerful technique to enhance laccase’s industrial potential. In this technology, appropriate support selection for laccase immobilization is a crucial step since the support could broadly affect the properties of the resulting catalyst system. Through the last decades, a large variety of inorganic, organic, and composite materials have been used in laccase immobilization. Among them, carbon-based materials have been explored as a support candidate for immobilization, due to their properties such as high porosity, high surface area, the existence of functional groups, and their highly aromatic structure. Carbon-based materials have also been used in culture media as supports, sources of nutrients, and inducers, for laccase production. This study aims to review the recent trends in laccase production, immobilization techniques, and essential support properties for enzyme immobilization. More specifically, this review analyzes and presents the significant benefits of carbon-based materials for their key role in laccase production and immobilization.

Photoenzymatic Activity of Artificial-Natural Bienzyme Applied in Biodegradation of Methylene Blue and Accelerating Polymerization of Dopamine.

Enzymes as biocatalysts have attracted extensive attention. In addition to immobilizing or encapsulating various enzymes for combating the easy loss of enzymatic activity, strengthening the enzymatic activity upon light irradiation is a challenge. To the best of our knowledge, the work of spatiotemporally modulating the catalytic activity of artificial-natural bienzymes with a near-infrared light irradiation has not been reported. Inspired by immobilized enzymes and nanozymes, herein a platinum nanozyme was synthesized; subsequently, the platinum nanozyme was grafted on the body of laccase, thus successfully obtaining the artificial-natural bienzyme. The three-dimensional structure of the artificial-natural bienzyme was greatly different from that of the immobilized enzyme or the encapsulated enzyme. The platinum nanozyme possessed excellent laccase-like activity, which was 3.7 times higher than that of laccase. Meanwhile, the coordination between the platinum nanozyme and laccase was proved. Besides, the cascaded catalysis of artificial-natural bienzyme was verified with hydrogen peroxide as a mediator. The enzymatic activities of artificial-natural bienzyme with and without near-infrared light irradiation were, respectively, 46.2 and 29.5% higher than that of free laccase. Moreover, the reversible catalytic activity of the coupled enzyme could be manipulated with and without a near-infrared light at 808 nm. As a result, the degradation rates of methylene blue catalyzed by the coupled enzyme and the platinum nanozyme were higher than that of laccase. Furthermore, accelerating polymerization of the dopamine was also demonstrated. Briefly, this facile strategy may provide a universal approach to control the catalytic activity of other natural enzymes.

A customizable 3D printed device for enzymatic removal of drugs in water

The infiltration of drugs into water is a key global issue, with pharmaceuticals being detected in all nearly aqueous systems at often alarming concentrations. Pharmaceutical contamination of environmental water supplies has been shown to negatively impact ecological equilibrium and pose a risk to human health. In this study, we design and develop a novel system for the removal of drugs from water, termed as Printzyme. The device, fabricated with stereolithography (SLA) 3D printing, immobilises laccase sourced from Trametes Versicolor within a poly(ethylene glycol) diacrylate hydrogel. We show that SLA printing is a sustainable method for enzyme entrapment under mild conditions, and measure the stability of the system when exposed to extremes of pH and temperature in comparison to free laccase. When tested for its drug removal capacity, the 3D printed device substantially degraded two dissolved drugs on the European water pollution watch list. When configured in the shape of a torus, the device effectively removed 95% of diclofenac and ethinylestradiol from aqueous solution within 24 and 2 h, respectively, more efficiently than free enzyme. Being customizable and reusable, these 3D printed devices could help to efficiently tackle the world's water pollution crisis, in a flexible, easily scalable, and cost-efficient manner.

A robust biocatalyst based on laccase immobilized superparamagnetic Fe3O4@SiO2–NH2 nanoparticles and its application for degradation of chlorophenols

The presence of chlorophenols in water and wastewater is considered a serious environmental issue. To eliminate these micropollutants, biodegradation of chlorophenols using enzyme-nanoparticle conjugated biocatalyst, is proposed as an economical and eco-friendly method. Herein, amino-functionalized superparamagnetic Fe3O4@SiO2–NH2 nanoparticles with core-shell structure were constructed as a promising carrier for immobilization of laccase from Trametes versicolor. Compared with free laccase, Fe3O4@SiO2–NH2-Laccase displayed remarkable outcomes in all major areas such as temperature and storage stabilities, and tolerance to organic solvents and metal ions. The biocatalytic performance and reusability of Fe3O4@SiO2–NH2-Laccase were evaluated for the degradation of 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in repeated cycles. Even after 10 successive reuses, the degradation rate of 2,4-DCP and 2,4,6-TCP were found to be 54.9% and 68.7%, respectively. The influences of solution pH, initial chlorophenol concentration, and temperature on the degradation rate of these two chlorophenols were evaluated. The degradation intermediate products including dimers, trimers, and tetramers of 2,4-DCP and 2,4,6-TCP were identified. Release of chloride ions was observed during the enzymatic degradation of these two chlorophenols. Based on the determination of intermediate products and released chloride ions, the degradation pathway that was involved in dehydrogenation, reactive radical intermediates formation, dechlorination, self-coupling and oligomers/polymers formation was proposed. The toxicity of these two chlorophenols and their intermediates was substantially reduced during the enzymatic degradation. The results of this study might present an alternative clean biotechnology for the remediation of 2,4-DCP and 2,4,6-TCP contaminated water matrices.

Bacterial laccase immobilized on a magnetic dialdehyde cellulose without cross-linking agents for decolorization

In the process of using cross-linking method to immobilize enzymes, cross-linker have a certain negative effect on immobilization efficiency. In this study, a magnetic dialdehyde cellulose (MDAC) was prepared by oxidizing hydroxyl groups in cellulose to aldehyde groups. The modified cellulose could be used for the immobilization of bacterial laccase without cross-linker. The morphology and structure of the MDAC–laccase complex (MDAC-L) confirmed that the laccase was immobilized on MDAC via -CHO in MDAC and -NH2 in laccase. Under conditions of pH 4 and 45 °C, the maximum loading capacity of MDAC-L reached 210.74 mg/g, and relatively high enzyme activities maintained,. Compared with free laccase and immobilized laccase with glutaraldehyde, MDAC-L exhibited a wide pH range, temperature stability, reusability, and is suitable for long-term storage at low temperature. MDAC-L also improved the tolerance of free laccase to the presence of organic solvents, inhibitors and metal ions. Furthermore, the maximum saturation magnetization of MDAC-L was 34.07 emu/g, allowing for rapid magnetic separation. The relative activity of the immobilized laccase remained about 70% after 10 cycles of reuse. In addition, MDAC-L showed good decolorization performance when applied to crystal violet. Under the conditions of 45 °C and pH 6 for 21 h, the decolorization rate can reach about 90%. This study developed a novel cross-linking method for enzyme immobilization.

Laccase producing bacteria influenced the high decolorization of textile azo dyes with advanced study

Recent year, bacterial laccases are increasing interest in the field of industry and environmental applications especially decolorization of azo dyes. In industry, the dyes are present in stable nature including chemicals and lights. Due to these defects, the novel approaches are needed to removal of dyes before discharging into the environment. Among the various technologies, biological treatment methods and their strategies are very important, because of the decolorization and detoxification. Consecutively, biological mediated dyes removal are emerged with high potential especially microbes. Microbial laccases creates up new opportunities for their commercial applications. In this study, laccases were produced from Bacillus cereus (B. Cereus) and Pseudomonas parafulva (P. parafulva) by sub merged fermentation. For immobilization, the produced laccases were subjected to purify using 80% saturated ammonium sulphate and followed by dialysis. Then, crude laccases were immobilized through copper-alginate entrapment method. The maximum immobilized enzyme activity of the immobilized laccases were shown pH 8 at 50 °C and pH 7 at 40 °C for B. Cereus and P. parafulva respectively. In contrast, the normal enzyme activity was pH 10 at 40 °C and pH 8 at 40 °C were indicated for Bacillus cereus and P. parafulva respectively. Next, the free and immobilized laccases were performed the decolorization of three azo dyes T-blue, yellow GR and orange 3R, and exhibited that the 91.69 and 89.21% of Orange 3R were completely decolorized by both the B. Cereus and P. parafulva laccases when compared with free laccases enzymes. The confirmation of decolorization was monitored by UV–vis spectroscopy and FTIR spectroscopy, which clearly confirm the changes of peaks when compared with normal laccases. Finally, we have concluded that the B. Cereus and P. parafulva laccases are very important in azo dye decolorization and these used in future biological treatment of dyeing effluents.

Synthesis of three-dimensional laccase-Cu3(PO4)2⋅3H2O microflowers via biomineralization for UV–vis epinephrine biosensing

A novel colorimetric biosensor for epinephrine (EP) was developed based on three-dimensional laccase-Cu3(PO4)2⋅3H2O microflowers (La-MFs) fabricated via biomineralization. La-MFs were constructed by self-assembly of laccase molecules with the assistance of Cu3(PO4)2⋅3H2O. The increased enzymatic activity and kinetic parameters of La-MFs could be due to the probably synergistic effect of laccase molecules and Cu2+ of Cu3(PO4)2⋅3H2O compared with free laccase. Further, the enhanced detection performance of La-MFs for EP was ascribed to the role of Cu2+ in Cu3(PO4)2⋅3H2O, which could accelerate the electron transfer among active centers of adjacent laccase molecules. La-MFs-based colorimetric biosensor exhibited wide linear detection range (0.4–400 µg mL−1) and good selectivity for EP detection. Moreover, La-MFs reserved 96.6% and 64.4% of initial activity after stored for 30 days and 5 recycles, respectively, demonstrating the satisfied storage stability and reusability of La-MFs. In addition, La-MFs-based colorimetric biosensor has favorable applicability for EP detection in human blood serum and urine samples.

Immobilization of Trametes versicolor laccase on chitosan/halloysite as a biocatalyst in the Remazol Red RR dye

In this study, the laccase obtained from Trametes versicolor was immobilized onto the chitosan(CTS)/halloysite (HNT) beads. In the immobilization step, the effects of chitosan (1–3% w/v), halloysite (0–2% w/v), glutaraldehyde (0.5–1.5% v/v) and enzyme concentrations (1–3%) on loading and immobilization efficiency were investigated. SEM, FT-IR, XRD, TGA and XPS analyses were performed to examine the structure of beads. In addition, the effects of parameters such as pH (4–10), temperature (25–55 °C), storage life on the activity of free and immobilized laccase were also investigated. The activities of free and immobilized laccase preserved 23% and 56% of its initial activity at the end of 59 days of storage. The effects of mediators such as 2.2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 1-Hydroxybenzotriazole hydrate (HBT), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO) and violuric acid (VLA) on the dye removal efficiency were investigated. Reusability of the CTS/HNT/Lac in the presence of HBT and VLA mediators, which enable the highest dye removal, was tested. After 15 cycles, 42% and 54% dye removal were achieved with the CTS/HNT/Lac in the medium containing HBT and VLA, and 42% and 49% of the activity is preserved, respectively. This study showed that CTS/HNT/Lac can be used repeatedly for Remazol Red RR dye removal.

The Biocatalytic Degradation of Organic Dyes Using Laccase Immobilized Magnetic Nanoparticles

Free laccase has limitations for its use in industrial applications that require laccase immobilization on proper support, to improve its catalytic activity. Herein, the nanoparticles of magnetic iron oxide (Fe3O4) and copper ferrite (CuFe2O4) were successfully used as support for the immobilization of free laccase, using glutaraldehyde as a cross-linker. The immobilization conditions of laccase on the surface of nanoparticles were optimized to reach the maximum activity of the immobilized enzyme. The synthesized free nanoparticles and the nanoparticle-immobilized laccase were characterized using different techniques, including X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), vibrating sample magnetometer (VSM), and thermogravimetric analysis (TGA). CuFe2O4 nanoparticles, as support, enhanced laccase activity compared to free laccase and Fe3O4 nanoparticle-immobilized laccase that appeared during the study of pH, temperature, and storage stability on free and immobilized laccase. The CuFe2O4 and Fe3O4 nanoparticle-immobilized laccase showed superior activity in a wide pH range, temperature range, and storage period, up to 20 days at 4.0 °C, when compared to free laccase. Additionally, the synthesized nanobiocatalysts were examined and optimized for the biodegradation of the anionic dye Direct Red 23 (DR23). HPLC analysis was used to confirm the dye degradation. The reusability of immobilized laccases for the biodegradation of DR23 dye was investigated for up to six successive cycles, with a decolorization efficiency over 70.0%, which indicated good reusability and excellent stability.

Facile synthesis of recyclable laccase-mineral hybrid complexes with enhanced activity and stability for biodegradation of Evans Blue dye

Two morphologies of laccase-mineral hybrid complexes, i.e., laccase-mineral hybrid nanoflowers (La-HNF) and nanopetals (La-HNP), were synthesized via biomineralization using Cu3 (PO4)2·3H2O as the mineral for Evans Blue (EB) dye biodegradation. XRD patterns and FT-IR spectra results revealed the successful immobilization of laccase via in-situ formed Cu3(PO4)2·3H2O crystals. Compared with free laccase, laccase-mineral hybrid complexes showed higher enzymatic activity due to the activation effect induced by copper ions of Cu3(PO4)2·3H2O, further, the improved kinetic parameters of laccase-mineral hybrid complexes could be ascribed to nanoscale-dispersed laccase molecules within hybrid complexes. For EB dye biodegradation, the reason why the biodegradation efficiency (94.9%) of La-HNF was higher than that (86.8%) of La-HNP could be synergistic effect of immobilized laccase within 3D hierarchical structure of La-HNF. In addition, the optimized biodegradation conditions (pH 4.6 and 40 °C) of La-HNF were obtained, moreover, 93.2% and 48.1% of EB dye were biodegraded by La-HNF after stored for 30 days and reused for 10 cycles, respectively, demonstrating La-HNF have good practicability.

The study of laccase immobilization optimization and stability improvement on CTAB-KOH modified biochar

BackgroundAlthough laccase has a good catalytic oxidation ability, free laccase shows a poor stability. Enzyme immobilization is a common method to improve enzyme stability and endow the enzyme with reusability. Adsorption is the simplest and common method. Modified biochar has attracted great attention due to its excellent performance.ResultsIn this paper, cetyltrimethylammonium bromide (CTAB)-KOH modified biochar (CKMB) was used to immobilize laccase by adsorption method (laccase@CKMB). Based on the results of the single-factor experiments, the optimal loading conditions of laccase@CKMB were studied with the assistance of Design-Expert 12 and response surface methods. The predicted optimal experimental conditions were laccase dosage 1.78 mg/mL, pH 3.1 and 312 K. Under these conditions, the activity recovery of laccase@CKMB was the highest, reaching 61.78%. Then, the CKMB and laccase@CKMB were characterized by TGA, FT-IR, XRD, BET and SEM, and the results showed that laccase could be well immobilized on CKMB, the maximum enzyme loading could reach 57.5 mg/g. Compared to free laccase, the storage and pH stability of laccase@CKMB was improved greatly. The laccase@CKMB retained about 40% of relative activity (4 °C, 30 days) and more than 50% of relative activity at pH 2.0–6.0. In addition, the laccase@CKMB indicated the reusability up to 6 reaction cycles while retaining 45.1% of relative activity. Moreover, the thermal deactivation kinetic studies of laccase@CKMB showed a lower k value (0.00275 min− 1) and higher t1/2 values (252.0 min) than the k value (0.00573 min− 1) and t1/2 values (121.0 min) of free laccase.ConclusionsWe explored scientific and reasonable immobilization conditions of laccase@CKMB, and the laccase@CKMB possessed relatively better stabilities, which gave the immobilization of laccase on this cheap and easily available carrier material the possibility of industrial applications.

Preparation and characterization of oxidized Multi-walled Carbon Nanotubes-Immobilized Aspergillus sp. Laccase Hybrid Materials

This work deals with preparation and characterization of immobilized laccase (Aspergillus sp.) over oxidized multi-walled carbon nanotubes (ox-MWCNTs) via simple mixing technique. The resulting materials were characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), transmission electron microscope (TEM) and particle size distribution analysis using dynamic light scattering technique (DLS). The results showed that the TEM images exhibited more separate individual carbon bundles with particle size around of 396 nm after enzyme immobilization rather than the spaghetti-like tubes with size about 180 nm in the case of ox-MWCNTs. Also, the lowering in the zeta potential negative value (-5 mv) proved that the free carboxyl groups at ox-MWCNT surface were decreased after enzyme immobilization. Moreover, the thermal stability was decreased after enzyme immobilization using TGA. These results confirmed that the laccase could be reacted at the side walls of the ox-MWCNTs without structure damage. The biocatalytic effect of the immobilized laccase was investigated after its incubation with silver nitrate solution for 1 and 24 h. It can be concluded that the biocatalytic efficiency of the immobilized laccase could be enhanced after its incubation with silver nitrate solution for 24 h at room temperature relative to the free form. On the other hand, the enzyme stability was improved after immobilization up to 50ºC and at pH 3.0, while no remarkable differences on the activity values were observed for immobilized and free laccases at acidic pH range (4-6).

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

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