Catalytic Activity Of Laccase Research Articles

Investigation of laccase activity in cholinium-based ionic liquids using experimental and molecular dynamics techniques

Laccases hold great potential for biotechnological applications, particularly in environmental pollutant remediation. Laccase activity is governed by the solvent environment, and ionic liquids (ILs) emerge as a versatile solvent for activation or stabilization of enzymes. Herein, effects of cholinium-based ILs formulated with carboxylic acids, inorganic acid, and amino acids as anionic species, on the catalytic activity of laccase from Trametes versicolor were investigated by experimental and computational approaches. Experimental results showed that laccase activity was enhanced by 21.39 % in 0.5 M cholinium dihydrogen citrate ([Cho][DHC]), in relation to the laccase activity in phosphate buffer medium. However, cholinium aminoate ILs negatively affected laccase activity, as evidenced by the partial deactivation of laccase in both cholinium glycinate and cholinium phenylalaninate, at concentrations of 0.1 M and 0.5 M, respectively. Molecular dynamics studies revealed that the enhancement of laccase activity in [Cho][DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. Enhanced catalytic performance of laccase in [Cho][DHC] was postulated to be driven by the high accumulation level of dihydrogen citrate anions around laccase's surface. [Cho][DHC] holds great promise as a cosolvent in laccase-catalyzed biochemical reactions.

Fabrication of superior laccase-mimicking enzyme with catalytic oxidative and photothermal properties for anti-bacterial and dual-mode glutathione S-transferase monitoring

A novel laccase mimic enzyme Cu–Mn with excellent photothermal properties was firstly prepared via a combination of hydrothermal and in situ synthesis. Cu–Mn nanozymes could catalyze the typical laccase substrate 2,4-dichlorophenol (2,4-DP) to generate the red quinone imine. Further, loading the MnO2 nanosheets with photothermal properties, Cu–Mn nanozymes possessed not only excellent laccase catalytic activity, but also high photothermal conversion efficiency. The presence of glutathione S-transferase (GST) recovered the glutathione (GSH)-induced weakness of the laccase activity and photothermal properties of Cu–Mn. Hence, a GST enzyme-regulated dual-mode sensing strategy was established based on Cu–Mn nanozymes. The detection limits of GST monitoring based on colorimetric and photothermal methods were 0.092 and 0.087 U/L with response times of 20 min and 8 min, respectively. Furthermore, the proposed method enabled the measuring of GST levels in human serum and was successfully employed in the primary evaluation of hepatitis patients. Another attraction, the impressive photothermal behavior also endowed the Cu–Mn nanozymes with promising antimicrobial properties, which exhibited significant antimicrobial effects against Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus). Unsurprisingly, multifunctional Cu–Mn nanozymes certainly explore new paths in biochemical analysis and antimicrobial applications.

Production and characterization of a highly stable laccase from extreme thermophile Geobacillus stearothermophilus MB600 isolated from hot spring of Gilgit Balitistan (Pakistan)

Extracellular thermophilic multicopper oxidase i.e. laccase was purified, identified and characterized from Geobacillus stearothermophilus MB600. Fast protein liquid chromatography (FPLC) of lacasse showed a prominent peak at 610 nm. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for laccase presented a molecular weight of 59.13 kDa. Analysis via Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) confirmed the presence of laccase similar to in Geobacillus thermoleovorans B23 and Geobacillus sp. LEMMY01. Laccase showed Km 0.532 mM for guaiacol and 0.307 mM for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)). Its optimum activity was witnessed at 90°C, at pH 5.0. Laccase catalytic activity was completely inhibited by sodium dodecyl-sulfate (SDS), sodium azide (NaN3), and ethylenediaminetetraacetic acid (EDTA). Among the metal salts against Cu, Ca, Cd, and Li enhanced while Mg inhibited the enzyme activity. It was stable against Cl, I and solvents (Ethanol > Methanol > Dimethyl sulfoxide > Acetonitrile). Thermophilic laccase effectively degraded 93% of Remazol brilliant blue R, 92% Bisphenol A diglycidyle ether without mediator.

Laccase catalytic activity shielded by SiO2 nanostructured materials: an in vitro and in silico approach

This study investigates the enhancement of enzymatic catalytic performance by immobilizing laccase on various nanostructured mesoporous silica materials (SBA-15, MCF, and MSU-F). The activity of immobilized laccase was evaluated under different hydrothermal, pH, and solvent conditions, with laccase@MSU-F showing a three-fold increase in stability. Laccase immobilized on these materials demonstrated stability in a pH range of 4.5 to 10.0, while free laccase was inactivated at pH higher than 7. Molecular dynamics simulations revealed that electrostatic interactions and protective confinement effects contribute to the improved stability of immobilized laccase. Overall, the findings suggest that nanomaterials can enhance the operational stability and recovery of enzymes. Communicated by Ramaswamy H. Sarma

Effect of UVB solar irradiation on Laccase enzyme: evaluation of the photooxidation process and its impact over the enzymatic activity for pollutants bioremediation.

The multi-copper Laccase enzyme corresponds to one of the most investigated oxidoreductases for potential uses in xenobiotic bioremediation. In this work, we have investigated the photo-degradation process of Laccase from Trametesversicolor induced by UVB light and the influence on its activity over selected substrates. Laccase undergoes photo-degradation when irradiated with UVB light, and the process depends on the presence of oxygen in the medium. With the kinetic data obtained from stationary and time resolved measurements, a photo-degradation mechanism of auto-sensitization was proposed for the enzyme. Laccase generates singlet oxygen, by UVB light absorption, and this reactive oxygen species can trigger the photo-oxidation of susceptible amino acids residues present in the protein structure. The catalytic activity of Laccase was evaluated before and after UVB photolysis over hydroxy-aromatic compounds and substituted phenols which represent potential pollutants. The dye bromothymol blue, the antibiotic rifampicin and the model compound syringaldazine, were selected as substrates. The values of the kinetic parameters determined in our experiments indicate that the photo-oxidative process of Laccase has a very negative impact on its overall catalytic function. Despite this, we have not found evidence of structural damage by SDS-PAGE and circular dichroism experiments, which indicate that the enzyme retained its secondary structure. We believe that, given the importance of Laccase in environmental bioremediation, the information found about the stability of this kind of biomolecule exposed to UV solar irradiation may be relevant in the technological design and/or optimization of decontamination strategies.

Construction of a bioinspired laccase-mimicking nanozyme for the degradation and detection of phenolic pollutants

Nanozymes, defined as nanomaterials with enzyme-like activity, have attracted extensive interest in both fundamental and applied research. Laccases are members of the multi-copper oxidases, which are utilized as green catalysts in the environmental catalysis and biochemical industry. In this paper, we report a facile strategy for the preparation of a new class of nanozyme (denoted as CH-Cu) with laccase-like activity inspired by the structure of the active site and the electron transfer pathway of laccase via the coordination of Cu+/Cu2+ with a cysteine (Cys)-histidine (His) dipeptide. The CH-Cu nanozymes exhibit excellent catalytic activity, recyclability and substrate universality and have a similar Km (Michaelis constant) and a higher vmax (maximum rate) than laccase at the same mass concentration. They are robust under a variety of conditions, such as extreme pH, high temperature, long-term storage and high salinity, which can cause severe loss in the catalytic activity of laccase. Higher efficacy of the CH-Cu nanozymes compared with laccase in the degradation of chlorophenols and bisphenols is also demonstrated in a batch reaction. Furthermore, a method for the quantitative detection of epinephrine by a smart phone is established based on the CH-Cu nanozymes. We believe that this nanozyme has promising applications in environmental catalysis and rapid detection and expect that combining key peptides as metal ligands with metal ions to mimic the structure of the catalytic center of a natural enzyme will be a general and important strategy for the design and synthesis of a new type of nanozyme that can be used in various applications.

Investigation of the role of central metal ion of Cyathus bulleri laccase 1 using guanidinium chloride-induced denaturation.

The structure and folding/unfolding kinetics of Cyathus bulleri laccase 1 (Lcc1), expressed in Pichia pastoris, were analyzed by spectroscopic methods to understand the role of central metal ion. Far UV CD structure analysis revealed major β-sheet and minor α helical segments present in the Lcc1. A significant change in the spectrum of Lcc1, indicative of unfolding of secondary structures, was observed with increasing concentrations of guanidinium chloride (GdnHCl) during Trp fluorescence, absorption and CD measurements. A similar trend was also noticed for enzyme activity with respect to GdnHCl concentrations. To establish the role of copper ion in the catalytic activity of laccase, a complete removal of copper was carried out and addition of copper was found to restore the structure and activity during the refolding process. The apo form was also reconstituted by addition of zinc ion which restored nearly 84% of enzyme activity, indicating non-essential role of copper in maintaining conformation and activity. Structural studies and inductively coupled plasma mass spectrometry data supported these observations.

Facile synthesis of laccase mimic Cu/H3BTC MOF for efficient dye degradation and detection of phenolic pollutants.

Herein, we report an effectual method for designing a novel form of nanozyme laccase mimic namely Cu/H3BTC, using copper ions and 1,3,5-benzene tricarboxylic acid (1,3,5-H3BTC). This Cu-based metal–organic framework (MOF) was synthesized through a simple procedure of mixing of two usual reagents at room temperature. Amido Black 10B (AB-10B) was chosen as a model dye for degradation consequences. Results showed that Cu/H3BTC MOF revealed significantly higher catalytic efficacy under certain conditions like high pH, extreme temperature and high salt conditions and it has long-term storage stability, which can lead to a significant decline in catalytic activity of laccase. In addition, the degradation of AB-10B was up to 60% after ten cycles, showing good recyclability of Cu/H3BTC MOF. The UV-visible spectral changes clearly showed that Cu/H3BTC MOF is an effective laccase mimic for the degradation of azo dye AB-10B, which was degraded more easily within the time duration of 60 min. The Cu/H3BTC MOF also possessed fundamental activities like laccase with regard to oxidation of the phenolic compounds. Moreover, a technique for the quantitative detection of epinephrine by Cu/H3BTC MOF was established. These findings help to understand the laccase-like reactivity and provide a basis for the future design and application of metal-based catalysts.

The second conserved motif in bacterial laccase regulates catalysis and robustness.

Laccase (EC1.10.3.2), an oxidase that binds multiple copper ions, is heterogeneous in different species, implying diversity in its function. Nevertheless, the four copper-binding motifs are conserved in most laccases, especially bacterial forms. In order to exploit laccase more widely and more effectively in industrial processes, we investigated the regulatory effects, if any, of the second conserved copper-binding motif in the bacterial laccases CAR2 and CAHH1. The data suggested that three critical amino acid residues His155, His157, and Thr/Ala158 in this motif strongly regulated laccase's catalysis, substrate range, and robustness. Indeed, these residues were essential for laccase's catalytic activity. The data also suggested that laccase's catalytic efficiency and activity are not completely consistent with its stability, and that the enzyme might have evolved naturally to its favor stability. This study provides important insights into the second conserved copper-binding motif and defines some of the previously undefined amino acid residues in this conserved motif and their significances.

Screening trials for the encapsulation of laccase enzymatic extract in silica sol-gel

The effects of selected process conditions for the sol-gel encapsulation of laccase enzymatic extract, obtained from Coriolus hirsutus, were investigated. Screening trials were carried out to identify the parameters having the most pertinent effects on the encapsulation efficiency (EE) and the residual laccase activity. These parameters included water/silane molar ratio (r), HCl content and protein loading, for the pre-gel silica sol as well as the required time for gel drying and for aging, for the sol-gel process. The experimental findings indicated that a sol-gel drying time of over 6 h resulted in a complete loss of laccase catalytic activity, while an increase in the gel aging time led to an enhancement of the residual enzyme activity. Except for r, the investigated parameters demonstrated no significant effect on the EE of the sol-gel encapsulated enzymatic extract. Overall, the encapsulation of laccase extract in the sol-gel matrix resulted in an enhancement of its catalytic activity, where its highest residual activity (349%) was obtained with an r-value of 4, an HCl content of 4 µmol and a protein loading of 1 mg/mL, using 6 and 24 h of drying and aging times, respectively.

Effect of Common Metal Ions and Anions on Laccase Catalysis of Guaiacol and Lignocellulosic Fiber

The effects of 12 common metal ionic compounds on the laccase catalytic activity in reactions using guaiacol as the substrate was determined using spectrophotometry. Furthermore, the influence of several metal ionic compounds on the generation of reactive oxygen species (ROS) by oxidation of lignin in jute fiber under laccase catalysis was studied by electron paramagnetic resonance (EPR) spectroscopy using N-tert-butyl-alpha-phenylnitrone (PBN) as the spin-trapping agent. Common metal cations, such as K+, Na+, Mg2+, Ca2+, and Cu2+ and the anion SO42- had almost no effect on laccase activity during the initial stage of the catalytic reactions. High concentrations of the Mn2+ ion exhibited weak inhibition of laccase; Ag+ and NO3- showed a moderate inhibitory effect on laccase activity during the initial stage of the catalytic reactions. Fe2+ had no direct effect on the binding of laccase to its substrate, but strongly retarded the progress of the catalytic reaction by reducing the intermediate free radicals. The ions Cl-, Fe3+, and Ag+ exhibited either strong inhibitory effects on the catalysis of the substrate or a destructive effect on the structure of laccase itself. Furthermore, the results showed that an appropriate concentration of Cu2+ helped to promote the thermal stability of laccase during the enzymatic reaction. This study could help researchers to avoid the use of inhibitory exogenous metal ions and anions in the application of laccase and to maximize the value of laccase.

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

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

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

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

Modeling of laccase inhibition by formetanate pesticide using theoretical approaches

The inhibition of laccase enzymatic catalytic activity by formetanate hydrochloride (FMT) was investigated by cyclic voltammetry and by quantum chemical calculations based on density functional theory with a protein fragmentation approach. The cyclic voltammograms were obtained using a biosensor prepared by enzyme immobilization on gold electrodes modified with gold nanoparticles and 4-aminophenol as the target molecule. The decrease in the peak current in the presence of FMT was used to characterize the inhibition process. The calculations identified Asp206 as the most relevant moiety in the interaction of FMT with the laccase enzymatic ligand binding domain. The amino acid residue Cys453 was important, because the Cys453–FMT interaction energy was not affected by the dielectric constant, although it was not a very close residue. This study provides an overview of how FMT inhibits laccase catalytic activity.

Visible-Light-Induced Effects of Au Nanoparticle on Laccase Catalytic Activity.

A deep understanding of the interaction between the nanoparticle and enzyme is important for biocatalyst design. Here, we report the in situ synthesis of laccase-Au NP (laccase-Au) hybrids and its catalytic activity modulation by visible light. In the present hybrid system, the activity of laccase was significantly improved (increased by 91.2% vs free laccase) by Au NPs. With a short time visible light illumination (λ > 420 nm, within 3 min), the activity of laccase-Au hybrids decreased by 8.1% (vs laccase-Au hybrid without light), which can be restored to its initial one when the illumination is removed. However, after a long time illumination (λ > 420 nm, over 10 min), the catalytic activity of laccase-Au hybrids consecutively decreases and is not reversible even after removing the illumination. Our experiments also suggested that the local surface plasma resonance effect of Au NPs causes the structure change of laccase and local high temperature near the Au NPs. Those changes eventually affect the transportation of electrons in laccase, which further results in the declined activity of laccase.

Tuning laccase catalytic activity with phosphate functionalized carbon dots by visible light.

The phosphate functionalized carbon dots (PCDs) with high biocompatibility and low toxicity can be used as efficient additives for the construction of laccase/PCDs hybrids catalyst. A series of experiments indicated that the activity of laccase/PCDs was higher than that of free laccase (increased by 47.7%). When laccase/PCDs hybrids catalyst was irradiated with visible light (laccase/PCDs-Light), its activity was higher than that of laccase/PCDs hybrids without light irradiation (increased by 92.1%). In the present system, the T1 Cu in laccase was combined with the phosphate group on PCDs, which can increase binding capacity of laccase/PCDs hybrids and substrate. Further, the visible light irradiation increased the donating and accepting electronic capability of the laccase/PCDs hybrids, improving their catalytic activity.

Gold nanoparticles tune the activity of laccase in anionic reverse micelles.

The interfacial property of reverse micelles is an important factor affecting the catalytic activity of enzymes hosted in the micelles. In this article, the effect of gold nanoparticles (GNPs) on the catalytic activity of laccase (non-surface-active enzyme) and the related mechanism are reported. It was found that laccase activity was dependent on the size of the particle and its concentration as well as on the water content and the concentration of AOT. It was shown that there existed several types of micelles in the present reverse micellar system in the presence of GNPs. The population of the various micelles depended on the concentrations of both GNPs and AOT. Fluorescence and circular dichroism spectra of laccase at different water contents and GNP concentrations indicated that the conformation of laccase and its activity were tuned by GNPs via changing the structure of the reverse micelles. Analysis showed that changes in the thickness of the water layer (Lw) and in the apparent occupied area of individual AOT molecules (AAOT) caused by GNPs were the main parameters affecting the activity of laccase. The present work extends and deepens the understanding of the tuning mechanism of GNPs on enzymatic performance in reverse micelles and provides guidance for rational design of the optimal microenvironment of laccase.

Controlling the Surface Chemistry of Multiwalled Carbon Nanotubes for the Production of Highly Efficient and Stable Laccase‐Based Biocatalysts

AbstractLaccase was immobilized over multiwalled carbon nanotubes (MWCNTs) with diameters ranging from 10 to 100 nm and different surface chemical groups. Oxygen‐containing groups were introduced selectively or removed by liquid‐phase oxidation and thermal treatments. The effect of pH on the immobilization efficiency and catalytic activity of laccase has been evaluated. Pristine MWCNTs show excellent immobilization capacity (100 %) and high enzyme activity, but low thermal stability (at 50 °C) owing to weak interaction (mostly hydrophobic) between laccase and the support. Immobilization capacity and catalytic activity of laccase on the modified MWCNTs were related to the surface electric charge of both the support and the enzyme. MWCNTs oxidized with HNO3 and posteriorly heated at 400 °C, mostly presenting hydroxyl surface groups, provided the best compromise between laccase activity and thermal stability, which has been attributed to the formation of hydrogen bonds between the support and the enzyme.

Identification, Purification and Characterization of a Novel Extracellular Laccase from Cladosporium Cladosporioides

ABSTRACTCladosporium cladosporioides was identified as a laccase producer when grown on malt extract media supplemented with 0.0 2% ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid). Extracellular laccase was purified to homogeneity from culture supernatant of Cladosporium cladosporioides by acetone precipitation, gel filtration chromatography and anion exchange chromatography, and was also characterized. Purified laccase (specific activity 1785.71 U·mg−1) is a monomeric protein with molecular weight of 50 kDa on SDS-PAGE. Purified laccase showed maximum activity at pH 5 and optimal temperature was observed 40°C. The enzyme showed a high relative activity over a wide range of pHfrom 3 to 6 and was stable in a temperature range of 40° to 70°C. The Km value of the purified enzyme was found to be 15 μmol·L−1, while the Vmax value was observed to be 4612 μmol·L−1·min−1 with ABTS as a substrate. The catalytic activity of laccase was completely inhibited by dithiothreitol, sodium azide and L-cysteine at a final concentration of 5 mmol·L−1, and slightly inhibited by EDTA and SDS at a final concentration of 10 mmol·L−1. The laccase was activated by Cu2+ and Mg2+, and certain metal ions such as Ni+2, Ba+2 and Ag+2 inhibit the catalytic activity of enzyme at a final concentration of 1.0 mmol·L−1. To the best of our knowledge this is the first report on the purification and characterization of laccase from Cladosporium cladosporioides.

Catalytic activity and stability of laccase entrapped in sol–gel silica with additives

This study investigated the effects of different additives and precursors on the catalytic activity of laccase entrapped in sol–gel silica. It was found that the laccase catalytic activity and stability of sol–gel laccase could be enhanced if the entrapment was performed in the presence of additives such as PVA, PEG and APTS. The use of TEOS as a precursor showed slightly higher laccase catalytic activity compared to TMOS. The PVA as an additive showed a better catalytic activity enhancement compared to the PEG and APTMS with the optimum PVA concentration of 0.03 mg/mL. The optimal temperatures of sol–gel laccase without and with additives were found to be at 40 and 27°C, respectively. After 70 days of storage at 27°C, the catalytic activity of the immobilized sol–gel laccase with additives maintained its catalytic activity compared to only 30% of its original catalytic activity for the sol–gel laccase without additives.