No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Investigation of laccase activity in cholinium-based ionic liquids using experimental and molecular dynamics techniques
... 104 [DHC] might be attributed to the highly stabilized and compact structure of laccase, facilitating a better internal electron transfer during the laccase-substrate interactions. 106 As shown in Fig. 12 In order to discuss the interaction of ILs towards an enzyme, the Hofmeister effect, that was originally observed during the salting-out process of protein aqueous solutions, has been frequently used (Fig.13). 68,75,107,108,109 Yan et al. proposed that kosmotropic anions, which have a strong hydration property, generally enhance protein stabilization, while hydrophobic chaotropic anions cause a significant destabilization of proteins. ...
Selective oxidative depolymerization of a lignocellulosic biomass is the first step in the valorisation process. Chemical oxidations generally require hazardous reagents and harsh reaction conditions; thus, the resulting produced compounds are low-value molecules or complicated mixtures. Laccases are copper ion-containing oxidases and catalyze the oxidation of polyphenol or amine derivatives using molecular oxygen; laccase-mediated reaction systems thus allow the depolymerization of lignocellulosic compounds and the decomposition of aromatic pollutants in wastewater. However, due to the short distance between the active site and the surface of the laccase, the reactivity of laccase is influenced by the reaction conditions, in particular, the solvent system. Ionic liquids (ILs) and deep eutectic solvents (DESs) have now been acknowledged as not only new reaction media but also as activating agents of biocatalysts. In order to improve the activity or increasing the tolerance of laccases against ILs or DESs, three methods have been developed: the first is the direct evolution of the enzyme that is a very powerful tool for tailoring the enzyme, the second is the design of supporting materials including ILs for the immobilization of a laccase, and the third is modification of the surface of a laccase protein by chemical methods or protein engineering. This review examines laccase-mediated reactions in ILs and DESs focusing on how laccase contributes to sustainable chemistry; using laccase-mediated reactions, the depolymerization of lignocellulosic compounds, phenolic compounds, and synthetic dyes has now been accomplished. Since the reactions were accomplished under hazardous chemical reagent-free conditions, it is expected that investigation in this field of laccase-mediated oxidation might become even more important in sustainable chemistry.
... [Cho][AA] were synthesized based on the methods reported by Chan et al. [31]. The corresponding amino acids (glycine, β-alanine, or Lphenylalanine) were added to a 250-ml round bottom flask containing an equimolar solution of aqueous ...
... A plausible explanation for this phenomenon might be the capacity of surfactants to modulate the microenvironment surrounding laccase, particularly affecting hydrophobic amino acid residues such as Tyrosine and Tryptophan. This modulation likely leads to a less polar surrounding or a confinement within the laccase structure, thereby fostering favorable folding and stabilization of the enzyme (Chan et al., 2024). ...
The emergence of environmental endocrine disruptor chlorobenzene (CB) in surface water and its potential environmental impacts have attracted serious global attention. It is still very difficult to achieve effective degradation of it by catalytic oxidation process under mild conditions. Here, an optimized method for degrading CB in aqueous solution using Trametes versicolor laccase and surfactant-assisted laccase-mediator (SALM) system was investigated. The use of a Tween 80 surfactant enhanced the solubility of CB and promoted its efficient degradation. Under favorable conditions, the SALM system yielded a degradation efficiency of 43.5% and a dechlorination efficiency of 41.55% for CB (25 mg/L) within 24 h. The possible degradation pathway of CB by this system was speculated by detecting the intermediates produced during the reaction. The outcome of the proliferation assays on MCF-7 human breast cancer cells demonstrated a reduction in the estrogenic activity of the CB solution following treatment with the SALM system. Furthermore, the influence of the quantity and positional variation of chlorine substituents on the degradation process was methodically investigated. Moreover, molecular analyses were employed to study the detailed interaction mechanism between laccase and CB, which revealed that the hydrophobic interaction contributed dominantly to binding process. These findings provide an efficient and environmentally friendly degradation system for the development of purification strategies for halogenated pollutants.
Candida antarctica lipase B (CALB) is widely used in biocatalysis with applications in plastics degradation and chemical synthesis. CALB is activated by hydrophobic matrices and, enigmatically, shows striking activation in polar, choline-based, Deep Eutectic Solvents (DES). Herein, we show that CALB activation and stabilisation by TAAs is caused by binding to choline’s tetraalkylammonium (TAA) moiety. Several related TAA salts also caused CALB activation which was proportional to the hydrophobicity of their alkyl substituents. Remarkably, tetraoctylammonium bromide showed activation of ∼500% even at low micromolar levels. These TAA salts represent a new class of enzyme activator. Molecular modelling identified the alkylammonium binding location as a hydrophobic patch centred around Asp-145 of CALB. Binding at this site explains lipase activation in polar DES solvents and its relationship to other pathways of CALB activation.
Herein, we also demonstrate that CALB, like many lipases, is activated by calcium. Intriguingly, mixed soluble activator experiments showed that calcium and choline bind to different CALB sites, suggesting a two-site model for CALB activation.
These observations, along with previous findings, show that TAA activation is a widespread property of enzymes and constitutes a novel and potent means to enhance enzyme turnover and stability.
Highlights
CALB is activated by choline
Several tetraalkylammonium salts cause activation of CALB
Hyperactivation of CALB (5-fold) by tetraoctylammonium ions occurs at low micromolar concentrations.
Two independent sites for CALB activation, by calcium and TAA ions, are identified
Activation at the choline binding site stabilises CALB while calcium binding destabilises the enzyme
A soluble activator is demonstrated, that can be used to probe the activation mechanism of CALB or other enzymes.
Ionic liquids (ILs) can stabilize or destabilize proteins, which motivates us to examine their effect on hemoglobin. The native state of hemoglobin (Hb) is disrupted at different physical conditions such as pressure, temperature, and solvents. Herein, we have monitored the stability of Hb in a nontoxic and biocompatible IL, i. e., choline amino acid‐based Ils (ChAAILs), using various spectroscopic techniques like UV‐Vis and fluorescence spectroscopy, circular dichroism (CD), and isothermal titration calorimetry (ITC) measurements. It was observed that Hb stays neither in its native state nor in its fully denatured state; rather, it achieves an intermediate state in the presence of ChAAILs. The research on the intermediate state of Hb is still unexplored. Research has been pursued to find a suitable ligand or IL that can stabilize the intermediate state of Hb. In that context, ChAAILs are among the best choices. Molecular docking studies unravel the binding of ChAAILs with Hb. The obtained binding energies of the docked complex are −7.2 kcal/mol and −8.7 kcal/mol for binding of Hb with [Chl][Gly] and [Chl][Met], respectively, which was in line with the ITC results. The quantum chemical calculations show that H‐bond plays a significant role for the interaction between Hb and ChAAILs.
As a new generation of green solvents, deep eutectic solvents (DESs) are considered a promising alternative to current harsh organic solvents and find application in many chemical processing methods such as extraction and synthesis. DESs, normally formed by two or more components via various hydrogen bond interactions, offer high potential as medium for biocatalysis reactions where they can improve efficiency by enhancing substrate solubility and the activity and stability of the enzymes. In the current study, the stabilization of Humicola insolens cutinase (HiC) in natural deep eutectic solvents (NADESs) was assessed. The best hydrogen bond donor among sorbitol, xylitol, erythritol, glycerol and ethylene glycol, and the best acceptor among betaine, choline chloride, choline acetate, choline dihydrogen citrate and tetramethylammonium chloride, were selected, evaluating binding energies and molecular orientations through molecular docking simulations, and finally used to prepare NADES aqueous solutions. The effects of component ratio and NADES concentration on HiC thermostability at 90 °C were also investigated. The choline dihydrogen citrate:xylitol, in a 1:1 ratio with a 20wt% concentration, was selected as the best combination in stabilizing HiC, increasing its half-life three-fold.
Ribonucleic acid (RNA) is exceedingly sensitive to degradation compared to DNA. The current protocol for storage of purified RNA requires freezing conditions below −20 °C. Recent advancements in biological chemistry have identified amino acid-based ionic liquids as suitable preservation media for RNA, even in the presence of degrading enzymes. However, the mechanistic insight into the interaction between ILs and RNA is unclear. To the best of our knowledge, no attempts are made so far to provide a molecular view. This work aims to establish a detailed understanding of how ILs enable structural stability to RNA sourced from Torula yeast. Herein, we manifest the hypothesis of multimodal binding of IL and its minimal perturbation to the macromolecular structure, with several spectroscopic techniques such as time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) aided with molecular dynamics at microsecond time scales. Relevant structural and thermodynamic details from biophysical experiments confirm that even long-term RNA preservation with ILs is a possible alternative devoid of any structural deformation. These results establish a unifying mechanism of how ILs are maintaining conformational integrity and thermal stability. The atomistic insights are transferable for their potential applications in drug delivery and biomaterials by considering the advantages of having maximum structural retention and minimum toxicity.
A series of hydrazide-hydrazones 1–3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM with the predicted and desirable competitive type of inhibition. The structure–activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.
Lipases are well‐known biocatalysts used in several industrial processes/applications. Thus, as with other enzymes, changes in their surrounding environment and/or their thermodynamic parameters can induce structural changes that can increase, decrease, or even inhibit their catalytic activity. The use of ionic compounds as solvents or additives is a common approach for adjusting reaction conditions and, consequently, for controlling the biocatalytic activity of enzymes. Herein, to elucidate the effects of ionic compounds on the structure of lipase, the stability and enzymatic activity of lipase from Aspergillus niger in aqueous solutions (at 0.05, 0.10, 0.50, and 1.00 M) of six cholinium‐based ionic liquids (cholinium chloride [Ch]Cl; cholinium acetate ([Ch][Ac]); cholinium propanoate ([Ch][Prop]); cholinium butanoate ([Ch][But]); cholinium pentanoate ([Ch][Pent]); and cholinium hexanoate ([Ch][Hex])) were evaluated over 24 hr. The enzymatic activity of lipase was maintained or enhanced in the lower concentrations of all the [Ch]⁺‐ILs (below 0.1 M). [Ch][Ac] maintained the biocatalytic behavior of lipase, independent of the IL concentration and incubation time. However, above 0.1 M, [Ch][Pent] and [Ch][Hex] caused complete inhibition of the catalytic activity of the enzyme, demonstrating that the increase in the anionic alkyl chain length strongly affected the conformation of the lipase. The hydrophobicity and concentration of the [Ch]⁺‐ILs play an important role in the enzyme activity, and these parameters can be controlled by adjusting the anionic alkyl chain length. The inhibitory effects of [Ch][Pent] and [Ch][Hex] may be of great interest to the pharmaceutical industry to induce pharmacological inhibition of gastric and pancreatic lipases.
Long term storage and stability of DNA is of paramount importance in biomedical applications. Ever since the emergence of ionic liquids (ILs) as alternate green solvents to aqueous and organic solvents, their exploration for the extraction and application of DNA need conscientious understanding of the binding characteristics and molecular interactions between IL and DNA. Choline amino acids ILs (CAAILs) in this regard seem to be promising due to their noncytotoxic, completely bio based and environment friendly nature. To unravel the key factors for the strength and binding mechanism of CAAILs with DNA, various spectroscopic techniques, molecular docking and molecular dynamics simulations have been employed in this work. UV-Vis spectra indicate multimodal binding of CAAILs with DNA whereas dye displacement studies through emission confirm the intrusion of IL molecules into the minor groove of DNA. Circular dichorism (CD) spectra show that DNA retains its native B-conformation in CAAILs. Both ITC and molecular docking studies provide an estimate of the binding affinity of DNA with CAAILs ~4 kcal/mol. The heterogeneity in binding modes of CAAIL-DNA system with evolution of time was established by molecular dynamics simulations. Choline cation while approaching DNA first binds at surface through electrostatic interactions, whereas a stronger binding at minor groove occurs via van der Waals and hydrophobic interactions irrespective of anions considered in this study. We hope, this result can encourage and guide the researchers in designing new bio-ILs for biomolecular studies in future.
Fungal pretreatment of sweet sorghum bagasse (SSB) by solid state fermentation (SSF) studied in a Mesh tray bioreactor. In pretreatment by using Coriolus versicolor, optimal mesh size in tray and humid airflow into bioreactor overcame the problems of SSF and improved fungal growth; increased the production of lignolytic enzymes laccase, lignin peroxidase, manganese peroxidase, polyphenol peroxidase, aryl alcohol oxidase by 1.9, 1.85, 2.6, 2.0, 1.9 folds respectively; hemicellulolytic enzyme xylanase by 1.8 folds and decreased cellulolytic enzymes production. Altered lignocellulolytic enzyme profiles resulted in high lignin degradation 46.09 ± 2.0% w w⁻¹, high selectivity value 5.98 and low cellulose loss 7.7 ± 0.3% w w⁻¹. Enzymatic hydrolysis of pretreated SSB yielded higher (~ 2.47 times) fermentable sugar. Characterizations of SSB by SEM, XRD, FTIR, TGA/DTG supported the results. Mesh tray bioreactor could be used for fungal pretreatment and enzyme productions by SSF of waste biomass.
Graphical Abstract
Open image in new window
Laccases are a family of copper-containing oxidases with important applications in bioremediation and other various industrial and biotechnological areas. There have been over two dozen reviews on laccases since 2010 covering various aspects of this group of versatile enzymes, from their occurrence, biochemical properties, and expression to immobilization and applications. This review is not intended to be all-encompassing; instead, we highlighted some of the latest developments in basic and applied laccase research with an emphasis on laccase-mediated bioremediation of pharmaceuticals, especially antibiotics. Pharmaceuticals are a broad class of emerging organic contaminants that are recalcitrant and prevalent. The recent surge in the relevant literature justifies a short review on the topic. Since low laccase yields in natural and genetically modified hosts constitute a bottleneck to industrial-scale applications, we also accentuated a genus of laccase-producing white-rot fungi, Cerrena, and included a discussion with regards to regulation of laccase expression.
PyMOL , a cross‐platform molecular graphics tool, has been widely used for three‐dimensional ( 3D ) visualization of proteins, nucleic acids, small molecules, electron densities, surfaces, and trajectories. It is also capable of editing molecules, ray tracing, and making movies. This Python‐based software, alongside many Python plugin tools, has been developed to enhance its utilities and facilitate the drug design in PyMOL . To gain an insightful view of useful drug design tools and their functions in PyMOL , we present an extensive discussion on various molecular modeling modules in PyMOL , covering those for visualization and analysis enhancement, protein–ligand modeling, molecular simulations, and drug screening. This review provides an excellent introduction to present 3D structures visualization and computational drug design in PyMOL . WIREs Comput Mol Sci 2017, 7:e1298. doi: 10.1002/wcms.1298
This article is categorized under: Structure and Mechanism > Molecular Structures
Computer and Information Science > Visualization
Molecular and Statistical Mechanics > Molecular Mechanics
Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment.
The use of a wide range of water miscible and immiscible ionic liquids (ILs) as reaction media for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) oxidation by Trametes versicolor laccase was studied. Thirteen ILs were shown to be suitable media for the laccase oxidation reaction, increasing the activity with respect to conventional media. Among them, the water-miscible IL choline dihydrogen phosphate [Chol][H2PO4] allowed over-laccase activity with an enhancement rate of 451% at 25°C and pH 7.0. This ionic liquid improved the stability of the enzyme in the face of high temperature and high pH, while storage at room temperature in aqueous medium was increased up to 4.5 times. Moreover, it was found that its use in the reaction medium for decolourizing dyes (antraquinonic and azoic) using laccase increased the decolourization rate by up to 216% and 137% for the azoic dyes Acid Black 1 and Remazol Brillant Blue R, respectively. A high decolorization rate was also obtained for a mix of dyes (80% within 8 h). To understand the effect of [Chol][H2PO4] on the secondary protein structure of the laccase, several spectroscopic techniques were used such as Circular Dichroism (CD), Fourier transform infrared (FT-IR) and Fluorescence, all of which demonstrated that the β sheets structure was affected. A shift to α-helix structure [Chol][H2PO4] could be responsible of the enhancement of the enzyme activity observed at 300 mM.
andida Antarctica Lipase B (CALB) is extensively studied in enzymatic production of biodiesel, pharmaceutical products, detergents and other chemicals. One drawback of using CALB is its relatively low optimum temperature at 313 K (40°C). The objective of this research is to design CALB mutant with improved thermostability by introducing extra disulfide bond. Molecular dynamic simulation was conducted to get better insight into the process of thermal denaturation or unfolding in CALB. Thermal denaturation of CALB was accelerated by conducting simulation at high temperature. Molecular dynamic simulation of CALB was performed with GROMACS software package at 300-700 K. Prediction of possible mutation was done using “Disulfide by DesignTM” software. Selection of mutated residues was based on flexibility analysis of CALB. From those analyses, three mutants were designed, which are Mutant-1 (73LeuCys/151AlaCys), Mutant-2 (155TrpCys/294GluCys) and Mutant-3 (43ThrCys/67SerCys). Parameters that were used to compare the thermostability of mutant with wild type enzyme were Root Mean Square Deviations (RMSD), Solvent Accessible Surface Area (SASA), Radius of gyration (Rg) and secondary structure. Molecular dynamic simulation conducted on those three mutants showed that Mutant-1 has better thermostability compared to wild type CALB. We proposed the order of mutant thermostability improvement as follows: Mutant-1, Mutant-2 and Mutant-3, with Mutant-1 having better potential thermostability improvement and Mutant-3, the least stable
Ionic liquids can be used for clean synthesis in a variety of reactions. Here we present a review of the type of chemical reactions that can be carried out in ionic liquids. These include commercially important reactions carried out in chloroaluminate(III) ionic liquids; the isomerisation of fatty acids and the cracking of polyethylene to low molecular weight alkanes. Also, a growing number of reaction types can be performed in neutral ionic liquids. These include the Heck reactions, Diels-Alder reaction, nucleophilic displacement reactions (S(N)2 reaction), and Friedel-Crafts reactions.
Laccase (p-diphenol:dioxygen oxidoreductase), one of the earliest discovered enzymes, contains four copper ions in two active sites and catalyzes a one-electron oxidation of substrates such as phenols and their derivatives, or aromatic amines, coupled to a four-electron reduction of dioxygen to water. The catalytic mechanism has been studied for decades but is still not completely elucidated, especially in terms of the reduction of dioxygen to water. The key structural features of this enzyme are under investigation in several groups using techniques such as X-ray diffraction, electron paramagnetic resonance (EPR) spectroscopy, and site-directed mutagenesis. The high interest in laccases is explained by the large number of biotechnological applications. In this review, the most recent research on the overall structural features as well as on the structures and properties of the active sites are summarized, along with currently proposed mechanisms of reaction.
Background
The Avogadro project has developed an advanced molecule editor and visualizer designed for cross-platform use in computational chemistry, molecular modeling, bioinformatics, materials science, and related areas. It offers flexible, high quality rendering, and a powerful plugin architecture. Typical uses include building molecular structures, formatting input files, and analyzing output of a wide variety of computational chemistry packages. By using the CML file format as its native document type, Avogadro seeks to enhance the semantic accessibility of chemical data types.
Results
The work presented here details the Avogadro library, which is a framework providing a code library and application programming interface (API) with three-dimensional visualization capabilities; and has direct applications to research and education in the fields of chemistry, physics, materials science, and biology. The Avogadro application provides a rich graphical interface using dynamically loaded plugins through the library itself. The application and library can each be extended by implementing a plugin module in C++ or Python to explore different visualization techniques, build/manipulate molecular structures, and interact with other programs. We describe some example extensions, one which uses a genetic algorithm to find stable crystal structures, and one which interfaces with the PackMol program to create packed, solvated structures for molecular dynamics simulations. The 1.0 release series of Avogadro is the main focus of the results discussed here.
Conclusions
Avogadro offers a semantic chemical builder and platform for visualization and analysis. For users, it offers an easy-to-use builder, integrated support for downloading from common databases such as PubChem and the Protein Data Bank, extracting chemical data from a wide variety of formats, including computational chemistry output, and native, semantic support for the CML file format. For developers, it can be easily extended via a powerful plugin mechanism to support new features in organic chemistry, inorganic complexes, drug design, materials, biomolecules, and simulations. Avogadro is freely available under an open-source license from
http://avogadro.openmolecules.net.
ACPYPE (or AnteChamber PYthon Parser interfacE) is a wrapper script around the ANTECHAMBER software that simplifies the generation of small molecule topologies and parameters for a variety of molecular dynamics programmes like GROMACS, CHARMM and CNS. It is written in the Python programming language and was developed as a tool for interfacing with other Python based applications such as the CCPN software suite (for NMR data analysis) and ARIA (for structure calculations from NMR data). ACPYPE is open source code, under GNU GPL v3, and is available as a stand-alone application at http://www.ccpn.ac.uk/acpype and as a web portal application at http://webapps.ccpn.ac.uk/acpype.
We verified the topologies generated by ACPYPE in three ways: by comparing with default AMBER topologies for standard amino acids; by generating and verifying topologies for a large set of ligands from the PDB; and by recalculating the structures for 5 protein-ligand complexes from the PDB.
ACPYPE is a tool that simplifies the automatic generation of topology and parameters in different formats for different molecular mechanics programmes, including calculation of partial charges, while being object oriented for integration with other applications.
The effect of the ionic liquid 1-butyl-3-methylimidazolium chloride ([bmim]Cl) and of high pressure on the activity of cellulase from Aspergillus niger were studied separately and in combination. The enzyme activity decreased with increasing concentrations of [bmim]Cl, reaching 50% the value in aqueous buffer with 20% [bmim]Cl. However, when the enzyme is held in 10% [bmirn]Cl and is then assayed in 1% [bmim]Cl, it showed only 8% reduction of activity. These results can be explained by the fact that the activity of the enzyme in [bmim]Cl is linearly correlated with the decrease of the thermodynamic water activity (a(w)). Under pressure the enzyme activity varied from less 60% (at 200MPa) to equal (at 400 MPa), compared to atmospheric pressure. In 10% [bmim]Cl under pressure, cellulase activity is improved compared to atmospheric pressure, varying from equal (at 600 MPa) to 1.7-fold higher (at 100 MPa). This opens the possibility to improve cellulase activity in ionic liquids, and possibly of other enzymes, by carrying out the reaction under pressure.
Ionic liquids were initially proposed as replacements for conventional organic solvents; however, their chemistry has developed remarkably and offers unexpected opportunities in numerous fields, ranging from electrochemistry to biology. As a consequence of ionic liquids advancing towards potential and actual applications, a comprehensive determination of their environmental, health and safety impact is now required. This critical review aims to present an overview of the current understanding of the toxicity and environmental impact of the principal ionic liquid groups, and highlights some emerging concerns. Each cation type is considered separately, examining the significance of the biological data, and identifying the most critical questions, some yet unresolved. The need for more, and more detailed, studies is highlighted (176 references).
Rhus laccase (RL) was covalently immobilised onto chitosan, and the effects of immobilisation on pH optimum, enzyme activity, thermostability, and re-use evaluated, using either N,N-dimethyl-p-phenylenediamine or 2,6-dimethoxyphenol as substrate. Immobilisation greatly enhanced enzyme thermostability, resulted in negligible loss of activity, and showed excellent re-use potential, with >80% relative activity retained after 15 cycles in aqueous solvent. Immobilised Rhus laccase (I-RL) was more catalytically active in both hydrophobic and hydrophilic organic solvents than free RL. With water-immiscible organic solvents, both free RL and I-RL required a minimum water content to achieve activity. With water-miscible organic solvents, in general a water content of ∼20-50% (v/v) was required to achieve activity using free RL, whereas with I-RL less water was generally required to achieve enzyme activity, and therefore considerably higher relative activity was exhibited at lower water contents. Kinetic investigations showed that the rate of substrate disappearance generally followed a pseudo-first-order law, and for evaluated water-immiscible organic solvents rate constants generally increased with decrease of hydrophobicity, however, in water-miscible organic solvents no such relationship was observed. Some discussion of the potential interactions between organic solvent molecules and enzyme active centres was provided to explain obtained results.
Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side-chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha-helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side-chain torsion potentials of these residues to match new, high-level quantum-mechanical calculations. Finally, we used microsecond-timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side-chain conformations. The new force field, which we have termed Amber ff99SB-ILDN, exhibits considerably better agreement with the NMR data.
We thank the North Carolina Supercomputing Center, the Pittsburgh Supercomputing Center and the National Cancer Institute Supercomputing Center for access to resources. LGP thanks the National Institutes of Health for HL-06350 and the National Institute of Environmental Health Sciences (NIEHS) for access to their facilities.
Laccase is a polyphenol oxidase, which belongs to the family of blue multicopper oxidases. These enzymes catalyze the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water. Laccases oxidize a broad range of substrates, preferably phenolic compounds. In the presence of mediators, fungal laccases exhibit an enlarged substrate range and are then able to oxidize compounds with a redox potential exceeding their own. Until now, only one crystal structure of a laccase in an inactive, type-2 copper-depleted form has been reported. We present here the first crystal structure of an active laccase containing a full complement of coppers, the complete polypeptide chain together with seven carbohydrate moieties. Despite the presence of all coppers in the new structure, the folds of the two laccases are quite similar. The coordination of the type-3 coppers, however, is distinctly different. The geometry of the trinuclear copper cluster in the Trametes versicolor laccase is similar to that found in the ascorbate oxidase and that of mammalian ceruloplasmin structures, suggesting a common reaction mechanism for the copper oxidation and the O(2) reduction. In contrast to most blue copper proteins, the type-1 copper in the T. versicolor laccase has no axial ligand and is only 3-fold coordinated. Previously, a modest elevation of the redox potential was attributed to the lack of an axial ligand. Based on the present structural data and sequence comparisons, a mechanism is presented to explain how laccases could tune their redox potential by as much as 200 mV.
We have synthesized biocompatible ionic liquids (ILs) with choline as cation and amino acids as anions to explore their potential towards prevention of fibrillation in insulin and the obtain corresponding mechanistic insights. This has been achieved by examining the effect of these ILs on insulin at the nucleation, elongation and maturation stages of the fibrillation process. A combination of high sensitivity isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) have been employed along with spectroscopy and microscopy to evaluate interaction of the ILs at each stage of fibrillation quantitatively. Choline glycinate is observed to provide maximum stabilization to insulin compared to that provided by choline prolinate, choline leucinate, and choline valinate. This increased thermal stabilization has direct correlation with the extent of reduction in the fibrillation of insulin by ILs determined using Thioflavin T and 8-anilinonaphthalene sulfonate based fluorescence assays. ITC has permitted understanding nature of interaction of the ILs with the protein at different fibrillation stages in terms of standard molar enthalpy of interaction whereas DSC has enabled understanding the extent of reduction in thermal stability of the protein at these stages. These ILs are able to completely inhibit formation of insulin aggregates at a concentration of 50 mM. Stabilization of proteins by ILs could be explained based on involvement of preferential hydration process. The work provides biocompatible IL based approach in achieving stability and prevention of fibrillation in insulin.
Dyes are being increasingly utilized across the globe, but there is no appropriate method of bioremediation for their full mineralization from the environment. Laccases are key enzymes that help microbes to degrade dyes as well as their intermediate metabolites. Various dyes have been reported to be degraded by bacteria, but it is still unclear how these enzymes function during dye degradation. To effectively eradicate toxic dyes from the system, it is essential to understand the molecular function of enzymes. As a result, the interaction of laccase with different toxic dyes was investigated using molecular docking. Based on the highest binding energy we have screened ten dyes with positive interaction with laccase. Evaluating the MD simulation results, three out of ten dyes were more stable as potential targets for degradation by laccase of Bacillus subtilis. As a result, subsequent research focused solely on the results of three substrates: pigment red, fuchsin base, and Sudan IV. Analysis of MD simulation revealed that pigments red 23, fuchsin base, and Sudan IV form hydrogen and hydrophobic bond as well as Vander Waals interactions with the active site of laccase to keep it stable in aqueous solution. The conformation of laccase is greatly altered by the inclusion of all three substrates in the active site. The MD simulation findings show that laccase complexes remain stable throughout the catalytic reaction. Therefore, this research provides a molecular understanding of laccase expression and its role in the bioremediation of the pigments red 23, fuchsin base, and Sudan IV.
Communicated by Ramaswamy H. Sarma
Four types of amino acid ionic liquids (AAILs) with chiral structure were used to modify Candida antarctica lipase B (CALB). The results showed that the catalytic activity at different temperatures and pH, thermostability, and tolerance to organic solvents of all modified lipases were improved. The composition and configuration of modifiers have great influence on the catalytic performance of the modified lipases. AAILs composed of l-proline exhibited better modification effect than those containing d-proline. The use of [N-AC-l-Pro] [Cl] led to the highest modification degree (47.92 %) of the lipase, which exhibited the highest hydrolytic activity (430.67 U/g), as well as enhanced thermal stability and tolerance to organic solvents. The structure of CALB was characterized by circular dichroism (CD) and fluorescence spectroscopy. It was found that the introduction of a modifier changes the secondary structure of CALB to a certain extent, and the microenvironment around the fluorescent group changed slightly. The structural stability of CALB modified with [N-AC-l-Pro] [Cl] and the mechanism of reaction were studied by molecular dynamics simulations. The molecular dynamics simulations of the native and modified CALB were performed for 20 ns at 300 and 328 K. The simulation results showed that the root mean square deviation (RMSD) and total energy of modified CALB were less than those of native CALB, indicating a more stable structure for the modified CALB. The root mean square fluctuation (RMSF) calculations showed that the rigidity of the modified CALB and the flexibility of the active center region were both enhanced. The solvent accessibility area (SASA) calculations showed that both hydrophilicity and hydrophobicity of the modified enzyme-protein were improved. The increase in radial distribution function (RDF) of water molecules confirmed that the number of water molecules around the active sites was also increased. Thus, the modified CALB has enhanced structural stability and higher hydrolytic activity towards the triglyceride substrates.
A new laccase gene from newly isolated Bacillus licheniformis TCCC 111219 was actively expressed in Escherichia coli. This recombinant laccase (rLAC) exhibited a high stability towards a wide pH range and high temperatures. 170% of the initial activity was detected at pH 10.0 after 10-d incubation, and 60% of the initial activity was even kept after 2-h incubation at 70 °C. It indicated that only single type of extreme environment, such as strong alkaline environment (300 K, pH 12) or high temperature (370 K, pH 7), did not show obvious impact on the structural stability of rLAC during molecular dynamics simulation process. But the four loop regions of rLAC where the active site is situated were seriously destroyed when strong alkaline and high temperature environment existed simultaneously (370 K, pH 12) because of the damage of hydrogen bonds and salt bridges. Moreover, this thermo- and alkaline-stable enzyme could efficiently decolorize the structurally differing azo, triphenylmethane, and anthraquinone dyes with appropriate mediator at pH 3.0, 7.0, and 9.0 at 60 °C. These rare characteristics suggested its high potential in industrial applications to decolorize textile dyeing effluent.
Conformational stability is an important criterion for proteins applied in food, biopharmaceutical, and biochemical industries. The fluctuations of temperature during production, transit, and storage of proteins tend to alter the inherent structure and biological activity of proteins. Among the chemical additives explored for the stabilization of proteins, cholinium-based ionic liquids (ILs) have emerged as a promising solvation medium to preserve the native structure of proteins. By pairing with the amino acids as an anionic counterpart, the cholinium aminoates have a good biocompatibility for biopharmaceutical applications. Using molecular dynamics simulations, we investigated the stability of insulin aspart, a protein-based drug, in cholinium aminoates made of different amino acids. Our simulation results revealed for the first time that the structural stability of insulin aspart in aqueous solution containing cholinium prolinate ([Ch][Pro]) was exceptionally high. The stabilizing effect was mainly contributed by the large size and hydrophilicity of the [Pro] anion as well as the interaction of the [Pro] anion with insulin aspart. The results also indicated that the conformational dynamics of insulin aspart were dependent on the concentration and type of ionic constituents in the IL medium. These positive observations highlight the possibility of utilizing “bio-ionic” liquids as preservation media for the formulation of protein-based products.
Herein, we performed meticulous experimental and computational studies to explore the stability, activity, and dynamics of laccase in the presence of various organic and inorganic solvents. It is well known that laccases are eco-friendly enzymes, which can quickly eliminate recalcitrant chemicals from contaminated media. We determined the Asp96 (COO⁻)---Arg43 (N-H2), Asp131 (COO⁻)---Arg197(N-H1), Asp138(COO⁻)---Arg195 (N-H2), Asp140(COO⁻)---Arg199(N-H2), Asp214(COO⁻)---Arg260(N-H2), Asp224(COO⁻)---Arg423(N-H2), Asp424(COO⁻)---Arg243(N-H1), Asp424(COO⁻)---Arg243(N-H2), Glu288(COO⁻)---Arg176(N-H1) and Glu288(COO⁻)---Arg176(N-H2) salt bridges as the crucial ones in maintaining the structural integrity of laccase. Furthermore, the fluorescence, circular dichroism (CD) spectroscopies, and molecular dynamics simulation outcomes highlighted that the secondary structure elements and tertiary structure of the enzyme did not change significantly in the presence of both selected organic (ethanol and hexane) and inorganic (1-Butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]) co-solvents. The obtained experimental results demonstrated that the enzyme was more stable in the hexane and aqueous ionic liquid solutions than in the ethanol solution. Additionally, laccase exhibited more activity in the presence of the hexane and aqueous ionic liquid solutions. Probably due to more accessibility of the substrate to the active site of laccase. Interestingly, the activity of laccase in the presence of the co-solvents was in decreasing order of hexane<[BMIM][PF6] < ethanol and the same order was observed for the number of water molecules in the enzyme's hydration shell. The results also indicated that the biocatalyst may keep a balance between the interactions with both water molecules and co-solvents to maintain its native conformation. The results also showed that there were some co-solvent molecules in the first hydration shell of the enzyme, but they were not enough to make a considerable change in the enzyme structure.
In this study, we report how similarly two serum albumins (bovine serum albumin (BSA) and human serum albumin (HSA)) respond in the presence of different concentration of aromatic amino acid based ionic liquids (AAILs), which are cholinium tryptophan [CHO][Trp]IL and tetraethylammonium tryptophan [TEA][Trp]IL. Extended results of thermodynamic stability indicate the extent to which both serum albumins differ in their thermal stability despite having structural similarity in presence of AAILs. To efficiently quantify the results, biomolecular interactions studies were carried out between serum albumins and AAILs with the help of differential scanning calorimetry (DSC), dynamic light scattering (DLS) and various spectroscopic techniques. DSC results illustrated that both AAILs are increasing the thermal stability of BSA and HSA, as per transition temperature (Tm) values, BSA (65.51 to 72.46 °C) and HSA (65.46 to 75.97 °C) have more thermal stability in the presence of [CHO][Trp]IL as compare to [TEA][Trp]IL, BSA (65.51 to 69.75 °C) and HSA (65.46 to 72.08 °C). Secondary structure results obtained using Dichroweb software and selcon calculations. Furthermore, to illustrate the specific binding of AAIL's cations or anions with the binding sites of BSA and HSA, the molecular docking studies were also performed using Molegro trail version v 6.0.
The demand for the long term storage and stability of proteins has increased substantially in the pharmaceutical industries, yet the sensitivity of proteins towards environment has become a cardinal task for researchers. To deal with, we have selected a multifunctional enzyme Cytochrome-c (Cyt-c) involved in many chemical and biochemical reactions as model protein, which is very sensitive and loses structural integrity on exposure to environment. The remarkable features of ionic liquids (ILs) have entitled them as alternatives to aqueous and organic solvents for solubility, storage and surrogate reaction medium. Hence, we have adapted the biocompatible and non-toxic cation and anion based amino acid ILs (CAAAILs) as potential solvents for storage and stability of Cyt-c. Herein, we report the molecular insights and thermodynamics of interaction between CAAAILs and Cyt-c with the help of isothermal titration calorimetry (ITC), transmission electron microscopy (TEM), UV-VIS, CD and fluorescence spectroscopy as well as molecular docking and molecular dynamics (MD) simulations. The structure and stability of Cyt-c remains unchanged in presence of CAAAILs. Both electrostatic and hydrophobic interactions are accountable for the binding of CAAAILs in the region between terminal helices and loop of Cyt-c through non-specific multiple binding sites, which can be exploited for storage and stability of proteins and will be helpful in designing new biobased ILs for biochemical applications.
This is a personalized URL providing free access to this article before April 09, 2020:
https://authors.elsevier.com/a/1abYy8jaZDlFng
The research on alternative solvents and co-solvents is a relevant aspect when envisioning the improvement of biocatalytic reactions. Among these solvents and co-solvents, deep eutectic solvents (DES) may be considered as customizable new reaction media for biocatalysis. Accordingly, in this work, sixteen DES aqueous solutions, as well as of the individual DES components at the same conditions have been investigated in laccase-catalyzed reactions. Cholinium- and betaine-based DES formed with polyols at different molar ratio and concentrations were evaluated. The results reported show that in presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen-bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in additional tests under extreme storage temperatures (60 ºC and -80 ºC). Although no significant protection to high temperatures was afforded by DES, an enhanced laccase activity when stored at low temperatures was found, at least up to 20 days. Combining experimental results and molecular docking this work shows that DES can be designed as co-solvents to improve biocatalytic reactions.
The major concerns about protein-based drugs are their stability i.e. maintaining the protein in the folded state throughout processing and storage, as well as the preparation of novel formulation. Stabilization of the monomeric form of insulin (In) under the condition of low pH has been a recent challenge. In our earlier investigation, we found that 1-butyl-3-methylimidazolium-based ionic liquids (ILs) containing acetate, trifluoroacetate or dicyanamide anions enhance In thermal stability and prevent protein aggregation. In the present study, six non-toxic ILs containing biocompatible cholinium cation [Chol] and an anion charged amino acid (asparginyl (Asp), glutaminyl (Glu), lysinyl (Lys) and arginyl (Arg)) were synthesized using a two-step procedure. Their effect of the ILs on the In secondary structures was evaluated using FTIR spectroscopy. Rearrangement in the protein molecules, an increase in the beta-structures on behalf of the α-helices, partial denaturation but no aggregation was observed in all In solutions containing ILs. Differential scanning calorimetry was applied to elucidate the effect of ILs on thermal stability of In. Interestingly, in presence of [Chol][Glu] and [Chol] 2 [Asp] the denaturation temperature of the In was shifted to higher temperatures with 9.6 and 4.1 °C, respectively.
Enzymes are very imperative components which are vital for existence of every cellular life. There is significant interest in the use of structurally stable and catalytically active enzymes in pharmaceutical, food, fine chemicals industries and various industrial processes as catalyst. Stem bromelain (BM) is a proteolytic enzyme which is widely used in chemical, medical and pharmaceutical field. However, harsh process conditions are main barriers to the effective use of this enzyme in different applications. To overcome these drawbacks, biocompatible bio-based ionic liquids (ILs), composed of the choline cation (an essential nutrient) and different anions. The ILs include choline chloride [Ch]+[Cl]−, choline acetate [Ch]+[Ac]−, choline dihydrogen phosphate [Ch]+[Dhp]−, choline bitartrate [Ch]+[Bit]−, choline iodide [Ch]+[I]− and choline hydroxide [Ch]+[OH]− are chosen for the current work. Therefore, in the present study, structural stability and activity of BM have evaluated in the presence of choline-based ILs using various biophysical techniques at different concentrations. Present work demonstrated that [Ch]+[OH]− is the strongest destabilizer, whereas [Ch]+[Cl]− is best stabilizer for the native structure of BM among all studied ILs. This work revealed that the suitability of some choline-based ILs as potential media for the sustained stability and activity of BM.
The potential of ionic liquids (ILs) as green solvent has opened burgeoning new areas of its application in various scientific and engineering fields. However, all ILs cannot be considered as “green solvent” due to toxicity and biodegradability issues associated with them. Therefore, the need of the hour is to synthesize green ILs in which efforts should be made to include cations and anions of the ILs from biological source. In this regard, choline glycine ([Ch][Gly]) has been synthesized in which both the ions belong to the bio-available sources. The stability and activity of stem bromelain (BM) have been studied in presence of choline bromide ([Ch][Br]) and [Ch][Gly]. The comparison of activity and stability of BM in both ILs have been performed using spectroscopic techniques and size parameter has been assessed using dynamic light scattering (DLS). On contrary to our expectation [Ch][Gly] proved to be a poor stabilizer for the BM when compared to [Ch][Br]. This can be due to the fact that in case of [Ch][Gly], Glycinate anions are forming strong H-bonds with the enzyme polypeptide backbone which dissociate the H-bonds that maintain the structural integrity of protein, while no such kind of interaction is present in case of [Ch][Br].
In recent years, the potential of α-chymotypsin (CT) as biocatalysts has mushroomed new areas of its application ranging from pharmaceutical to chemicals industries. However, high thermal stability is one of the major challenges to the use of this enzyme in biocatalysis. In this regards, ionic liquids (ILs) have been used as promising media for the stabilization and preservation of proteins, enzymes, DNA and other biomolecules. In present study, it was found that a series of cholinium-based ILs such as choline acetate ([Ch][Ac]), choline chloride ([Ch][Cl]), and choline dihydrogen phosphate ([Ch][Dhp]) stabilised the CT structure against thermal denaturation. The transition temperature (Tm) of CT was increased from ~48.9 °C (in buffer) to 58 °C (in ILs media). Enzymatic activity of CT in the presence of ILs was also monitored by using casein as the substrate. It was found that choline dihydrogen citrate ([Ch][Dhc]) and choline hydroxide ([Ch][OH]) dramatically decrease the enzyme activity. While both structural stability and enzymatic activity was retained in [Ch][Ac], [Ch][Cl] and [Ch][Dhp] indicating the suitability of these ILs as a high temperature bio-catalytic reactor. Our results revealed that [Ch][Ac] is best stabilizer among all studied ILs for the native structure of CT whereas [Ch][OH] is strongest destabilizer for CT structure. The outcome of our results can be helpful to overcome some of the major limitations found in the development of biocatalysis processes
The ever-increasing demand for determining compounds at low concentration levels in complex matrices requires a preliminary step of analytes isolation/enrichment in order to employ a detection technique characterised by high sensitivity at low LOQ. Sample preparation is considered as crucial part of analytical procedures. Previously the parameter of „greenness” is as important as selectivity in order to avoid using harmful organic solvents in sustainable extraction techniques. These solvents can generate hazardous, toxic waste while consuming large resources volume. Developing new green solvents is one of the key subjects in Green Chemistry in order to reduce the intensity of anthropogenic activities related to analytical laboratories. A lot of new, more eco-friendly media have been employed as extractant phases. These media, besides of being more eco-friendly, provide shorter extraction times, simplicity, low cost, better selectivity in some cases. The most promising, most widely used green extraction solvents are described in this review.
A series of phenolic/non-phenolic biphenyl model compounds mimicking 5-5′ type “condensed” lignin substructures were subjected to the oxidation with laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole (HBT) or [SiW11VO40]5− (SiW11V) as mediators. Phenolic models suffered a significant degradation in both the laccase-mediator systems (LMS), which was more pronounced, however, in the case of SiW11V-mediated oxidation. This result was explained, at least partially, by HBT decomposition and by the increased extent of competing radical coupling reactions of phenolic models in the HBT–laccase reaction system. The non-phenolic biphenyl model was non-reactive in the presence of SiW11V and degraded substantially in the presence of HBT. The main degradation pathways of lignin model compounds were deduced based on the analysis of the detected oxidation products.
Previous attempts to simulate phospholipid bilayers using the General Amber Force Field (GAFF) yielded many bilayer characteristics in agreement with experiment, however when using a tensionless NPT ensemble the bilayer is seen to compress to an undesirable extent resulting in low areas per lipid and high order parameters in comparison to experiment. In this work, the GAFF Lennard-Jones parameters for the simulation of acyl chains are corrected to allow the accurate and stable simulation of pure lipid bilayers. Lipid bilayers comprised of six phospholipid types were simulated for timescales approaching a quarter of a microsecond under tensionless constant pressure conditions using Graphics Processing Units. Structural properties including area per lipid, volume per lipid, bilayer thickness, order parameter and headgroup hydration show favourable agreement with available experimental values. Expanding the system size from 72 to 288 lipids and a more experimentally realistic 2 × 288 lipid bilayer stack induces little change in the observed properties. This preliminary work is intended for combination with the new AMBER Lipid11 modular force field as part of on-going attempts to create a modular phospholipid AMBER force field allowing tensionless NPT simulations of complex lipid bilayers.
The participation of biological agents in pulp bleaching systems has received a lot of attention from research teams around the world, driven by the environmental benefits that biobleaching could bring. Nature showed us the ability of some of its agents, such as wood-decaying fungi, to delignify and bleach wood and wood pulp. What we need to do is to enhance the efficiency of such agents to make them cope with the fast pace of our modern pulp mills. To do so, a profound understanding of the biobleaching system is required. Our efforts to discover new efficient mediators for the laccase-mediator system (LMS) brought us to use several techniques to analyse the reactions involved in mediated enzymatic delignification. Mostly based on electrochemistry, these techniques are reviewed in this paper, along with key results. Cyclic voltammetry was used to characterize electron transfer rates between each element of the LMS. We found, along with other authors, that the mediator redox potential has a great influence on its efficiency. We used bulk electrolysis to simulate the oxidative action of laccase on mediators and model compounds of lignin. Such electrolysis techniques allowed us to study mediated lignin oxidation outside of normal laccase working conditions. Finally, an electrolysis-based method for mediated pulp delignification that we developed, based upon our research on biobleaching, is presented.
The activity, stability and conformation of laccase were first investigated in an aqueous solution of ionic liquids 1-butyl-3-methylimidazolium trifluoromethanesulfonate ([Bmim]TfO), 1-butyl-1-methylpyrrolidinium trifluoromethanesulfonate ([Bmpyr]TfO) or tetramethylammonium trifluoromethanesulfonate ([TMA]TfO). Compared with control system, high level of [Bmim]TfO or [Bmpyr]TfO destabilizes laccase while [TMA]TfO stabilizes laccase. These effects are more pronounced with the extension of the incubation time. The activity variations are well correlated with the changes of the conformation of laccase evidenced by fluorescence and circular dichroism spectra under specified conditions. The effects of the three ionic liquids on laccase are associated with the chaotropicity of the cations in Hofmeister series. For laccase, [TMA]TfO is not a good activating agent but it greatly enhances the stability of laccase in addition to maintaining the catalytic efficiency of laccase, showing its great potential in real application.
The stability and reactivity of five different thermostable fungal laccases from the species Trametes hirsuta,
Melanocarpus albomyces, Thielavia arenaria (two laccases) and Chaetomium thermophilum were investigated
in the presence of organic solvents. Oxidations of small organic phenolic compounds, matairesinol
and 7-hydroxymatairesinol lignans, as well as synthetic lignin dehydrogenation polymer DHP in aqueous
solutions of ethanol and propylene glycol solvents were investigated using analysis of oxidation rates,
high performance liquid chromatography and size-exclusion chromatography. The laccases showed variability
in their solvent tolerance. The redox potential of the laccases appeared not to be the main factor
determining the efficiency of the polymerization reactions of complex phenolic model compounds in
aqueous organic solutions. Nuclear magnetic resonance spectroscopic analysis of laccase treated DHP
in 50% propylene glycol indicated that the formation of new biphenylic 5–5� structures was favored in
laccase-catalyzed radical coupling reactions over the other possible reactions through the phenolic groups
forming new 5 O 4 ether bonds. The polymerization reactions took place even at high concentrations
of solvents, which already inhibited the enzyme activity, encouraging enzymatic upgrading of lignin in
organic solvents to be studied further. Thus, it was confirmed that thermostable laccases are potential
enzymes for various industrial applications where organic solvents are required for the reaction systems.
The stability and catalytic activity during oxidation of syringaldazine (SGZ) by laccase from Cerrena unicolor (CUL) was studied in aqueous solutions of ethanol, acetone and dimethyl sulfoxide (DMSO). Additional, the oxidation of other two substrates, catechol (CAT) and 2,6-dimethoxyphenol (DMOP), was studied in ethanol and in DMSO solutions, respectively. It was observed that in 3 M solutions of the studied solvents CUL was practically stable for 30 min of the experiments, which show its good applicability in solutions of water-miscible organic solvents. Moreover, it was observed that the effect of the tested organic solvents on the maximum rate of oxidation values (Vmax) of different substrates was not dependent on the substrate. Within the studied range of concentrations (up to 8 M) ethanol did not affect Vmax values and the presence of DMSO reduced it at roughly the same rate for the studied substrates. In the case of SGZ as a substrate, the effect of the solvent on Vmax values increased in the order ethanol < acetone < DMSO and in the case of KM in the order DMSO < acetone < ethanol. The experimental relationships between the content of organic solvents and the observed kinetic parameters were numerically described using the exponential relationships on the solvent concentrations, in the first approximations replacing the variability of the activity coefficients of the reactants. The fitted exponential coefficients were observed with the values observed for other fungal laccases and discussed.
This paper describes electrochemical behavior of laccase from the fungus Trametes versicolor. The issues related to discrimination of the redox potentials corresponding to copper centers T1 and T2/T3 in the active site and possible mechanism of intramolecular electron transfer have been discussed. The electron-transfer rate constant for laccase immobilized on carbon electrode is 3.4 s−1. The bioelectrocatalytic activity of the enzyme was studied in the presence of 1,4-hydroquinone (HQ). The kinetics of HQ oxidation is very fast (KM=3.8 μM). However, the catalytic activity of laccase in the presence of high concentration of HQ decreases drastically. It is suggested that the T2/T3 copper center is able to accept electrons from HQ molecules directly via intramolecular channel.
Laccase (E.C. 1.10.3.2) from Trametes versicolor was immobilized (adsorbed) by drying on various supports (glass, glass powder, silica gel, and Nylon 66 membrane). The enzyme activity and stability were determined in diethyl ether, ethyl acetate, and methylene chloride. The initial rate for the oxidation of syringaldazine varied up to 245-fold depending on the solvent and support, the best results being obtained with Nylon 66 membrane. No inactivation of immobilized laccase over 72h was observed in diethyl ether and ethyl acetate, while exposure to methylene chloride resulted in significant activity decreases regardless of the support material.
VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more "representations," in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.