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Today, due to the excessive use of plastic products, the pollution caused by them is increasing, and an efficient management system is needed to reduce their environmental effects. It is noteworthy that the risk of human ingestion can increase when plastic waste degrades into microplastics (MPs) and nanoplastics (NPs). Additionally, as plastic ages and decomposes, common additives will continue to be released into the environment, having an incalculable and long-lasting harmful effect on the environment. As a result, several treatment strategies, such as recycling or upcycling, and degrading, have been evolved for the cleanup of this waste. Additionally, techniques for identifying and removing MPs, NPs, and plasticizers have been created. Researchers working in this area have recently become interested in carbon dots (CDs), a novel class of carbon-based nanomaterial, due to their simple and inexpensive synthesis from a variety of sources, fluorescence properties, and advantageous characteristics such as excellent biocompatibility, distinctive physicochemical properties, an abundance of functional groups (such as amino, hydroxyl, and carboxyl), high stability, and charge transport. Therefore, this article reviews the role of innovative CDs in the fate of plastics (waste, MPs, and plasticizers), including upcycling, sorting, detection, and photocatalytic degradation. Finally, a brief description of the issues and potential directions for future research in this area is provided. [Display omitted] • Plastic waste and their harmful substances are abundant in different environments. • The role of CDs for plastic waste remediation are reviewed. • Upcycling and sorting of plastic waste are explained. • Detection and photocatalytic degradation of microplastics and plasticizers are presented. [ABSTRACT FROM AUTHOR]

The glycolysis is a common characteristic of cancer and it is responsible for providing enough energy to ensure growth. The glycolysis suppression is beneficial in tumor growth reduction. The stimulation/inhibition of glycolysis in cancer is tightly regulated by ncRNAs. The regulation of glycolysis by ncRNAs can influence proliferation and therapy response of tumor. The miRNAs are capable of inactivating enzymes responsible for glycolysis and suppressing signaling networks resulting in glycolysis induction. By regulation of glycolysis, miRNAs can affect therapy response. The lncRNAs and circRNAs follow a same pathway and by targeting glycolysis, they affect progression and therapy response of tumor. Noteworthy, lncRNAs and circRNAs sponge miRNAs in glycolysis mechanism control in tumor cells. Furthermore, ncRNA-mediated regulation of glycolysis mechanism can influence metastasis to organs of body. The ncRNAs regulating glycolysis are reliable biomarkers in cancer patients and more importantly, exosomal ncRNAs due to their presence in body fluids, are minimally-invasive biomarkers. [Display omitted] [ABSTRACT FROM AUTHOR]

Low oxygen level at tumor microenvironment leads to a condition, known as hypoxia that is implicated in cancer progression. Upon hypoxia, HIF-1α undergoes activation and due to its oncogenic function and interaction with other molecular pathways, promotes tumor progression. The HIF-1α role in regulating breast cancer progression is described, Overall, HIF-1α has upregulation in breast tumor and due to its tumor-promoting function, its upregulation is in favor of breast tumor progression. HIF-1α overexpression prevents apoptosis in breast tumor and it promotes cell cycle progression. Silencing HIF-1α triggers cycle arrest and decreases growth. Migration of breast tumor enhances by HIF-1α signaling and it mainly induces EMT in providing metastasis. HIF-1α upregulation stimulates drug resistance and radio-resistance in breast tumor. Furthermore, HIF-1α signaling induces immune evasion of breast cancer. Berberine and pharmacological intervention suppress HIF-1α signaling in breast tumor and regulation of HIF-1α by non-coding RNAs occurs. Furthermore, HIF-1α is a biomarker in clinic. [Display omitted] [ABSTRACT FROM AUTHOR]

G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV–vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures. [ABSTRACT FROM AUTHOR]

Abstract: The effect of proline on refolding and unfolding kinetics as well as activity of lipase from Pseudomonas fluorescens was determined using stopped-flow fluorescence and UV/Vis absorbance spectroscopy. Enzyme assay at different concentrations of proline revealed that activity of enzyme reaches maximum at 0.6M concentration of proline. Kinetic measurements showed that refolding rate is considerably accelerated in the presence of 0.6M proline. Unfolding kinetic traces were fitted to double exponential function, and it was revealed that lipase molecules were unfolded via two different pathways. Fast unfolding rate constant decreased, while slow one did not change significantly upon addition of proline. [Copyright &y& Elsevier]

Abstract: Like many other bacterial lipases Pseudomonas aeruginosa lipase has a very strong structure–function relationship. Herein, the effect of structural compactness of lipase has been investigated on its activity. P. aeruginosa HR59 was isolated from burn infection as a new and inexpensive source. Bacterial lipase was extracted from this strain and purified by ion-exchange chromatography. Two divalent metal ions (calcium and magnesium) and three polar organic solvents (methanol, ethanol and iso-propanol) were used to study the structure–function relationship. Results of this study revealed that these additives activated enzyme by reducing the helix content of enzyme. Moreover, thermo stability of the enzyme decreased in the presence of calcium and magnesium ions; whereas it increased upon interaction with polar organic solvents under heat-induced denaturation. Results of this investigation encourage utilization of these activity enhancers for various medical and industrial applications. [Copyright &y& Elsevier]

Abstract: The unique morphology of anisotropic rod-shaped gold nanostructures has offered new prospects for biomedical and biosensing applications. This study investigates the interaction of two types of rod-shaped nanostructures, gold nanorods and gold nanorices with lysozyme as a model protein, comparing the probable structural, activity and kinetic stability alterations. Circular dichroism spectropolarimeter revealed that lysozyme retains high fraction of its native conformation in the presence of both nanostructures, with a slight increase in the helical and beta content. Upon the protein adsorption on both types of nanorods, kinetic studies showed maintenance of enzymatic activity, together with increase in the enzymatic affinity and kinetic stability at high temperature. Comparatively, gold nanorice induced better effect on the activity and stability of enzyme than that of gold nanorod. This study might open new insight into potential applications of gold nanorods as nanocarriers for genes and drugs; provided that the toxicological aspect of cationic surfactant-coated nanostructure is taken into consideration. [Copyright &y& Elsevier]

Abstract: The stability of enzymes with no reduction in their catalytic activity still remains a critical issue in industrial applications. Naturally occurring osmolytes are commonly used as protein stabilizers. In this study we have investigated the effects of sorbitol and trehalose on the structural stability and activity of Pseudomonas cepacia lipase (PCL), using UV–visible, circular dichroism (CD) and fluorescence spectroscopy. Surface plasmon resonance (SPR) technique was used to trace changes in the refractive index and dielectric constant of the environment. The results revealed that catalytic activity and intrinsic fluorescence intensity of PCL increased in the presence of both osmolytes. Far-UV CD spectra indicated that the protein has undergone some conformational changes upon interacting with these osmolytes. Increasing the concentration of sorbitol led to changes in the refractive index and consequently the dielectric constant of environment; whereas in the case of trehalose, such changes were not significant. Unfavorable interactions of trehalose with protein surface induced higher preferential exclusion from the enzyme–water interface than that of sorbitol. Results of this report could give further insights about the stabilization mechanism of osmolytes. [Copyright &y& Elsevier]

Abstract: Gold nanorods, with their unique morphology and optical properties have offered new prospects for biomedical and biosensing applications. Herein, the interaction between gold nanorods and a model protein has been monitored using spectroscopic techniques. The enzyme retains high fraction of its native structure with a slight increase in the helical content at the expense of β-turns. Kinetic investigations revealed notable increase of enzyme affinity for substrate without significant decrease in the V max. Emission spectra of tryptophan residues in the presence of chaotropic agent highlighted the maintenance of internal quenching due to the induced compactness. Comparison of the gold nanorod treated lysozyme with free enzyme revealed higher thermodynamic stability under denaturing condition. Results from this study encourage the possibility of utilizing gold nanorods as promising nanocarrier candidates for a new generation of drug delivery applications. [Copyright &y& Elsevier]

Abstract: Firefly luciferase is widely used in many analytical techniques. However, the enzyme is unstable, so that its relative inactivation results in low sensitivity of those techniques. In this study, we have investigated the effects of MgSO4 and trehalose on the structural stability and function of luciferase from Photinus pyralis using circular dichroism (CD), conventional and stopped-flow fluorescence spectroscopy and bioluminescence assay. The secondary structural content, compactness and its melting temperature are also studied, which showed that the stability of luciferase increased in the presence of additives. Measurements of refolding rate constants under conditions that favor folding, show that MgSO4 accelerates the folding of enzyme, on the contrary, refolding rate constant decreases in the presence of trehalose which can be attributed to its high viscosity. Finally, combined with remaining activity assay we concluded that magnesium sulfate and trehalose can be used for short- and long-term stabilization, respectively. [Copyright &y& Elsevier]

Abstract: Horseradish peroxidase is an important heme-containing plant enzyme with enormous medical diagnostic, biosensing, bioremediation and biotechnological applications. Any improvement in the stability of the enzyme will greatly enhance its application in mentioned areas. In the present study, the stabilizing effects of certain additives and chemical modification by citraconic anhydride on the thermal behavior of HRP were investigated. Both strategies brought about dramatic enhancement of the thermostability of the enzyme. Results obtained on T m, changes in the circular dichroism (CD) spectra and kinetic parameters of HRP and its modified form are discussed in terms of contributions to the mechanism of the thermal stability and the activity enhancement. Polyols were very effective in providing protection against the irreversible thermoinactivation of the native and the modified forms of the enzyme. Our results reveal that a combination of medium change and surface modification may provide an effective strategy for the enhancement of the thermodynamic and the kinetic stability of the enzyme. [Copyright &y& Elsevier]

We had previously investigated the role of a loop on the activity and conformational stability of chondroitinase ABC Ι (cABC Ι) by constructing some representative mutants in which a network interaction around Asp 689 was manipulated. Here we extended our study by measuring the proteolytic resistance, long term and thermal stability as well as unfolding kinetics of these variants. Long term stability data at 4 and 25 °C for 3 weeks indicates that all mutants remain considerably active at 4 °C. Thermoinactivation rates for all variants shows that the wild type (WT) enzyme retained 50% of its activity after 2 min keeping at 40 °C, while L701T, H700N and H700N/L701T as conformationally stabilized variants, have slower inactivation rate. It was also found that compact and thermodynamically stabilized variants are more resistant to tryptolytic digestion. Also, kinetic curves of chemical unfolding of the enzyme variants from stopped-flow fluorescence measurements were best fitted into a three-exponential function with three rate constants and corresponding amplitudes. We found that the energy barrier of the fast unfolding phase is lower in stabilized variants; while the amplitude of this phase to the whole amplitude of the unfolding reaction is lower than that of destabilized variants, indicating more population of stabilized mutants unfold via slower unfolding phase. We concluded that the rate of local conformational change alone is not the same that is expected from global thermodynamic stability; however the corresponding amplitude can compensate the rate constant toward thermodynamic stability. [ABSTRACT FROM AUTHOR]

Highlights: [•] In vitro refolding of recombinant bacterial laccase from Bacillus HR03 is studied. [•] We have shown that the drop dilution as a powerful method for refolding of laccase. [•] Differences in kinetic parameters of soluble and refolded enzyme were also observed. [•] Structural changes during the refolding monitored using fluorescence spectroscopy [Copyright &y& Elsevier]

Abstract: Laccases (benzenediol oxygen oxidoreductases, EC 1.10.3.2) are used in many biotechnological processes, including removal of polyphenols in beverages, decolorizing and detoxifying effluents, drug analysis and bioremediation. In the present work, we have tried to increase thermal stability of laccase from Bacillus HR03 using site directed point mutations. Glu188 was substituted with 2 positive (Lys and Arg) and one hydrophobic (Ala) residues. All mutations showed improved thermal stability. Thermal activation of laccase was also increased after introducing the mutations. Remarkably, the Glu188Lys variant showed 3-fold higher thermal activation and higher T 50 (5°C) with respect to the native enzyme. Furthermore steady-state k cat and K m values were influenced despite the distance between the mutated position and the catalytic site. In Glu188Arg mutation, the k cat was improved 3-fold and K m reduced by 25%. Interestingly, all three variants showed higher stability against urea as a chemical denaturant. Structural analyses of the native and mutated variants were carried out using fluorescence and far-UV circular dichroism. [Copyright &y& Elsevier]

Abstract: A thermophilic strain, L14, isolated from Iranian hot springs, produces an extracellular pullulanase upon growth on optimized liquid medium. According to 16S rDNA analysis and biochemical tests, L14 seems to be a Geobacillus stearothermophilus strain. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion-exchange and gel filtration chromatographies with a yield of 30%. The purified enzyme showed a single band on SDS-PAGE with an estimated molecular mass of about 100kDa for monomeric enzyme. Optimal conditions for the activity of the purified pullulanase were calculated as pH 5.5 and T opt =65°C. A reasonable stability was also observed at 60–70°C. The enzyme could hydrolyze pullulan, starch and malto-oligosaccharides and was unable to degrade α-cyclodextrin and panose. The apparent K m and V max for the enzyme activity on pullulan were 0.48g/l and 44μmolmin−1 and for soluble starch were 5g/l and 80μmolmin−1, respectively. The products of enzymatic reaction on pullulan were glucose, maltose and maltotriose, whereas conversion of starch produced glucose and maltose. This enzyme could also hydrolyze different types of malto-oligosaccharides like maltotriose, maltotetraose, maltopentaose, maltohexaose and maltoheptaose. Consequently, the enzyme attacked both α-d-(1→4) and α-d-(1→6) glycosidic linkages of pullulan and branched polysaccharides like starch and produced glucose and maltose. Therefore, it is assumed that the newly purified amylopullulanase from Geobacillus sp. L14 is the first report on the presence of a specific debranching enzyme which can produce glucose from pullulan. [Copyright &y& Elsevier]

Abstract: The industrial utilization of savinase is mainly constrained by its stability limitations. In the present study, the irreversible thermoinactivation of savinase has been evaluated at 70°C, and various possible mechanisms for irreversible thermoinactivation of savinase were examined. The main process seemed to be autodigestion of savinase at higher temperatures. To improve the thermal stability of the enzyme, the effect of two co-solvents (sorbitol and trehalose) on the enzyme''s activity and stability was investigated. Both osmolytes prevented the autolysis of savinase at 70°C without inactivating the enzyme; furthermore, the structural and kinetic stabilities of the enzyme increased in the presence of additives. [Copyright &y& Elsevier]

Abstract: Bacillus licheniformis α-amylase (BLA) is an industrially important extracellular enzyme with a number of applications. In the present work, an investigation was carried out on the tryptolytic digestion of BLA which produced two fragments, TF18K and TF38K, and no further fragments could be seen after 6h incubation of BLA with trypsin. The fragments were isolated by preparative gel electrophoresis and reverse phase HPLC. The N-terminal sequencing of fragments showed that trypsin attacks on Arg127–Val128 peptide bond in BLA. Intrinsic and acrylamide quenching fluorescence experiments and Far-UV circular dichroism studies showed that substantial changes in the secondary and tertiary structures of the TF18K and TF38K have occurred. Subsequently, polyclonal antibody was raised against TF18K. After purification of the antibody by protein A Sepharose, thermal stability of BLA in the presence of this antibody was determined. Results showed that the presence of antiTF18K leads to significant stabilization of BLA. For example, after 30min incubation at 90°C, residual activity of the enzyme in the presence of antibody (40μg/ml) was determined as 40% while the enzyme showed no activity in the absence of antibody after incubating in the same condition. In addition, it has been proved that calcium enhances the thermal stability of BLA and a synergistic stabilization of BLA has been seen with antiTF18K and calcium, simultaneously. [Copyright &y& Elsevier]

Abstract: Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213–216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure. [Copyright &y& Elsevier]

Detailed circular dichroism (CD), scattering and quenching studies, 1-anilinonaphthalene-8-sulfonate (ANS) binding, irreversible thermoinactivation, activity measurements and proteolytic digestion of bacterial α-amylases have been carried out to elucidate the effect of trifluoroethanol (TFE) on the structure of these enzymes. Under high concentrations of TFE both of the α-amylases, a thermostable α-amylase from Bacillus licheniformis (BLA) and its mesophilic counterpart from Bacillus amyloliquefaciens (BAA), acquire partially folded state characterized by an enhanced content of the secondary structure (helix) and reduced tertiary structures. According to ANS binding studies, we suggest that the TFE states induced by TFE/water mixture are not the molten globule state in the α-amylase folding pathway. In addition, data shows significant reversible aggregation of both enzymes in TFE/water mixtures with concentration between 10 and 60% (v/v). However, reversibility is more in case of BAA. As expected, in the absence of TFE, the thermophilic enzyme compared to mesophilic enzyme, shows a greater resistance to digestion by thermolysin. With respect to fluorescence quenching by acrylamide and potassium iodide, the thermophilic enzyme, BLA, is characterized by higher structural flexibility as compared to the BAA. On the other hand, in the presence of TFE, the enzymes are digested by protease to produce large protein fragments. It is proposed that highly helical secondary structures, acquired by BAA and BLA when dissolved in aqueous TFE, prevent binding and adaptation of the protein substrate at the active site of the protease. [Copyright &y& Elsevier]

Considering importance and several industrial applications of lysozyme, including natural antibiotic and preservative, identifier for the diagnosis of diseases, and extraction purposes, its reversibility and stability studies can be very important. In this paper, the role that buffer and osmolytes concentrations play on the thermodynamic stability of lysozyme denaturation process, that is a new simple and inexpensive method, was evaluated by Nano-DSC III, far- and near-UV CD and fluorescence techniques. In thermal denaturation study, RI and ΔG of protein increased from 25.62% to 58.82% and 48.87 to 63.63 kJ mol−1 with the increment of buffer and osmolytes concentrations, respectively. These changes showed a significant increase of 129.59% in RI and 28.16% in ΔG. The effect of buffer and osmolytes concentrations on the secondary and tertiary structures of protein was also investigated. The results indicated that increment of buffer and osmolytes concentrations increase rigidity and thermodynamic stability of protein. Also, structure of protein may be changed by its interaction with GNPs. Hence, interaction of lysozyme with GNPs was studied at the buffer and osmolytes concentrations that gives the maximum RI and ΔG, respectively. The results showed that molten globule-like state was formed by lysozyme in the presence of GNPs. • The effect of buffer, osmolytes and gold nanoparticles concentrations on lysozyme has been reported. • The role of buffer and osmolytes concentrations in thermodynamic stability has been studied. • Increment of buffer and osmolytes concentrations leads to increase of reversibility index and Gibbs free energy change. • Formation of molten globule-like state in the presence of gold nanoparticles has been investigated. [ABSTRACT FROM AUTHOR]

Laccases are multicopper oxidases that catalyze the oxidation of variety of substrates. The specificity and efficiency of laccases are clearly the important components leading to their remarkable uses. To develop an improved biocatalysts, site directed mutagenesis of laccase from Bacillus HR03 was carried out in the current study. Based on the ABTS-bound crystal structure of CotA from B. subtilis and alignment with closely related enzymes, T415 and T418 at the vicinity of the type 1 copper site were chosen and several mutants (T415I, T418I, T415G, T415G/T418I) were made. Kinetic parameters of the constructs were then determined using ABTS and SGZ as substrates. In comparison with the wild-type, catalytic efficiency toward ABTS was improved by 4 fold in T415I and 1.5 fold in T418I and T415G. T415I and T418I variants were identified to be almost 11 and 27 times more specific for ABTS than for SGZ compared with the wild type. T415I was also found to acquire enhanced thermal stability with the half-life of 60 min at 80 °C. Secondary and tertiary structure of mutants were analyzed by CD and fluorescence spectroscopy. Our result illustrated that replacement of residues in the substrate-binding pocket would change the specificity and efficiency of variants. [ABSTRACT FROM AUTHOR]

Chondroitinase ABC I (cABC I) cleaves glycosaminoglycan chains which are responsible for most of the inhibition of axon regrowth in spinal cord injury. The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is found to prune glycosaminoglycan chains of proteoglycans and facilitate axon regeneration. However, a limiting factor for such application is the enzyme’s instability. In this study, the structure and activity of cABC I have been investigated upon interaction with various concentrations of Gold nanorods. The enzyme preserved its major activity with increase in substrate affinity in the presence of the nanostructures. Analysis of circular dichroism spectropolarimetry data showed that secondary structural content of the enzyme slightly increased. The complex form of the enzyme also showed higher storage stability. Fluorescence studies indicated that enzyme obtained more rigidity in its structure. Taking higher stability of enzyme upon interaction, result of this investigation interaction paves the way for utilizing tiny plasmonic nanostructures for fruitful applications in biomedicine. [ABSTRACT FROM AUTHOR]

Mnemiopsin 1 is a member of photoprotein family, made up of 206 amino acid residues. These Ca 2 + -regulated photoproteins are responsible for light emission in a variety of marine cnidarians and ctenophores. They composed of an apoprotein, a single polypeptide chain of 25 kDa, molecular oxygen and the non-covalently bound chromophore. In this study, we examined whether three mutations, namely R39K, S128G and V183T affect the thermodynamic stability as well as refolding and unfolding kinetics of mnemiopsin 1. Conformational stability measurements using fluorescence and far-UV CD spectroscopies revealed that all variants unfold in multi-step manner in which the secondary and tertiary structures are lost in different steps. However kinetic studies showed that point mutation S128G destabilizes both kinetic intermediate and native conformation; while, these structural elements are stabilized in V183T. We also found that the stability of folded and intermediate states increases in R39K. We concluded that the initial packing of helical segments within the protein structure is more facilitated when Lys with smaller side chain is present in the protein chain. [ABSTRACT FROM AUTHOR]

Although considerable effort has been devoted to understanding the molecular mechanism of endostatin's anti-cancer activity, the role of its Zn bound N-terminal loop has not been completely clarified. To investigate whether Zn binding or the N-terminal loop is involved in the anti-cancer properties of endostatin, we compared the structure and biological activity of a native Zn binding endostatin peptide (ES-Zn) with three variants: a Zn free variant (ES), a variant containing both a Zn binding site and a disulfide bond (ES-SSZn), and a variant including a disulfide loop but incapable of Zn binding (ES-SS). Spectroscopic studies indicated that ES-Zn and ES-SS consist of random coil and β structures, whereas ES-SSZn and ES fold into random coils. Theoretical analysis proposed that ES-Zn and ES-SS have a similar binding site to α V β 3 integrin. The anti-proliferative activity of endostatin was retained by all peptides except ES, and the in vitro anti-angiogenic property was preserved in ES-Zn and ES-SS. Remarkably, breast tumor growth and CD31 activity were inhibited more effectively by ES-SS than by ES-Zn. Therefore, a correlation exists between the N-terminal loop and anti-cancer properties of endostatin fragment and a disulfide loop may be more promising than a Zn binding loop for inhibiting tumor growth. [ABSTRACT FROM AUTHOR]

Laccases are particularly promising enzymes for biotechnology and bioremediation purposes. They are among the most effective enzymes capable of catalyzing the degradation of phenolic compounds with poor water solubility. The technological utility of lacasses can be enhanced greatly by their use in ionic liquids rather than in conventional organic solvents or in their natural aqueous reaction media. In the current study, a laccase from Bacillus HR03 has been engineered through a semi rational method. By screening a library of 450 clones, Glu188Tyr and Glu188Phe showed a distinct improvement in thermal stability and ionic liquid tolerance. In comparison with the wild type, selected mutants exhibited higher k cat / K m against ABTS in the imidazolium based ionic liquids, (1-ethyl-3-methyl imidazolium chloride [EMIm][Cl], butyl-3-methyl imidazolium chloride [BMIm][Cl] and hexyl-3-methyl imidazolium chloride [HMIm][Cl]). Glu188Tyr had a catalytic efficiency, two times greater when compared to the wild type in [HMIm][Cl]. Far-UV circular dichroism (CD) exhibited no significant changes in the secondary structure of the mutants and wild type. Glu188Tyr revealed a more compact structure using Near-UV CD and fluorescence spectroscopy that could account for its high thermal stability. According to bioinformatic analysis, π–π and anion–π interactions played the dominant role in stabilizing both variants. [ABSTRACT FROM AUTHOR]