Biocatalytic Properties Research Articles

Novel Basidiomycetous Alcohol Oxidase from Cerrena unicolor—Characterisation, Kinetics, and Proteolytic Modifications

Intracellular alcohol oxidase (AOX) was isolated from the basidiomycetous white rot fungus Cerrena unicolor FCL139. The enzyme was semi-purified (13-fold) using two-step chromatography with 30% activity recovery. The identity of the protein was confirmed by LC-MS/MS analysis, and its MW (72 kDa) and pI (6.18) were also determined. The kinetics parameters of the AOX reaction towards various substrates were analysed, which proved that, in addition to methanol (4.36 ± 0.27% of the oxidised substrate), AOX most potently oxidises aromatic alcohols, such as 4-hydroxybenzyl alcohol (14.0 ± 0.8%), benzyl alcohol (4.2 ± 0.3%), anisyl alcohol (7.6 ± 0.4%), and veratryl alcohol (5.0 ± 0.3%). Moreover, the influence of selected commercially available proteases on the biocatalytic properties of AOX from C. unicolor was studied. It was proved that the digested enzyme lost its catalytic potential properties except when incubated with pepsin, which significantly boosted its activity up to 123%.

Constructing Electron-Rich Ru Clusters on Non-Stoichiometric Copper Hydroxide for Superior Biocatalytic ROS Scavenging to Treat Inflammatory Spinal Cord Injury.

Traumatic spinal cord injury (SCI) represents a complex neuropathological challenge that significantly impacts the well-being of affected individuals. The quest for efficacious antioxidant and anti-inflammatory therapies is both a compelling necessity and a formidable challenge. Here, in this work, the innovative synthesis of electron-rich Ru clusters on non-stoichiometric copper hydroxide that contain oxygen vacancy defects (Ru/def-Cu(OH)2), which can function as a biocatalytic reactive oxygen species (ROS) scavenger for efficiently suppressing the inflammatory cascade reactions and modulating the endogenous microenvironments in SCI, is introduced. The studies reveal that the unique oxygen vacancies promote electron redistribution and amplify electron accumulation at Ru clusters, thus enhancing the catalytic activity of Ru/def-Cu(OH)2 in multielectron reactions involving oxygen-containing intermediates. These advancements endow the Ru/def-Cu(OH)2 with the capacity to mitigate ROS-mediated neuronal death and to foster a reparative microenvironment by dampening inflammatory macrophage responses, meanwhile concurrently stimulating the activity of neural stem cells, anti-inflammatory macrophages, and oligodendrocytes. Consequently, this results in a robust reparative effect on traumatic SCI. It is posited that the synthesized Ru/def-Cu(OH)2 exhibits unprecedented biocatalytic properties, offering a promising strategy to develop ROS-scavenging and anti-inflammatory materials for the management of traumatic SCI and a spectrum of other diseases associated with oxidative stress.

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

Genetically Decorating Microbial Surface Curli Fiber with Biocatalytic Properties

Exploring the Structurally Conserved Regions and Functional Significance in Bacterial N-Terminal Nucleophile (Ntn) Amide-Hydrolases.

The initial adoption of penicillin as an antibiotic marked the start of exploring other compounds essential for pharmaceuticals, yet resistance to penicillins and their side effects has compromised their efficacy. The N-terminal nucleophile (Ntn) amide-hydrolases S45 family plays a key role in catalyzing amide bond hydrolysis in various compounds, including antibiotics like penicillin and cephalosporin. This study comprehensively analyzes the structural and functional traits of the bacterial N-terminal nucleophile (Ntn) amide-hydrolases S45 family, covering penicillin G acylases, cephalosporin acylases, and D-succinylase. Utilizing structural bioinformatics tools and sequence analysis, the investigation delineates structurally conserved regions (SCRs) and substrate binding site variations among these enzymes. Notably, sixteen SCRs crucial for substrate interaction are identified solely through sequence analysis, emphasizing the significance of sequence data in characterizing functionally relevant regions. These findings introduce a novel approach for identifying targets to enhance the biocatalytic properties of N-terminal nucleophile (Ntn) amide-hydrolases, while facilitating the development of more accurate three-dimensional models, particularly for enzymes lacking structural data. Overall, this research advances our understanding of structure-function relationships in bacterial N-terminal nucleophile (Ntn) amide-hydrolases, providing insights into strategies for optimizing their enzymatic capabilities.

The influence of near-infrared carbon dots on the conformational variation and enzymatic activity of glucose oxidase: A multi-spectroscopic and biochemical study with molecular docking

In recent years, the exceptional biocatalytic properties of glucose oxidase (GOx) have spurred the development of various GOx-functionalized nanocatalysts for cancer diagnosis and treatment. Carbon dots, renowned for their excellent biocompatibility and distinctive fluorescence properties, effectively incorporate GOx. Given the paramount importance of GOx's enzymatic activity in therapeutic efficacy, this study conducts a thorough exploration of the molecular-level binding dynamics between GOx and near-infrared carbon dots (NIR-CDs). Utilizing various spectrometric and molecular simulation techniques, we reveal that NIR-CDs form a ground-state complex with GOx primarily via hydrogen bonds and van der Waals forces, interacting directly with amino acid residues in GOx's active site. This binding leads to conformational change and reduces thermal stability of GOx, slightly inhibiting its enzymatic activity and demonstrating a competitive inhibition effect. In vitro experiments demonstrate that NIR-CDs attenuate the GOx's capacity to produce H2O2 in HeLa cells, mitigating enzyme-induced cytotoxicity and cellular damage. This comprehensive elucidation of the intricate binding mechanisms between NIR-CDs and GOx provides critical insights for the design of NIR-CD-based nanotherapeutic platforms to augment cancer therapy. Such advancements lay the groundwork for innovative and efficacious cancer treatment strategies.

Antibacterial and photo dye degradative ability of copper oxide nanoparticles from Pleurotus cystidiosus

The present study deals with in vitro experiments for to develop synthesized copper oxide nanoparticles using Pleurotus cystidiosus. Mushroom-based nanoparticles, such as copper nanoparticles have become more significant because of potential biocatalytic properties, including antibacterial activity. The blue-to-brown color shift suggests CuONPs, as confirmed by absorbance maxima at 246 nm in ultraviolet spectroscopy. FTIR spectra reveal the existence of carboxyl, alcohol, and aldehyde carbon; the amine group represents the myco-synthesized copper nanoparticle. Scanning electron microscopy (SEM) nanoparticles show spherical to oval nano aggregates of 80–110 nm in size. The EDX spectrum confirms the presence of Cu at 8.00 keV in myco-synthesized copper oxide nanoparticles. The antimicrobial efficacy of produced copper oxide nanoparticles on human pathogens showed a maximum zone of inhibition against coliform bacteria such as Enterococcus faecalis (32 ± 2), Enterobacter aerogenes (30 ± 1), E. coli (28 ± 1) and least zone of inhibition about 25 ± 1 mm recorded in Streptococcus mutans. The photocatalytic activity of Congo red dye, Malachite green dye, and Remazol blue dye by copper oxide nanoparticles from Pleurotus cystidiosus was investigated, and the results showed a maximum degradation efficiency of 99.2% in Congo red within 90 min. However, moderate degradation was noted in malachite green, and Remazol blue dye has a low degradation efficiency. Thus, myco-synthesized copper oxide nanoparticles from Pleurotus cystidiosus have potent antimicrobial and photocatalytic dye degradation efficacy.

Accelerated peroxidase-like activity of ultrathin amine-tagged bimetallic MOF-74 nanozyme for construction of versatile bioassays from small molecules to enzymes and bacteria

The exquisite design of nanostructures for enzyme-biocatalyzed active sites mimicking has become a prospective method to significantly boost biocatalytic properties, which is profitable to response amplification for bioanalysis scenarios. Metal-organic frameworks (MOFs) with customizable compositions, diverse microstructures and open catalytic sites are considered to deal with challenges in conventional materials of unfriendly microenvironment and inexact coordination. Herein, by combining a metal doping and post-synthetic modification approach, amine-tagged Fe-Ni bimetallic MOF-74 nanozyme (FeNi-MOF-74-NH2) with ultrathin structure and improved peroxidase-mimicking bioactivity is synthesized through a layered double hydroxide (LDH) directed method. The boosted biocatalytic property largely benefits from the synergistic effects of Fe-Ni species, attributing to the dual mechanism (electron transfer and generation of hydroxyl radicals). On the basis of the enhanced enzyme-mimicking property and accessible amino functionalization of FeNi-MOF-74-NH2, versatile applications were carried out for the ultrasensitive detection of analytes from bioactive small molecules (H2O2 and ascorbic acid), biomacromolecules (alkaline phosphatase, ascorbic acid oxidase, and α-glucosidase), and pathogenic bacteria (Staphylococcus aureus). The nanozyme-based biosensing platform without complex enzymatic techniques serves as a proof-of-concept to facilitate the application of MOF nanozymes in biochemical analysis, clinical diagnosis, and biomedical research.

Plasmonic nanozyme Cu3P with significantly enhanced peroxidase-like activity for Cr (VI) colorimetric detection

Plasmonic nanozymes with both plasmonic and biocatalytic properties stand out due to their distinctive localized surface plasmon resonance (LSPR) feature and adjustable catalytic activity, while the enhancement effect of present reported plasmon excitation remains relatively low. Here, a plasmonic nanozyme of Cu3P nanoparticles (Cu3P NPs) with LSPR characteristics and intrinsic peroxidase (POD)-like activity was prepared via simple gas-solid reaction strategy, which presents significantly enhanced POD-like activity under plasmon stimulation than that without LSPR excitation, with catalytic constants for TMB and H2O2 83 and 127 times higher than those without LSPR excitation respectively. On the one hand, the Cu3P plasmonic nanozyme generated abundant high-energy hot carriers under near-infrared (NIR) stimulation to promote cracking of H2O2 to produce plentiful reactive oxide species (ROS); on the other hand, the LSPR-induced photothermal effect on Cu3P plasmonic nanozyme greatly improves the reaction kinetics, thus making a significantly boosts POD-like activity. According to the excellent POD-like activity of Cu3P plasmonic nanozyme and 8-hydroxyquinoline (8-HQ)-mediated chromaticity switch, a highly sensitive Cr (VI) chrominance sensing platform was developed with a detection limit of 9.6 nM within a range from 0.01 to 20 μM. This study spotlights the high utilization efficiency of plasmon stimulation for enzyme-like activity tuning.

Heterologous expression and characterization of the carveol dehydrogenase from Klebsiella sp. O852

Carvone has been widely used in pharmaceutical, food, cosmetic, and chemical industries due to its pleasant odor with high economic value and pharmacological activities. Carveol dehydrogenase is an enzyme that catalyzes the oxidation of carveol into carvone in the presence of NAD(P)+. In this study, two dehydrogenase genes from Klebsiella sp. O852 (KlebADH1 and KlebADH2) were cloned and expressed in Escherichia coli BL21(DE3), and the purified enzymes were characterized for their biocatalytic properties. KlebADH1 and KlebADH2 displayed optimal catalytic activity at 40 °C and 30 °C, respectively, at pH 9. In addition to carveol, KlebADH1 had the potential to catalyze the oxidation of perillyl alcohol, nerol and geraniol, while borneol and geraniol could serve as potential substrates for KlebADH2. The substrate kinetic parameters of enzymes were performed by changing the concentration of carveol, and the results indicated that KlebADH2 exhibited a high affinity for carveol. Additionally, KlebADH1 and KlebADH2 were confirmed to possess carveol dehydrogenase activity through a double-enzyme-coupled (DEC) biotransformation system. The investigation of the biocatalytic properties of KlebADH1 and KlebADH2 laid a solid foundation for further study of carvone sustainable synthesis.

Gadolinium Doping Modulates the Enzyme-like Activity and Radical-Scavenging Properties of CeO2 Nanoparticles.

Their unique physicochemical properties and multi-enzymatic activity make CeO2 nanoparticles (CeO2 NPs) the most promising active component of the next generation of theranostic drugs. When doped with gadolinium ions, CeO2 NPs constitute a new type of contrast agent for magnetic resonance imaging, possessing improved biocatalytic properties and a high level of biocompatibility. The present study is focused on an in-depth analysis of the enzyme-like properties of gadolinium-doped CeO2 NPs (CeO2:Gd NPs) and their antioxidant activity against superoxide anion radicals, hydrogen peroxide, and alkylperoxyl radicals. Using an anion-exchange method, CeO2:Gd NPs (~5 nm) with various Gd-doping levels (10 mol.% or 20 mol.%) were synthesized. The radical-scavenging properties and biomimetic activities (namely SOD- and peroxidase-like activities) of CeO2:Gd NPs were assessed using a chemiluminescent method with selective chemical probes: luminol, lucigenin, and L-012 (a highly sensitive luminol analogue). In particular, gadolinium doping has been shown to enhance the radical-scavenging properties of CeO2 NPs. Unexpectedly, both bare CeO2 NPs and CeO2:Gd NPs did not exhibit SOD-like activity, acting as pro-oxidants and contributing to the generation of reactive oxygen species. Gadolinium doping caused an increase in the pro-oxidant properties of nanoscale CeO2. At the same time, CeO2:Gd NPs did not significantly inhibit the intrinsic activity of the natural enzyme superoxide dismutase, and CeO2:Gd NPs conjugated with SOD demonstrated SOD-like activity. In contrast to SOD-like properties, peroxidase-like activity was observed for both bare CeO2 NPs and CeO2:Gd NPs. This type of enzyme-like activity was found to be pH-dependent. In a neutral medium (pH = 7.4), nanoscale CeO2 acted as a prooxidant enzyme (peroxidase), while in an alkaline medium (pH = 8.6), it lost its catalytic properties; thus, it cannot be regarded as a nanozyme. Both gadolinium doping and conjugation with a natural enzyme were shown to modulate the interaction of CeO2 NPs with the key components of redox homeostasis.

Fabrication of iron nanoparticles using different bioactive precursors, their characterization and bioactivity evaluation

This study deals with the green synthesis of Iron nanoparticles (FeNPs) using plant extracts of Polygonum plebeium (Knotweed) and Camellia sinensis (Green tea), focusing on their antibacterial potential, biocatalytic action as well as prospective applications as nanofertilizers. Plant materials underwent a thorough cleaning and were processed into aqueous extracts. The synthesis of FeNPs involved blending these extracts with ferric chloride solution, resulting in a versatile nanomaterial that was subjected to various characterization techniques, such as UV-Vis spectroscopy, FT-IR, XRD, SEM, and DLS-Zeta unveiling critical nanoparticle attributes of the synthesized nanoparticles, including the size, crystallinity, morphology, stability, and elemental composition. The synthesized FeNPs exhibited substantial antibacterial activities, against Escherichia coli and Staphylococcus aureus. Additionally, the biocatalytic properties of the synthesized FeNPs were investigated through dye degradation. These nanoparticles further, were demonstrated for their potential as nanofertilizers, enhancing the germination and growth of tomato and radish seeds. The merging of nanotechnology and plant-based synthesis represents a promising intersection with the capacity to revolutionize diverse fields.

L-Asparaginase Conjugates from the Hyperthermophilic Archaea Thermococcus sibiricus with Improved Biocatalytic Properties.

This study investigated the effect of polycationic and uncharged polymers (and oligomers) on the catalytic parameters and thermostability of L-asparaginase from Thermococcus sibiricus (TsA). This enzyme has potential applications in the food industry to decrease the formation of carcinogenic acrylamide during the processing of carbohydrate-containing products. Conjugation with the polyamines polyethylenimine and spermine (PEI and Spm) or polyethylene glycol (PEG) did not significantly affect the secondary structure of the enzyme. PEG contributes to the stabilization of the dimeric form of TsA, as shown by HPLC. Furthermore, neither polyamines nor PEG significantly affected the binding of the L-Asn substrate to TsA. The conjugates showed greater maximum activity at pH 7.5 and 85 °C, 10-50% more than for native TsA. The pH optima for both TsA-PEI and TsA-Spm conjugates were shifted to lower pH ranges from pH 10 (for the native enzyme) to pH 8.0. Additionally, the TsA-Spm conjugate exhibited the highest activity at pH 6.5-9.0 among all the samples. Furthermore, the temperature optimum for activity at pH 7.5 shifted from 90-95 °C to 80-85 °C for the conjugates. The thermal inactivation mechanism of TsA-PEG appeared to change, and no aggregation was observed in contrast to that of the native enzyme. This was visually confirmed and supported by the analysis of the CD spectra, which remained almost unchanged after heating the conjugate solution. These results suggest that TsA-PEG may be a more stable form of TsA, making it a potentially more suitable option for industrial use.

L-asparaginase Conjugates of Rhodospirillum Rubrum with Polymers of Different Structures with Improved Biocatalytic Properties

L-asparaginase from the mesophyllic bacterium Rhodospirillum rubrum shows promise as a potential biomedicine treatment for tumor diseases. To enhance the enzyme's catalytic parameters, we have synthesized covalent conjugates with chitosan-PEG, chitosan-glycol and polyethylenimine. Interestingly, binding to these polymers had minimal impact on the enzyme's substrate binding constant. The RrA-PEI conjugate showed the most significant changes in secondary structure content. The success of the previous modification was evaluated by IR spectroscopy, which indicated that the polycations used in the work have a positive effect on the catalytic activity of the enzyme. The RrA-chitosan-PEG conjugate demonstrated the highest enzymatic activity.

Biotransformation of essential oil composition of Zanthoxylum limonella by the fungus Pleopunctum pseudoellipsoideum provides the products with enhanced antimicrobial activities

Microbial biotransformation presents an alternative technology for enhancing more valuable bioproducts. This study utilized three fungi including Pleopunctum pseudoellipsoideum, Stagonosporopsis pini, and Thyrostroma jaczewskii to biotransform Zanthoxylum limonella essential oil composition. The essential oil composition of Z. limonella treated with these fungi was analyzed by gas chromatography-mass spectrometry. Z. limonella oil treated with the fungus T. jaczewskii gave cryptone, neoiso-verbanol, and dehydro-linalool as the major compounds. P. pseudoellipsoideum treatment resulted in the transformation of linalool, cryptone, and thymol, while treatment with the fungus S. pini yielded major transformed compounds such as citronellic acid, cis-limonene oxide, and eucarvone. Varied enzymes produced by each fungus led to diverse transformed volatile profiles. Principal Coordinate Analysis (PCoA) was employed to analyze the volatile profiles of the transformed essential oils, effectively highlighting the distinct nature of fungal biotransformation. Antibacterial activities of the essential oils obtained from all treatments were tested. The Z. limonella oil treated with P. pseudoellipsoideum exhibited the highest antibacterial activity, followed by S. pini and T. jaczewskii, in comparison to pure Z. limonella oil. The results suggest the potential biocatalytic properties of these fungi as tools for enhancing novel value-added bioactive compounds for food and pharmaceutical applications.

Gold Nanoclusters for Tumor Diagnosis and Treatment

Nanomedicine shows remarkable potential to improve the efficacy of diagnosis and treatment of tumors, utilizing nanotechnologies and nanomaterials. Gold nanoclusters (AuNCs) have emerged as a highly promising option due to their exceptional optical and enzyme‐mimicking catalytic activities as well as good biocompatibility. The renal clearable clusters can enrich in the tumors upon their enhanced permeability and retention properties, which benefits the tumor‐related applications. The fluorescence of AuNCs in the second near‐infrared region possesses extraordinary penetration depth and high temporal–spatial resolution, enabling excellent in vivo imaging and real‐time monitoring of pathological process. AuNC‐based nanoplatforms represent a paradigm of integrated, efficient, intelligent, and safe treatment strategy, extending personalized tumor therapy. Meanwhile, the optical and biocatalytic properties can be modulated by adopting the atom/ligand engineering, which further enhances the efficacy of AuNCs. Herein, the advances of AuNCs in the field of diagnosis and treatment of tumors are summarized and the directions to be improved are proposed to promote the clinical translation of the AuNCs.

Immobilization of Horseradish Peroxidase on Ca Alginate-Starch Hybrid Support: Biocatalytic Properties and Application in Biodegradation of Phenol Red Dye.

In this study, horseradish peroxidase was extracted, purified, and immobilized on a calcium alginate-starch hybrid support by covalent bonding and entrapment. The immobilized HRP was used for the biodegradation of phenol red dye. A 3.74-fold purification was observed after precipitation with ammonium sulfate and dialysis. An immobilization yield of 88.33%, efficiency of 56.89%, and activity recovery of 50.26% were found. The optimum pH and temperature values for immobilized and free HRP were 5.0 and 50°C and 6.5 and 60°C, respectively. The immobilized HRP showed better thermal stability than its free form, resulting in a considerable increase in half-life time (t1/2) and deactivation energy (Ed). The immobilized HRP maintained 93.71% of its initial activity after 45days of storage at 4°C. Regarding the biodegradation of phenol red, immobilized HRP resulted in 63.57% degradation after 90min. After 10 cycles of reuse, the immobilized HRP was able to maintain 43.06% of its initial biodegradative capacity and 42.36% of its enzymatic activity. At the end of 15 application cycles, a biodegradation rate of 8.34% was observed. In conclusion, the results demonstrate that the immobilized HRP is a promising option for use as an industrial biocatalyst in various biotechnological applications.

Влияние рН и состава питательной среды на дегидрогеназную активность экстрактов, полученных из Escherichia coli

“Crude” extracts are a new perspective type of bioelectrocatalysts for biofuel cells. The method of obtaining a “crude” extract by disintegrating bacterial cell membranes (for example, by ultrasound) is simpler and more economical in comparison with pure enzymes. From a scientific point of view, the study of the biocatalytic properties of “crude” extracts is very interesting, since the disintegrated biomass contains a cascade of enzymes involved in the metabolism of microorganisms and coenzymes necessary for oxidative/reductive transformation. This set of bioactive substances makes it possible to simulate the processes occurring during the operation of a “natural” fuel cell in an artificial environment. The effect of the pH and composition of the LB nutrient medium used in the cultivation of E.coli on the dehydrogenase activity of protein extracts obtained from this culture was studied. It is shown that by selecting the pH and modifying the composition of the LB nutrient medium, it is possible to increase the value of the specific dehydrogenase activity of extracts by almost 9 times (from 0.25 to 2.2 mg/mg).

A Versatile G-Quadruplex (G4)-Coated Upconverted Metal-Organic Framework for Hypoxic Tumor Therapy.

Given the complexity of the tumor microenvironment, multiple strategies are being explored to tackle hypoxic tumors. The most efficient strategies combine several therapeutic modalities and typically requires the development of multifunctional nanocomposites through sophisticated synthetic procedures. Herein, the G-quadruplex (G4)-forming sequence AS1411-A (d[(G2 T)4 TG(TG2 )4 A]) is used for both its anti-tumor and biocatalytic properties when combined with hemin, increasing the production of O2 ca. two-fold as compared to the parent AS1411 sequence. The AS1411-A/hemin complex (GH) isgrafted on the surface and pores of a core-shell upconverted metal-organic framework (UMOF) to generate a UMGH nanoplatform. Compared with UMOF, UMGH exhibits enhanced colloidal stability, increased tumor cell targeting and improved O2 production (8.5-fold) in situ. When irradiated by near-infrared (NIR) light, the UMGH antitumor properties are bolstered by photodynamic therapy (PDT), thanks to its ability to convert O2 into singlet oxygen (1 O2 ). Combined with the antiproliferative activity of AS1411-A, this novel approach lays the foundation for a new type of G4-based nanomedicine.

Topology of DNA G-Quadruplexes Can Be Harnessed in Holliday Junction-Based DNA Suprastructures to Control and Optimize Their Biocatalytic Properties

Topology of DNA G-Quadruplexes Can Be Harnessed in Holliday Junction-Based DNA Suprastructures to Control and Optimize Their Biocatalytic Properties

Unveiling the Influence of Hydrated Deep Eutectic Solvents on the Dynamics of Water-Soluble Proteins.

Deep eutectic solvents (DESs) are mixtures of two or more pure compounds (e.g., Lewis or Brønsted acids and bases, anionic and/or cationic species) in a well-defined stoichiometric proportion, with a melting point lower to that of an ideal liquid mixture. These neoteric solvents are highly tunable through varying the structure or relative ratio of parent components and have been evaluated as solvents able to improve biomolecules' performance, specifically their stability and biocatalytic properties. Inspired by a recent crystallographic study, we have explored through molecular dynamics (MD) simulations the dynamic properties of two different proteins (hen egg-white lysozyme and the human VH antibody fragment HEL4) in a (20% w/w) hydrated solution of choline chloride-glycerol (1:2). We have developed proper force fields to account for DES, protein, and DES-protein interactions, which have been calibrated using pair distribution function measurements of pure DES solutions. MD results show that the presence of DES quenches the protein motion, increasing the rigidity of the overall protein structure. Specific interactions among DES components and protein residues, such as those between choline ions and two Tryptophan residues of lysozyme, may amplify the protein-DES interactions and lead to protein crystallization in the presence of hydrated DES. These findings open new horizons to improve or achieve control on protein properties by a proper choice of hydrated DESs used as solvents.