Hydrophilic Organic Solvents Research Articles

Biocatalytic hydrolysis of di-urethane model compounds in ionic liquid reaction media

The suitability of different enzymes to carry out the hydrolysis of two-different toluene-based urethane model compounds (i.e. bis(2-methoxyethyl) (4-methyl-1,3-phenylene)dicarbamate, and bis(2-methoxyethyl) (2-methyl-1,3-phenylene)dicarbamate) has been demonstrated for the first time by taking advantage of ionic liquid (IL) technologies. Toluene-based urethane compounds were prepared from usual substrates in polyurethane industrial synthesis. Afterwards, their carbamate groups were target of a biocatalytic hydrolysis by means of different commercial hydrolases (i.e. lipase, urease and proteases) in either water, hydrophilic organic solvents (i.e. ethylene glycol or 1,2-dimethyl-1,3-dioxolane-4-methanol, (solketal)), or hydrophobic ILs (e.g. [C4mim][NTf2], etc.) as reaction media. Because of the insolubility of these compounds in water, most of the enzymes were unable to catalyse the hydrolysis of the di-urethane substrates in pure water, being clearly improved (up to 31.6 mU/mg for the urease case) in solketal:water (90:10, v/v) reaction media. When hydrophobic ILs were added into this reaction medium, the urease activity increased by more than twice (74.1 mU/mg). The most promising results for the hydrolysis of these urethane compounds were obtained by combining lipase and urease biocatalysts in a IL:solketal:H2O (70:25:5, v/v/v) reaction medium. These results demonstrate a possible biocatalytic approach for the hydrolytic depolymerization of polyurethane foam wastes.

Enhanced Activity and Stability of an Acetyl Xylan Esterase in Hydrophilic Alcohols through Site-Directed Mutagenesis.

Current demands for the development of suitable biocatalysts showing high process performance is stimulated by the need to replace current chemical synthesis with cleaner alternatives. A drawback to the use of biocatalysts for unique applications is their low performance in industrial conditions. Hence, enzymes with improved performance are needed to achieve innovative and sustainable biocatalysis. In this study, we report the improved performance of an engineered acetyl xylan esterase (BaAXE) in a hydrophilic organic solvent. The structure of BaAXE was partitioned into a substrate-binding region and a solvent-affecting region. Using a rational design approach, charged residues were introduced at protein surfaces in the solvent-affecting region. Two sites present in the solvent-affecting region, A12D and Q143E, were selected for site-directed mutagenesis, which generated the mutants MUT12, MUT143 and MUT12-143. The mutants MUT12 and MUT143 reported lower Km (0.29 mM and 0.27 mM, respectively) compared to the wildtype (0.41 mM). The performance of the mutants in organic solvents was assessed after enzyme incubation in various strengths of alcohols. The mutants showed improved activity and stability compared to the wild type in low strengths of ethanol and methanol. However, the activity of MUT143 was lost in 40% methanol while MUT12 and MUT12-143 retained over 70% residual activity in this environment. Computational analysis links the improved performance of MUT12 and MUT12-143 to novel intermolecular interactions that are absent in MUT143. This work supports the rationale for protein engineering to augment the characteristics of wild-type proteins and provides more insight into the role of charged residues in conferring stability.

Highly reversible zinc metal anode enabled by zinc fluoroborate salt-based hydrous organic electrolyte

Although Zn(BF4)2 contains favorable ingredients of fluorine for the formation of ZnF2 rich-Zn2+ ion conducting solid electrolyte interface (SEI), the aqueous electrolyte based on inorganic Zn salt of Zn(BF4)2⋅xH2O shows high Hammett acidity with pH value <1, which gives rise to severe corrosion of metallic Zn electrode and thermodynamically spontaneous hydrogen evolution reaction (HER). Meanwhile, an uneven and arbitrarily aggregated SEI will result in uneven distribution of Zn2+ ion flux and electric field, leading to rampant dendrite growth. Here, we employ a hydrophilic organic solvent of vinylene carbonate (VC) and hydrate Zn(BF4)2⋅4H2O salt for a hydrous organic electrolyte, denoted as ZnBF-VC. With the ZnBF-VC electrolyte used, the VC molecules preferably adsorb on the Zn surface to block H2O molecules and Zn metals. Meanwhile, the unique Zn2+-solvation sheath of Zn(VC)2.89(H2O)1.28(BF4)1.83 is formed, which forms an organic/inorganic hybrid SEI in-situ with ZnF2 and ZnCO3 as inorganic species. The distinct SEI enables favorable Zn2+ ion transport and can effectively protect metallic Zn electrode from corrosion, side reactions and dendrite formation. Consequently, the ZnǀǀZn cells cycled over 2200 h at 0.5 mA cm−2 and the ZnǀǀCu asymmetric cells maintained an excellent Coulombic efficiency (CE) of ∼99.7% over 550 cycles at 1 mA cm−2. Whereas, the ZnǀǀZn cells broke after only 74 cycles in aqueous electrolyte. Additionally, the full cell we assembled with manganese hexacyanoferrate (MnHCF) in ZnBF-VC electrolyte demonstrates excellent cycling stability, achieving a high specific capacity of 146.2 mAh g − 1, and a high retention rate of 85.3% over 1300 cycles at 0.4 A g − 1, while the cell using the referred ZnBF-H2O electrolyte survived only ∼6 cycles. This work proposes a feasible direction for the study of Zn(BF4)2-based organic electrolyte for Zn batteries.

Specific Extraction of Aspartic Acid and Tyrosine with Aqueous Two-phase Systems Formed by Hydrophilic Organic Solvent and Monomeric Sugars

Specific Extraction of Aspartic Acid and Tyrosine with Aqueous Two-phase Systems Formed by Hydrophilic Organic Solvent and Monomeric Sugars

Partition of Amino Acids in Aqueous Two-Phase Systems Comprising Hydrophilic Organic Solvents and Salts

An aqueous two-phase system (ATPS) comprising a hydrophilic organic solvent and inorganic salt is advantageous because of its high polarity, low viscosity, and low cost. In this work, nine amino acids were partitioned in an ATPS that comprised hydrophilic organic solvents (2-methyl-2-propanol, tetrahydrofuran, and 2-propanol) and salts (Na2SO4, K2HPO4, and (NH4)2SO4). Amino acids were successfully extracted using different combinations of solvents and salts. The distribution ratio of amino acids in the ATPS containing ammonium sulfate increased with an increase in their hydrophobicity, except for that of alanine. The specific extraction of alanine was observed because of its salting-out effect in ammonium sulfate solution. The sulfate salts of amino acids exhibited higher extractability than amphoteric amino acids.

Glycosylation of luteolin in hydrophilic organic solvents and structure-antioxidant relationships of luteolin glycosides.

An effective approach was developed to biotransform luteolin glycosides in hydrophilic organic solvents. Bacillus cereus A46 cells showed high activity and stability in 5–20% (v/v) DMSO with 90–98% conversion rates of luteolin glycosides. Five glycosides of luteolin 7-O-β-glucoside, luteolin 4′-O-β-glucoside, luteolin 3′-O-β-glucoside, luteolin 7,3′-di-O-β-glucoside and luteolin 7,4′-di-O-β-glucoside were obtained. The addition of DMSO greatly promoted the solubility of luteolin and further regulated the formation of the main products from five luteolin glycosides to luteolin 7-O-β-glucoside (931.2 μM). Fourteen flavonoids and anthraquinones were used as tentative substrates. Glycosylation positions were located at the C-7, C-3′ or C4′ hydroxyl groups of flavonoids and C-5 hydroxyl group of anthraquinones. The 3′,4′-dihydroxy arrangement played the key role for the antioxidant activity of luteolin.

Низкотемпературное разделение и концентрирование в условиях образования гетерогенных систем (обзор)

В обзоре рассмотрены современные подходы, используемые в методах разделения и концентрирования биологически активных соединений в условиях образования гетерогенных систем при низких температурах. Обсуждены факторы, позволяющие повысить эффективность низкотемпературной жидкостно-жидкостной экстракции, криоконцентрирования, экстракционного вымораживания. Рассмотрены возможные механизмы физико-химических сепарационных процессов, реализуемых в условиях низких температур.

Biochemical characterization and application of a new lipase and its cognate foldase obtained from a metagenomic library derived from fat-contaminated soil

LipMF3 is a new lipase isolated from a metagenomic library derived from a fat-contaminated soil. It belongs to the lipase subfamily I.1 and has identities of 68% and 67% with lipases of Chromobacterium violaceum and C. amazonense, respectively. Genes encoding LipMF3 and its cognate foldase, LifMF3, were cloned and co-expressed in Escherichia coli. The highest hydrolytic activity of purified Lip-LifMF3 was at 40 °C and pH 6.5. Under these conditions, the highest activity was against tributyrin (1650 U mg−1), but it also had high activity against olive oil (862 U mg−1). It was stable in hydrophilic organic solvents (25%, v/v in water) with residual activity around 100% after 24 h. It also showed stability over a wide pH range (5.5 to 11) with residual activity above 80% after 24 h. Lip-LifMF3 was immobilized by covalent bonding onto Immobead 150P and by adsorption onto Sepabeads FP-BU. The latter preparation gave the best results, producing 94% conversion after 5 h for the synthesis of ethyl oleate and a 90% enantiomeric excess of the product (R)‑1‑phenylethyl acetate for the kinetic resolution of (R,S)‑1‑phenyl‑1‑ethanol. The results obtained in this work provide a basis for the development of applications of Lip-LifMF3 in biocatalysis.

Al[18F]F-complexation of DFH17, a NOTA-conjugated adrenomedullin analog, for PET imaging of pulmonary circulation

IntroductionAdrenomedullin receptors are highly expressed in human alveolar capillaries and provide a molecular target for imaging the integrity of pulmonary microcirculation. In this work, we aimed to develop a NOTA-derivatized adrenomedullin analog (DFH17), radiolabeled with [18F]AlF, for PET imaging of pulmonary microcirculation. MethodsHighly concentrated [18F](AlF)2+ (15 μL) was produced from purified fluorine-18 in NaCl 0.9%. Various complexation experiments were carried out at Al-to-NOTA molar ratios ranging from 1:1 to 1:40 to assess optimal radiolabeling conditions before using the peptide. DFH17 peptide (2 mM, pH 4) was radiolabeled with [18F](AlF)2+ for 15 min at 100 °C in a total volume of 60 μL. As part of the radiolabeling process, parameters such as fluorine-18 activity (~37 and 1480 MBq), concentration of AlCl3 (0.75, 2, 3, 6 or 10 mM) and the effects of hydrophilic organic solvent (aqueous vs ethanol 50%) were studied. The final formulation was tested for purity, identity and stability in saline. Initial in vivo evaluation of [18F]AlF-DFH17 was performed in normal rats by PET/CT. ResultsThe scaled-up production of [18F]AlF-DFH17 was performed in high radiochemical and chemical purities in an overall radiochemical yield of 22–38% (at end-of-synthesis) within 60 min. The final formulation was stable in saline at different radioactive concentrations for 8 h. PET evaluation in rats revealed high lung-to-background ratios and no defluorination in vivo up to 1 h post-injection. ConclusionThe novel radioconjugate [18F]AlF-DFH17 appears to be a promising PET ligand for pulmonary microcirculation imaging.

Biodiesel production from microalgae oil by lipase from Pseudomonas aeruginosa displayed on yeast cell surface

Yeast surface display has become a powerful technology in recent decades and one of the promising areas in this field is the biodiesel synthesis by microbial lipases. Hence, in this study the optimized lipase A (Lip A) gene from Pseudomonas aeruginosa was fused to GPI-anchored protein Gcw61 and successfully displayed on the surface of Pichia pastoris X33. A lipase activity of 85.2 U/mg dry cell weight was obtained from recombinant P. pastoris. The copy numbers of inserted lipase gene were determined 2.09 ± 0.06 by real time PCR absolute quantification method. The enzyme showed the best stability in pH 7.0–10.0 and at temperature 37 °C–40 °C and was also stable in hydrophilic organic solvents. Ca2+, Mg2+, Mn2+ and Cu2+ ions enhanced enzyme activity, whereas Fe2+ and Zn2+ ions and some detergents like SDS, CTAB, Tween 20 and 80 dramatically decreased the activity of the enzyme. The results demonstrate that our whole cell biocatalyst exhibited a good potential for biodiesel production from microalgae oil in 10 repeated batch cycles.

Characterization of a new nitrilase from Hoeflea phototrophica DFL-43 for a two-step one-pot synthesis of (S)-β-amino acids.

A nitrilase from Hoeflea phototrophica DFL-43 (HpN) demonstrating excellent catalytic activity towards benzoylacetonitrile was identified from a nitrilase tool-box, which was developed previously in our laboratory for (R)-o-chloromandelic acid synthesis from o-chloromandelonitrile. The HpN was overexpressed in Escherichia coli BL21 (DE3), purified to homogeneity by nickel column affinity chromatography, and its biochemical properties were studied. The HpN was very stable at 30-40°C, and highly active over a wide range of pH values (pH 6.0-10.0). In addition, the HpN could tolerate against several hydrophilic organic solvents. Steady-state kinetics indicated that HpN was highly active towards benzoylacetonitrile, giving a KM of 4.2mM and a kcat of 170s-1, the latter of which is ca. fivefold higher than the highest record reported so far. A cascade reaction for the synthesis of optically pure (S)-β-phenylalanine from benzoylacetonitrile was developed by coupling HpN with an ω-transaminase from Polaromonas sp. JS666 in toluene-water biphasic reaction system using β-alanine as an amino donor. Various (S)-β-amino acids could be produced from benzoylacetonitrile derivatives with moderate to high conversions (73-99%) and excellent enantioselectivity (> 99% ee). These results are significantly advantageous over previous studies, indicating a great potential of this cascade reaction for the practical synthesis of (S)-β-phenylalanine in the future.

Sustainable approach in recycling of phase components of large scale aqueous two-phase flotation for lipase recovery

Abstract Recently, aqueous two-phase flotation system has become a powerful method for separation of biomolecules. However, the application of this technique for large scale operation elevates a concern on the environmental issue. This is due to the large amount of chemical consumption for phase components formation. Recycling of hydrophilic organic solvent and inorganic salt in aqueous two-phase flotation is an efficient and green technique for biomolecules recovery. Up to date, there is limited research concerning the recycling of the phase components. In this present work, recycling capability of pilot scale aqueous two-phase flotation composing alcohol/salt was employed to separate lipase from fermentation broth. The drive of this research is to economize the usage of chemicals, to reduce environmental pollution and to establish this approach for sustainable recovery of lipase. The aqueous two-phase flotation system investigated was composed of 1-propanol and ammonium sulphate whereby both phase components went through complete recycling process. Recycling of entire phase components is found to be a sustainable approach as it able to reduce the consumption of chemicals and it is an eco-friendly technique. From the result obtained, it was exhibited that by reusing the bottom phase, separation efficiency was sustained beyond 70%. As for the lipase yield, 80% of yield was achieved from recycling the crystallized salt that was recovered by dilution crystallization with methanol.

Characterization of high-molar-mass fractions in a Scots pine (Pinus sylvestris L.) knotwood ethanol extract

AbstractThe identification of compounds in Scots pine (Pinus sylvestrisL.) knotwood (KnW), obtained by extracting with hydrophilic organic solvents, has been performed previously almost exclusively by gas chromatography-mass spectrometry (GS-MS) equipped with long GC columns (≥25 m). That means that the molar mass (MM) of the majority of the identified compounds was below 500–600 Da, and the analytical data accounted for only about half of the dry extract weight. In the present work, high-molar-mass (HMM) fractions in a Scots pine KnW-EtOH extract were isolated and chemically characterized by means of several advanced analytical techniques, such as high performance size-exclusion chromatography-evaporative light scattering detector (HPSEC-ELSD), high performance liquid chromatography (HPLC)-electrospray ionization-ion trap-mass spectrometry [(HPLC)ESI-IT-MS], ESI-quadrupole time of flight-mass spectrometry (QTOF-MS), pyrolysis-gas chromatography-mass spectrometry (Py-GC-MS), thermally assisted hydrolysis and methylation-gas chromatography-mass spectrometry (THM-GC-MS), nuclear magnetic resonance (NMR) and GC-MS. The results indicate that the MM maxima of the HMM fractions ranged from approximately 500 to 2200 Da, and that the compounds consist mainly of oligomers of hydroxylated resin acids (RAs), especially dehydroabietic acid, but also of fatty acids (FAs), stilbenes and sterols. A large number of RA dimers were tentatively identified in the HMM fractions. However, it remains unknown how the monomer units are linked together, as it was not possible to isolate a RA dimer fraction pure enough for NMR characterization. RA dimers in native KnW have not been identified previously.

Screening, purification, and characterization of a novel organic solvent-tolerant esterase, Lip2, from Monascus purpureus strain M7.

Organic solvent-tolerant esterases are proven to be excellent biocatalysts in chemical and pharmaceutical industries. A novel organic solvent-tolerant esterase gene, lip2, was isolated from filamentous fungi Monascus purpureus M7. The sequence analysis suggested that lip2 has a conserved "GDSL" motif near the active center. The multiple-sequence alignment and phylogenetic analysis revealed that Lip2 displayed two unique amino-acid sequence motifs that clearly separate it from any other previously described lipase family. After incubation in 20% methanol and ethanol for 3h, the Lip2 displayed 190 and 180% residual activities, respectively. It retained 99-110% relative activity in 20% (v/v) hydrophilic organic solvents after incubation for 1day. This esterase showed optimal activity at 40 °C and retained about 70% maximal activity at 60 °C. The enzyme also displayed more than 50% residual activity over a range of pH 5-11. In the presence of most of metal ions or additives, Lip2 retained most of the activity. These unique properties of Lip2 make it a promising as biocatalyst for industrial processes.

Extraction and purification of IgG by hydrophilic organic solvent salting-out extraction.

The distribution of IgG in different salting-out extraction systems (SOESs), including ethanol/K2HPO4, Na2CO3, and trisodium citrate, was investigated. The SOESs that were composed of 20% K2HPO4 (w/w)-14% ethanol (w/w), 19% Na2CO3 (w/w)-13% ethanol (w/w) and 25% trisodium citrate (w/w)-19% ethanol (w/w) showed satisfactory results, with IgG recovery rates as high as 97.4%, 93.1%, and 90.2%, respectively. ELISA, CD and IR analyses confirmed the active retention and structural constant of IgG in the K2HPO4-ethanol system. An optimized system that consisted of 14% ethanol (w/w)-20% K2HPO4 (w/w) (pH 7.0) was selected for extracting active plasma proteins directly from pig plasma. As a result, recovery rates as high as 95.7% for IgG and 93.0% for albumin were achieved. In addition, some contaminating proteins were also removed by this system, which may provide a new alternative method for the separation and purification of immunoglobulin.

親水性有機溶媒を用いる&lt;i&gt;in situ&lt;/i&gt;抽出剤生成―ミクロ溶媒抽出法によるクロム（III）とクロム（VI）の分別定量

親水性有機溶媒を用いる&lt;i&gt;in situ&lt;/i&gt;抽出剤生成―ミクロ溶媒抽出法によるクロム（III）とクロム（VI）の分別定量

Preparation of small-sized graphene oxide sheets and their biological applications.

By using carbon nanohorns as starting materials, small- and uniform-sized graphene oxide (S-GO) sheets can be prepared in high yields via an oxidation method. The obtained S-GO sheets have a band-like structure with a length of 20-50 nm, a width of 2-10 nm, and a thickness of 0.5-5 nm. S-GO sheets are hydrophilic due to abundant oxygenated groups on the surfaces and edges; hence, this nanomaterial is highly dispersive in aqueous solutions and some hydrophilic organic solvents. Additionally, like other S-GO samples, the S-GO sheets prepared here are strongly fluorescent over the visible light wavelength region. These characteristics underscore the high potential of S-GO sheets for nanomedical and diagnostic applications. In proof-of-concept experiments, the S-GO sheets were conjugated with an arginine-glycine-aspartic acid derivative for tumour-targeting drug delivery applications, and with an immunoglobulin G antibody for immunoassay applications.

Highly stable water-soluble magnetic nanoparticles synthesized through combined co-precipitation, surface-modification, and decomposition of a hybrid hydrogel

The synthesized magnetic nanoparticles were stable not only in aqueous media but also in mixtures of water and hydrophilic organic solvents.

Ethylene Glycol Quenching of Nitrogenase Catalysis: An Electron Paramagnetic Resonance Spectroscopic Study of Nitrogenase Turnover States and CO Bonding.

Most hydrophilic organic solvents inhibit enzymatic activity. Nitrogenase is shown to be approximately 3 times more sensitive to organic inhibition than most other soluble enzymes. Ethylene glycol (EG) is demonstrated to rapidly inhibit nitrogenase activity without uncoupling ATP hydrolysis. Our data suggest the mechanism of inhibition is EG's blocking of binding of MgATP to the nitrogenase Fe protein. EG quenching allows, for the first time, the observation of the relaxation of the intermediate reaction states at room temperature. Electron paramagnetic resonance (EPR) spectroscopy is used to monitor the room-temperature decay of the nitrogenase turnover states following EG quenching of catalytic activity. The return of the intermediate states to the resting state occurs in multiple phases over 2 h. During the initial stage, nitrogenase still possesses the ability to generate CO-induced EPR signals even though catalytic activity has ceased. During the last phase of relaxation, the one-electron reduced state of the MoFe protein (E1) relaxes to the resting state (E0) in a slow first-order reaction.

In vitro screening of inhibition of PPAR-γ activity as a first step in identification of potential breast carcinogens.

Alcohol consumption and increased estrogen levels are major risk factors for breast cancer, and peroxisome proliferator-activated receptor γ (PPAR-γ) plays an important role in alcohol-induced breast cancer. PPAR-γ activity is inhibited by ethanol, leading to increased aromatase activity and estrogen biosynthesis ultimately leading to breast cancer. If other organic solvents inhibit PPAR-γ activity, they should also lead to increased oestrogen biosynthesis and thus be potential breast carcinogens. Ten commonly used hydrophilic organic solvents were first tested in a cell-based screening assay for inhibitory effects on PPAR-γ transactivation. The chemicals shown to inhibit PPAR-γ were tested with vectors encoding PPAR-γ with deleted AB domains and only the ligand-binding domain to rule out unspecific toxicity. Next, the effects on biosynthesis of estradiol, testosterone and oestrone sulphate were measured in the H295R steroidogenesis assay after incubation with the chemicals. Ethylene glycol, ethyl acetate, and dimethyl sulphoxide inhibited PPAR-γ transactivation in a dose-dependent manner. The inhibitory effect on PPAR-γ was specific for PPAR-γ since the AB domain of PPAR-γ was required for the inhibitory effect. In the second step, ethylene glycol significantly increased production of oestradiol by 19% (p < 0.05) and ethyl acetate inhibited production of testosterone (p < 0.05). We here show that screening of 10 commonly used organic solvents for the ability to inhibit PPAR-γ transactivation followed by a well-established steroidogenesis assay for production of sex hormones in exposed H295 R cells may provide a screening tool for potential breast carcinogens. This initial screening thus identified ethylene glycol and possibly ethyl acetate as potential breast carcinogens.