Modified enzymes for reactions in organic solvents

Recent studies on biocatalysis in water—organic solvent biphasic systems have shown that many enzymes retain their catalytic activities in the presence of high concentrations of organic solvents. However, not all enzymes are organic solvent tolerant, and most have limited and selective tolerance to particular organic solvents. Protein modification or protein tailoring is an approach to alter the characteristics of enzymes, including solubility in organic solvents. Particular amino acids may play pivotal roles in the catalytic ability of the protein. Attaching soluble modifiers to the protein molecule may alter its conformation and the overall polarity of the molecule. Enzymes, in particular lipases, have been chemically modified by attachment of aldehydes, polyethylene glycols, and imidoesters. These modifications alter the hydrophobicity and conformation of the enzymes, resulting in changes in the microenvironment of the enzymes. By these modifications, newly acquired properties such as enhancement of activity and stability and changes in specificity and solubility in organic solvents are obtained. Modified lipases were found to be more active and stable in organic solvents. The optimum water activity (a _w ) for reaction was also shifted by using modified enzymes. Changes in enantioselective behavior were also observed.     

Growth kinetics for AgI nanoparticles in AOT reverse micelles: effect of molecular length of hydrocarbon solvents

The growth kinetics for AgI nanoparticles formed in the solutions of water/AOT reverse micelles in n-hexane, n-octane, n-decane, and n-dodecane were investigated. In small micelles, the rate of nanoparticles growth was found to be independent of the type of solvent, while in large micelles the growth rate grew with increasing length of solvent molecules. The effect was explained by a different amount of free water in the micelle pools of the same size.     

Optimization of hydrolase efficiency in organic solvents

The application of hydrolases in organic solvents for synthetic purposes is a procedure routinely adopted in organic chemistry, especially for the preparation of chiral building blocks. Numerous studies have shed light on several aspects of the mechanism of hydrolase action in low-water environments. Procedures suitable to improve the catalytic efficiency of enzymes and productivity of the synthetic processes have been reported. These fundamental and applied investigations have made hydrolase-catalyzed reactions in organic solvents of industrial interest. In this article we describe and discuss various approaches adopted to optimize the performance of hydrolases in organic media, with special emphasis on the formulation of the biocatalysts which, under proper conditions, can display an activity equal to that displayed in aqueous buffers.     

Micellar Solubilization of Proteins in Aprotic Solvents and their Spectroscopic Characterisation

The quaternary ammonium salt methyl-trioctylammonium chloride enables the transfer of α-chymotrypsin, trypsin, pepsin and glucagone from water to cyclohexane. Reversed micelles, whose polar core solubilizes both protein and water, are probably formed in the apolar phase. The influence of various parameters on the phase transfer (concentration, pH, solvent, temperature, etc.) has been investigated. Absorption, fluorescence and circular dichroism studies of the biopolymers in the cyclohexane system have been carried out. For trypsin and chymotrypsin, the CD. signal in the 200 nm region is very similar in water and in cyclohexane, which suggests that the polypeptide folding is not substantially different in the two phases. The fluorescence quantum yield is always much larger in the cyclohexane phase than in water. The longer wavelength region of the UV. absorption spectrum is slightly red-shifted relative to water, and a band at 225 nm, probably arising from the aromatic chromophore, is apparent in the organic phase. Reasons for these spectral perturbations are discussed. The enzymes transferred from water into cyclohexane phases can be continuously retransferred into a second water phase. The possible relevance of this ‘double transfer’ as a model for the vectorial transport of biopolymers or a separation technique is discussed.     

A comparison of ionic liquids to molecular organic solvents as additives for chiral separations in micellar electrokinetic chromatography

In this study, we report the effects of adding ionic liquids (ILs), as compared to adding conventional molecular organic solvents (MOSs), to aqueous buffer solutions containing molecular micelles in the separation of chiral analyte mixtures in micellar EKC (MEKC). The molecular micelle used in this study was polysodium oleyl-L-leucylvalinate (poly-L-SOLV). The ILs were 1-alkyl-3-methylimidazolium tetrafluoroborate, where the alkyl group was ethyl, butyl, hexyl, or octyl. These ILs were chosen due to their hydrophobicity, good solvating, and electrolyte properties. Thus, it was expected that these ILs would have favorable interactions with chiral analytes and not adversely affect the background current. Common CE buffers, mixed with a molecular micelle, and an IL or a MOS, were used for these chiral separations. The buffers containing an IL in the concentration range of 0.02-0.1 v/v were found to support a reasonable current when an electric field strength of 500 V/cm was applied across the capillary. However, a current break down was observed for the buffers containing more than 60% v/v MOS on application of the above-mentioned electric field. The chiral resolution and selectivity of the analytes were dependent on the concentration and type of IL or MOS used.     

Process development for production of medium chain triglycerides using immobilized lipase in a solvent-free system

The synthesis of tricaprylin, tricaprin, trilaurin, and trimyristin in a solvent-free system was conducted by mixing a commercial immobilized lipase with the organic reagents (glycerol and fatty acid) in a 20-mL batch reactor with constant stirring. The effects of temperature, fatty acid/glycerol molar ratio, and enzyme concentration on the reaction conversion were determined. The reactions were carried out for 26 h and the nonpolar phase was analyzed by gas chromatography. Appreciable levels of medium chain triglycerides were achieved, except for tricaprylin. The higher selectivity values for the production of triglycerides were attained under the following conditions: a fatty acid/glycerol molar ratio of 5; enzyme concentration of 5 or 9% (w/w); and temperatures of 70°C (tricaprin), 80°C (trilaurin), and 90°C (trimyristin). After completion of the esterification reaction under these conditions, the recovery of the triglyceride and fatty acids, and the reusability of the enzyme were studied. The unreacted fatty acid and the produced triglyceride were satisfactorily recovered. The commercial immobilized lipase was used in 10 consecutive batch reactions at 80°C, with 100% selectivity in the trilaurin and trimyristin synthesis. The possibility of enzyme reuse and the recovery of residual fatty acid are relevant results that contribute to increasing the viability of the process.     

One-pot access to pyridocoumarins via Povarov-hydrogen transfer cascade under auto-tandem catalysis of iodine in aqueous micelles

A cascade of inverse electron demand Diels–Alder reaction of 6-aminocoumarin-derived aldimines with an excess of styrene and oxidative aromatization of the formal Povarov adducts under autotandem iodine catalysis in aqueous micellar conditions provides direct access to a small library of substituted pyridine annulated coumarins in good to acceptable yields (12 examples). The unusual formation of linearly annulated pyridocoumarins under essentially neutral conditions is a remarkable feature of the protocol.     

Development of Microdroplet Generation Method for Organic Solvents Used in Chemical Synthesis

Recently, chemical operations with microfluidic devices, especially droplet-based operations, have attracted considerable attention because they can provide an isolated small-volume reaction field. However, analysis of these operations has been limited mostly to aqueous-phase reactions in water droplets due to device material restrictions. In this study, we have successfully demonstrated droplet formation of five common organic solvents frequently used in chemical synthesis by using a simple silicon/glass-based microfluidic device. When an immiscible liquid with surfactant was used as the continuous phase, the organic solvent formed droplets similar to water-in-oil droplets in the device. In contrast to conventional microfluidic devices composed of resins, which are susceptible to swelling in organic solvents, the developed microfluidic device did not undergo swelling owing to the high chemical resistance of the constituent materials. Therefore, the device has potential applications for various chemical reactions involving organic solvents. Furthermore, this droplet generation device enabled control of droplet size by adjusting the liquid flow rate. The droplet generation method proposed in this work will contribute to the study of organic reactions in microdroplets and will be useful for evaluating scaling effects in various chemical reactions.     

Structural and catalytic aspects of cutinase in w/o microemulsions

 Structural and catalytic properties of cutinase were studied in bis(2-ethylhexyl) sodium sulfo-succinate (AOT)-isooctane microemulsion systems. The effect of the water content of the microemulsions on the cutinase activity on an esterification reaction of lauric acid with pentanol showed that cutinase followed a bell-shaped profile presenting a maximum at w _o=9, with w _o=[H₂O]/[AOT]. Kinetic studies allowed the determi-nation of the apparent parameters K _m and V _max. Electron paramagnetic resonance (EPR) spectroscopy studies of active site labeled cutinase in microemulsions with varying w _o values showed that in all microemulsions, the mobility of the label is higher than in the aqueous solution. Furthermore, it was found that the maximum of the enzyme activity did not correspond to a reduced active site mobility. Up to w _o=9 there was an increase of both activity and active site mobility. As the water content of the system became higher, the mobility of the bound spin label further increased whereas the enzymatic activity dropped considerably. Received: 20 December 1996 Accepted: 24 February 1997     

锂及锂离子蓄电池有机溶剂研究进展

从有机溶剂对电池安全性的影响，氧化稳定性，与负极的相容性及对电解液电导率的影响四个方面，论述了锂及锂离子蓄电池有机溶剂的化学和电化学，介绍了碳酸酯类，醚类和羧酸酯类溶剂的性质与电极的相容性及在有机电解液中的应用，对含硫，硼基及胺类有机溶剂等也作了论述。     

Highly Selective Methodology for Entrapment and Subsequent Removal of Cobalt (II) Ions under Optimized Conditions by Micellar-Enhanced Ultrafiltration

Micellar-enhanced ultrafiltration (MEUF), being a separation technique, was used to remove cobalt metal ion (Co²⁺) from their aqueous solutions in an application to reduce the toxicity level from industrial effluents using a micellar solution of anionic and cationic surfactants. The metal ions were first adsorbed by using anionic surfactants, i.e., sodium dodecyl sulfate (SDS) and sodium oleate (SO). The calculations for partition (K_x) and binding constants (K_b) and their respective free energy of partition and binding (ΔG_p and ΔG_b kJmol⁻¹) helped significantly to find out the extent of binding or interaction of Co²⁺ with the surfactant and ΔG_p and ΔG_b were found to be −29.50 and −19.38 kJmol⁻¹ for SDS and −23.95 and −12.67 kJmol⁻¹ in the case of SO. MEUF work was also performed to find out the optimal conditions to remove metal pollutants from the aqueous system. For the said purpose, various factors and concentrations effect were studied, such as the concentration of the surfactant, concentration of the electrolyte (NaCl), transmembrane pressure, RPM, and pH. The efficiency of this process was checked by calculating various parameters, such as rejection percentage (R%) and permeate flux (J). A maximum rejection of 99.95% with SDS and 99.99% with SO was attained.     

Application of design-of-experiments procedures to optimize efficiently pretreatment of lipase for use in a nonaqueous reaction

A variety of different pretreatments can improve the performance of enzymes in nonpolar reaction media. These pretreatments have primarily been studied in isolation; however, interactions between some pairs of pretreatments are known to exist. The presence of these interactions complicates the design of an optimum multifactor pretreatment. Modern design-of-experiments techniques allow the simultaneous optimization of two or more variables. To improve the performance of lipase in a model reaction, we used a technique called the method of steepest ascent to optimize three variables simultaneously: pretreatment pH and sodium phosphate concentration, and the concentration of acetic acid (one of the reactants) in the reaction mixture. In only 26 experimental runs, this optimization process determined a combination of variable settings that yielded a reaction product approx 180 times faster than achieved with untreated enzyme. Evidence is presented to demonstrate that locating this optimum with single-factor experiments would be inefficient. This article demonstrates the efficiency of the method of steepest ascent particularly for evaluation of enzymatic reaction conditions exhibiting significant interactions.     

锂离子电池有机电解液材料研究进展

综述了锂离子电池有机电解液材料的研究现状。锂离子电池有机电解液主要由电解质锂盐、有机溶剂和添加剂三个部分组成,新型电解质锂盐的研究开发可分为三个方面：（1）LiTFSI及其类似物;（2）络合硼酸锂化合物;（3）络合磷酸锂化合物。有机溶剂的研究工作主要集中在新型有机溶剂的开发上。最重要的添加剂主要有三类：（1）主要用以改善碳负极SEI膜性能的添加剂;（2）过充电保护添加剂;（3）配体添加剂。     

Design of a composite amperometric enzyme electrode for the control of the benzoic acid content in food

A graphite–Teflon–tyrosinase composite biosensor for the determination of benzoic acid in foodstuffs is reported. The biosensor functioning is based on the inhibition effect of benzoic acid on the biocatalytic activity of the enzyme in a reversed micelle working medium formed with ethyl acetate as the continuous phase, a 0.05 mol l⁻¹ phosphate buffer solution of pH 7.4 (5%) as the aqueous dispersed phase, and 0.10 mol l⁻¹ dioctyl sulfosuccinate (AOT) as the emulsifying agent. A potential value of −0.10 V, and a constant enzyme-substrate (phenol) concentration of 2.0×10⁻⁴ mol l⁻¹ were selected to carry out the amperometric inhibition measurements. The tyrosinase inhibition process by benzoic acid is reversible and of the competitive type, with an apparent inhibition constant of 0.016 mmol l⁻¹. The composite bioelectrodes allow the regeneration of the electrode surface by polishing and exhibit long-term operation and stability. A limit of detection of 9.0×10⁻⁷ mol l⁻¹ benzoic acid was obtained. An interference study from other substances which can be found in foodstuffs together with benzoic acid was performed. Taking advantage of the capabilities of reversed micelles as universal solubilization media, the composite tyrosinase electrode was used for the determination of benzoic acid in two different kind of samples: mayonnaise sauce, which is a highly hydrophobic matrix, and Cola soft drinks, a hydrophilic matrix for which practically no sample treatment is necessary.     

The HLD–NAC equation of state for microemulsions formulated with nonionic alcohol ethoxylate and alkylphenol ethoxylate surfactants

The net–average curvature (NAC) model (Langmuir, 19, 186, 2003) is a simple, yet powerful, equation of state to fit and predict the phase behavior of microemulsions (μEs) formulated with ionic surfactants. However, its use for nonionic surfactants systems has not been evaluated until now. The objective of this work is to use the NAC equation of state to model the phase behavior of μEs formulated with pure alcohol ethoxylate (CiEj) and commercial nonylphenol ethoxylate (NPEj) surfactants with a range of alkanes (Cn). The NAC model requires three basic parameters: the characteristic curvature of the nonionic surfactant (Ccn), the characteristic length (ξ), and the scaling length parameter (L). The first part of this contribution presents a brief review of the NAC model and other thermodynamic models of μEs. Later, five example applications of the NAC model are presented and discussed: (I) fit/prediction of phase inversion temperature (PIT) values, (II) prediction of phase transition temperatures and characteristic lengths, (III) fit/prediction of interfacial tensions, (IV) prediction of “fish” phase diagrams, and (V) prediction of the composition of bicontinuous systems. The NAC model was able to fit the phase behavior observed in all the experimental scenarios, and predict within 30% the value of phase volumes and transition temperatures for CiEj systems. The largest deviations can be attributed to a poor estimation of ξ obtained through empirical correlations. It was also noted that for commercial NPEj surfactants, the NAC model can also fit and predict the phase behavior of these systems, only that more complex empirical correlations are needed for ξ and Ccn due to the partition of different surfactant n-mers into the excess oil and aqueous phases. In all cases, a good agreement was found when the length parameter (L) was estimated as 1.4 times the extended length of the surfactant tail.     

A Cofactor‐Substrate‐Based Supramolecular Fluorescent Probe for the Ultrafast Detection of Nitroreductase under Hypoxic Conditions

Identifying the location and expression levels of enzymes under hypoxic conditions in cancer cells is vital in early‐stage cancer diagnosis and monitoring. By encapsulating a fluorescent substrate, L‐NO₂ , within the NADH mimic‐containing metal–organic capsule Zn‐ MPB , we developed a cofactor‐substrate‐based supramolecular luminescent probe for ultrafast detection of hypoxia‐related enzymes in solution in vitro and in vivo. The host–guest structure fuses the coenzyme and substrate into one supramolecular probe to avoid control by NADH, switching the catalytic process of nitroreductase from a double‐substrate mechanism to a single‐substrate one. This probe promotes enzyme efficiency by altering the substrate catalytic process and enhances the electron transfer efficiency through an intra‐molecular pathway with increased activity. The enzyme content and fluorescence intensity showed a linear relationship and equilibrium was obtained in seconds, showing potential for early tumor diagnosis, biomimetic catalysis, and prodrug activation.     

Low‐density‐solvent‐based air‐assisted liquid–liquid microextraction followed by gas chromatography with flame ionization detection for the determination of synthetic phenolic antioxidants in milk samples

A simple and rapid sample pretreatment technique termed low‐density‐solvent‐based air‐assisted liquid–liquid microextraction has been developed for the extraction and preconcentration of three synthetic phenolic antioxidants including butylated hydroxyanisole, butylated hydroxytoluene, and tert‐butyl hydroquinone from milk samples prior to their analysis by gas chromatography with flame ionization detection. In this method, initially trichloroacetic acid as a proteins precipitation agent is added to the sample, and then it is sonicated and centrifuged. The obtained aqueous phase is removed and the analytes extracted by the proposed method using a low‐density organic solvent. Some important parameters such as type and volume of extraction solvent, ionic strength, pH, and centrifugation rate and time were studied. Under the optimum conditions, enrichment factors were obtained in the range of 501–614. LODs and quantification were between 0.76–1.16 and 2.66–3.96 ng mL^–1, respectively. This method is rapid and requires less than 15 min for sample preparation.     

Synthesis of Sulfonated Porous Organic Polymers with a Hydrophobic Core for Efficient Acidic Catalysis in Organic Transformations

Synthesis of sulfonated porous polymers with improved hydrophobicity and stability is of extreme importance in both academic research and industrial applications. However, there is often a trade-off between acidity and surface hydrophobicity of sulfonated polymers. In this study, we report a strategy for the synthesis of sulfonated porous organic polymers (S-PT) with improved hydrophobicity via free radical polymerization method by using a rigid and large multidentate monomer, 1,3,5-tri(4-vinylphenyl)-benzene, having a hydrophobic core. The results of vapor adsorption measurement show that S-PT has more hydrophobic properties than sulfonated poly(divinylbenzene) (S-PD), attributed to the hydrophobic core of its multidentate monomer. Furthermore, the optimization of sulfonation time established a balance between surface acidity and hydrophobicity. Under optimized conditions, S-PT afforded up to 113 mmol g⁻¹ h⁻¹ TOF in the esterification of oleic acid with methanol, more active than commercial Amberlyst-15 with TOF of 15 mmol g⁻¹ h⁻¹ and Nafion NR50 with TOF of 7 mmol g⁻¹ h⁻¹. We believe that the findings of this study will provide useful insights to advance the design and synthesis of solid acid catalysts for organic transformations.