Detergentless Microemulsions Research Articles

The Synthetic Potential of Fungal Feruloyl Esterases: A Correlation with Current Classification Systems and Predicted Structural Properties

Twenty-eight fungal feruloyl esterases (FAEs) were evaluated for their synthetic abilities in a ternary system of n-hexane: t-butanol: 100 mM MOPS-NaOH pH 6.0 forming detergentless microemulsions. Five main derivatives were synthesized, namely prenyl ferulate, prenyl caffeate, butyl ferulate, glyceryl ferulate, and l-arabinose ferulate, offering, in general, higher yields when more hydrophilic alcohol substitutions were used. Acetyl xylan esterase-related FAEs belonging to phylogenetic subfamilies (SF) 5 and 6 showed increased synthetic yields among tested enzymes. In particular, it was shown that FAEs belonging to SF6 generally transesterified aliphatic alcohols more efficiently while SF5 members preferred bulkier l-arabinose. Predicted surface properties and structural characteristics were correlated with the synthetic potential of selected tannase-related, acetyl-xylan-related, and lipase-related FAEs (SF1-2, -6, -7 members) based on homology modeling and small molecular docking simulations.

Evolution of the feruloyl esterase MtFae1a from Myceliophthora thermophila towards improved catalysts for antioxidants synthesis.

The chemical syntheses currently employed for industrial purposes, including in the manufacture of cosmetics, present limitations such as unwanted side reactions and the need for harsh chemical reaction conditions. In order to overcome these drawbacks, novel enzymes are developed to catalyze the targeted bioconversions. In the present study, a methodology for the construction and the automated screening of evolved variants library of a Type B feruloyl esterase from Myceliophthora thermophila (MtFae1a) was developed and applied to generation of 30,000 mutants and their screening for selecting the variants with higher activity than the wild-type enzyme. The library was generated by error-prone PCR of mtfae1a cDNA and expressed in Saccharomyces cerevisiae. Screening for extracellular enzymatic activity towards 4-nitrocatechol-1-yl ferulate, a new substrate developed ad hoc for high-throughput assays of feruloyl esterases, led to the selection of 30 improved enzyme variants. The best four variants and the wild-type MtFae1a were investigated in docking experiments with hydroxycinnamic acid esters using a model of 3D structure of MtFae1a. These variants were also used as biocatalysts in transesterification reactions leading to different target products in detergentless microemulsions and showed enhanced synthetic activities, although the screening strategy had been based on improved hydrolytic activity.

Optimized Enzymatic Synthesis of Feruloyl Derivatives Catalyzed by Three Novel Feruloyl Esterases from Talaromyces wortmannii in Detergentless Microemulsions

Three novel feruloyl esterases (Fae125, Fae7262 and Fae68) from Talaromyces wortmannii overexpressed in the C1 platform were evaluated for the transesterification of vinyl ferulate with two acceptors of different size and lipophilicity (prenol and L-arabinose) in detergentless microemulsions. The effect of reaction conditions such as the microemulsion composition, the substrate concentration, the enzyme load, the pH, the temperature and the agitation were investigated. The type A Fae125 belonging to the subfamily 5 (SF5) of phylogenetic classification showed highest yields for the synthesis of both products after optimization of reaction conditions: 81.8% for prenyl ferulate and 33.0% for L-arabinose ferulate. After optimization, an 8-fold increase in the yield and a 12-fold increase in selectivity were achieved for the synthesis of prenyl ferulate.

Optimization of enzymatic synthesis of l-arabinose ferulate catalyzed by feruloyl esterases from Myceliophthora thermophila in detergentless microemulsions and assessment of its antioxidant and cytotoxicity activities

The feruloyl esterases FaeA1, FaeA2, FaeB1, FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464 were used as biocatalysts for the transesterification of vinyl ferula ...

Optimized synthesis of novel prenyl ferulate performed by feruloyl esterases from Myceliophthora thermophila in microemulsions

Five feruloyl esterases (FAEs; EC 3.1.1.73), FaeA1, FaeA2, FaeB1, and FaeB2 from Myceliophthora thermophila C1 and MtFae1a from M. thermophila ATCC 42464, were tested for their ability to catalyze the transesterification of vinyl ferulate (VFA) with prenol in detergentless microemulsions. Reaction conditions were optimized investigating parameters such as the medium composition, the substrate concentration, the enzyme load, the pH, the temperature, and agitation. FaeB2 offered the highest transesterification yield (71.5 ± 0.2%) after 24 h of incubation at 30 °C using 60 mM VFA, 1 M prenol, and 0.02 mg FAE/mL in a mixture comprising of 53.4:43.4:3.2 v/v/v n-hexane:t-butanol:100 mM MOPS-NaOH, pH 6.0. At these conditions, the competitive side hydrolysis of VFA was 4.7-fold minimized. The ability of prenyl ferulate (PFA) and its corresponding ferulic acid (FA) to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was significant and similar (IC50 423.39 μM for PFA, 329.9 μM for FA). PFA was not cytotoxic at 0.8–100 μM (IC50 220.23 μM) and reduced intracellular reactive oxygen species (ROS) in human skin fibroblasts at concentrations ranging between 4 and 20 μM as determined with the dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay.

Determination of nitrogen-containing polycyclic aromatic compounds in diesel and gas oil by reverse-phase high performance liquid chromatography using introduction of sample as detergentless microemulsion

Carbazoles, quinolines, indoles, pyrroles, anilines and acridines are nitrogen-containing polycyclic aromatic compounds (NPAC) that contribute to deteriorate fuels affecting storage stability, causing problems to motor engines and poisoning automotive catalysts. Carbazole, 3-ethylcarbazole, 9-ethylcarbazole, 9-methylcarbazole, quinoline, benzo[h]quinoline, indole, 3-methylindole, acridine and N,N-dimethylaniline were separated and quantified in diesel and gas oil by reverse-phase high performance liquid chromatography with fluorimetric detection and molecular absorption photometric detection using introduction of fuel samples as detergentless microemulsion (DME). The DME is composed by ethanol, propan-2-ol, water and iso-octane in specific proportions to stabilize the organic samples. The introduction of sample in the DME simplifies the procedure since it avoids extraction of the analytes and enables the determination of basic and neutral NPAC altogether. The limits of quantification for the analytes in the DME were in μgL−1 order (ng range for 10μL of injected volume). Satisfactory analyte recoveries were achieved (between 89.5% and 108.7%). Results were found to be comparable using or not using 7-methylindole and N-methylpyrrole as internal standards (since these were not found in the analyzed samples). Studies showed homogeneity of the analytes in the DME after mechanical stirring (at 7200rpm for 5min). Stability studies indicated results with coefficients of variation below 3%. The method is adequate for a rapid screening of NPACs in petroleum samples.

Detergentless ultrasound-assisted extraction of trace elements from edible oils using lipase as an extractant

A new method for the extraction and preconcentration of trace elements (Al, Ba, Cd, Cu, Fe, Mn, Mo, Ni, Ti, V and Zn) from edible oils by producing detergentless micro-emulsions via an ultrasound-assisted extraction using a water phase containing Lipase at pH 3 as an extractant was developed. The trace elements in the water phase post-extraction were determined against matrix matched standards using ICP-MS. In the first step of the work, the parameters that affect extraction, such as pH, the volume of 1% lipase in the water phase and the ultrasonic and centrifugation times were optimized. Under the optimal conditions, the detection limits (µgkg–1) were 0.46, 0.03, 0.007, 0.028, 0.67, 0.038, 0.022, 0.14, 0.17, 0.05 and 0.07 for Al, Ba, Cd, Cu, Fe, Mn, Mo, Ni, Ti, V and Zn respectively for edible oils (3Sb/m). A certified reference material (EnviroMAT HU-1 Used oil) was analysed to check the accuracy of the developed method. Results obtained were in agreement with certified values with a t-test showing that no significant differences at the 95% confidence levels were found. The proposed method was applied to different edible oils such as sunflower oil, rapeseed oil, olive oil and salmon oil.

Extraction of trace elements by ultrasound-assisted emulsification from edible oils producing detergentless microemulsions

The aim of this study is to develop a new method for the extraction and preconcentration of trace elements from edible oils via an ultrasound-assisted extraction using ethylenediaminetetraacetic acid (EDTA) producing detergentless microemulsions. These were then analyzed using ICP-MS against matrix matched standards. Optimum experimental conditions were determined and the applicability of the proposed ultrasound-assisted extraction method was investigated. Under the optimal conditions, the detection limits (μgkg−1) were 2.47, 2.81, 0.013, 0.037, 1.37, 0.050, 0.049, 0.47, 0.032 and 0.087 for Al, Ca, Cd, Cu, Mg, Mn, Ni, Ti, V and Zn respectively for edible oils (3Sb/m). The accuracy of the developed method was checked by analyzing certified reference material. The proposed method was applied to different edible oils such as sunflower seed oil, rapeseed oil, olive oil and cod liver oil.

Development of an analytical method for the determination of arsenic in gasoline samples by hydride generation–graphite furnace atomic absorption spectrometry

The purpose of the present work was to optimize the conditions for the determination of arsenic in gasoline with hydride generation–graphite furnace atomic absorption spectrometry after acid digestion using a full two-level factorial design with center point. The arsine was generated in a batch system and collected in a graphite tube coated with 150μg Ir as a permanent modifier. The sample volume, the pre-reduction conditions, the temperature program and modifier mass were kept fixed for all experiments. The estimated main effects were: reducing agent concentration (negative effect), acid concentration (negative effect) and trapping temperature (positive effect). It was observed that there were interactions between the variables. Moreover, the curvature was significant, indicating that the best conditions were at the center point. The optimized parameters for arsine generation were 2.7molL−1 hydrochloric acid and 1.6% (w/v) sodium tetrahydroborate. The optimized conditions to collect arsine in the graphite furnace were a trapping temperature of 250°C and a collection time of 30s. The limit of detection was 6.4ngL−1 and the characteristic mass was 24pg. Two different systems for acid digestion were used: a digester block with cold finger and a microwave oven. The concentration of arsenic found with the proposed method was compared with that obtained using a detergentless microemulsion and direct graphite furnace determination. The results showed that the factorial design is a simple tool that allowed establishing the appropriate conditions for sample preparation and also helped in evaluating the interaction between the factors investigated.

Direct determination of arsenic in petroleum derivatives by graphite furnace atomic absorption spectrometry: A comparison between filter and platform atomizers

In the present work a direct method for the determination of arsenic in petroleum derivatives has been developed, comparing the performance of a commercial transversely heated platform atomizer (THPA) with that of a transversely heated filter atomizer (THFA). The THFA results in a reduction of background absorption and an improved sensitivity as has been reported earlier for this atomizer. The mixture of 0.1% (m/v) Pd + 0.03% (m/v) Mg + 0.05% (v/v) Triton X-100 was used as the chemical modifier for both atomizers. The samples (naphtha, gasoline and petroleum condensate) were stabilized in the form of a three-component solution (detergentless microemulsion) with the sample, propan-1-ol and 0.1% (v/v) HNO 3 in a ratio of 3.0:6.4:0.6. The characteristic mass of 13 pg found in the THFA was about a factor of two better than that of 28 pg obtained with the THPA; however, the limits of detection (LOD) and quantification (LOQ) were essentially the same for both atomizers (1.9 and 6.2 μg L −1, respectively, for THPA, and 1.8 and 5.9 μg L −1, respectively, for THFA) due to the increased noise observed with the THFA. A possible explanation for that is a partial blockage of the radiation from the hollow cathode lamp by the narrow inner diameter of this tube and the associated loss of radiation energy. Due to the lack of an appropriate certified reference material, recovery tests were carried out with inorganic and organic arsenic standards and the results were between 89% and 111%. The only advantage of the THFA found in this work was a reduction of the total analysis time by about 20% due to the ‘hot injection’ that could be realized with this furnace. The arsenic concentrations varied from < LOQ to 43.3 μg L −1 in the samples analyzed in this work.

Novel sol-gel immobilization of horseradish peroxidase employing a detergentless micro-emulsion system

A novel sol-gel immobilization method employing a detergentless micro-emulsion system that consisted ofn-hexane/iso-propanol/water was developed and used to immobilize a horseradish peroxidase (HRP). Micro-sized gel powder containing enzymes was generated in the ternary solution without drying and grinding steps or the addition of detergent, therefore, the method described in this study is a simple and straightforward process for the manufacture of gel powder. The gel powder made in this study was able to retain 84% of its initial enzyme activity, which is higher than gel powders produced through other immobilization methods. Furthermore, the HRP immobilized using this method, was able to maintain its activity at or above 95% of its initial activity for 48h, whereas the enzyme activities of free HRP and HRP that was immobilized using the other sol-gel method decreased dramatically. In addition, even when in the presence of excess hydrogen peroxide, the enzyme immobilized using the novel sol-gel method described here was more stable than enzymes immobilized using the other method.

Determination of Mo, Zn, Cd, Ti, Ni, V, Fe, Mn, Cr and Co in crude oil using inductively coupled plasma optical emission spectrometry and sample introduction as detergentless microemulsions

A procedure to prepare crude oil samples as detergentless microemulsions was optimized and applied for the determination of Mo, Zn, Cd, Si, Ti, Ni, V, Fe, Mn, Cr and Co by ICP OES. Propan-1-ol was used as a co-solvent allowing the formation of a homogeneous and stable system containing crude oil and water. The optimum composition of the microemulsion was crude oil / propan-1-ol / water / concentrated nitric acid, 6 / 70 / 20 / 4 w/w/w/w. This simple sample preparation procedure together with an efficient sample introduction (using a Meinhard K3 nebulizer and a twister cyclonic spray chamber) allowed a fast quantification of the analytes using calibration curves prepared with analyte inorganic standards. In this case, Sc was used as internal standard for correction of signal fluctuations and matrix effects. Oxygen was used in the nebulizer gas flow in order to minimize carbon building up and background. Limits of detection in the ng g − 1 range were achieved for all elements. The methodology was tested through the analysis of one standard reference material (SRM NIST 1634c, Residual Fuel Oil) with recoveries between 97.9% and 103.8%. The method was also applied to two crude oil samples and the results were in good agreement with those obtained using the acid decomposition procedure. The precision ( n = 3) obtained was below 5% and the results indicated that the method is well suited for oil samples containing low concentrations of trace elements.

Application of multivariate technique in method development for the direct determination of copper in petroleum condensate using graphite furnace atomic absorption spectrometry

A method has been developed for the direct determination of copper in petroleum condensate using detergentless micro-emulsions (three-component solutions) and graphite furnace atomic absorption spectrometry. The optimization was performed using a three-level full factorial design, resulting in pyrolysis and atomization temperatures of 920 °C and 2600 °C, respectively. Using the secondary copper line at 327.4 nm, which is less affected by the magnetic field of the Zeeman-effect background correction system, a characteristic mass of 39 pg, and detection limit of 0.4 μg l−1 was obtained using 20 μl of micro-emulsion, which corresponds to 1.2 μg l−1 in petroleum condensate. The precision, expressed as relative standard deviation, was 2.0% or 2.8% for copper concentrations of 30 μg l−1 and 5.0 μg l−1, respectively. The spike recovery of 105% for copper added to a petroleum condensate sample proved that this procedure could be applied satisfactorily to a determination in this matrix.

Electrothermal atomic absorption spectrometric method for the determination of vanadium in diesel and asphaltene prepared as detergentless microemulsions

A method based on electrothermal atomic absorption spectrometry for the determination of vanadium in diesel and asphalthene fractions is proposed. In order to avoid analyte losses observed at the microgram per liter range for metal traces in organic solutions, diesel samples were stabilized as detergentless microemulsions by mixing with propan-1-ol and nitric acid solution. The solid asphaltene oil fraction was separated and dissolved in dichloromethane before mixing these solution with propan-1-ol and nitric acid solution. Wall atomization as well as no modifier was used. For diesel, aqueous analytical solutions could be used for calibration. For asphaltene, calibration was performed with analytical solutions prepared at the dichloromethane+propan-1-ol+nitric acid medium, spiked with inorganic standard solution. Linear ranges up to 200 μg l −1 were observed, as well as limit of detection of 5 μg l −1 and 4 μg g −1 for diesel and asphaltene, respectively. Good recoveries were obtained for V-cyclohexanebutyrates spiked diesel samples, as well as coherent results for the asphaltene fraction of the NIST 1634c (trace elements in fuel oil) certified reference material.

Evaluation of electrothermal atomic absorption spectrometry for trace determination of Sb, As and Se in gasoline and kerosene using microemulsion sample introduction and two approaches for chemical modification

Studies aimed at the development of analytical methods based on electrothermal atomic absorption spectrometry for the determination of antimony, selenium and arsenic in gasoline and kerosene were performed. The goal was to develop sensitive methods that allow a simple calibration procedure to be used through direct correlation between analyte signals measured in the sample and signals from aqueous standards. This was possible by optimization of both the sample preparation procedure and furnace conditions. The platform atomization technique was used together with chemical modification (ruthenium permanent modifier or Pd/Mg nitrate chemical modifier). Better results were obtained when samples were introduced as microemulsions prepared with a surfactant (Triton X-100) rather than sample introduction as detergentless microemulsions (three component solution). Good sensitivity was achieved, which allowed the determination of analytes in gasoline and kerosene samples in the µg L−1 range. Linear ranges up to three decades were observed for the analytical curves. Laboratory-prepared samples were analyzed with good precision and recovery.

Kinetics of reactions of ethylp-nitrophenyl ethylphosphonate with anionic nucleophiles in a detergentless microemulsion

The kinetics of reactions of ethylp-nitrophenyl ethylphosphonate with anionic nucleophiles in a detergentless water—oil microemulsion, formed in then-hexane—water—isopropyl alcohol system, was studied. The rate constants of the reactions in the microemulsion are higher than those in aqueous solutions and increase with increasing isopropyl alcohol: water ratio.

The influence of detergentless microemulsion on the kinetics of hydrolysis of di(p-nitrophenyl) methylphosphonate catalyzed by amines

The influence of the detergentless water/oil microemulsion formed in then-hexane-water-2-propanol system on the kinetics of the hydrolysis of di(p-nitrophenyl) methylphosphonate under the conditions of general base catalysis by primary, secondary, and tertiary aliphatic amines was investigate. The leveling of the catalytic rate constants and basicities of the amines was found for this reaction in a microemulsion. The catalytic rate constants for the amines containing hydrophilic groups increase in microemulsions as compared to those in an aqueous medium, while the catalytic constants for the amines containing hydrophobic groups decrease.

Bile Salt Stimulated Human Milk Lipase Catalyzed Ester Hydrolysis in Detergentless Microemulsion Media

Bile salt stimulated human milk lipase (E.C.3.1) has been used to catalyze the hydrolysis of 4-nitrophenylalkanoates (alkyl C-chain length, n = 2, 3, 4, 6, 10, 12 and 16) in a detergentless microemulsion medium of n-hexane/iso-propanol/water (71.1:27.7:1.2 vol%) containing 2 mM taurocholate at 37°C. The rate of hydrolysis of the esters with n = 2, 3, or 4 was nearly equal but the rate then fell rapidly with increasing alkyl-chain length. No activity was observed with the palmitate ester. Three dimensional surfaces were built up to represent both the catalytic activity and the rate constant of inactivation for the enzyme catalyzed hydrolysis of 4-nitrophenylpropionate in a range of compositions of the ternary system n-hexane/wo-propanol/water at 37 °C. Maximum activation and maximum inactivation were observed in the ternary region of the phase diagram. In the stable; transparent micro-emulsion region another smaller maximum in activity was observed and, at this same solvent composition, minimum inactivatio...

Aggregation of unsaturated long‐chain fatty alcohols in nonaqueous systems

AbstractAggregation and related phenomena in nonaqueous binary and ternary solutions containing unsaturated long‐chain fatty alcohol amphiphiles have been studied. Six C18 fatty alcohols were studied—oleyl alcohol (9Z‐octadecen‐1‐ol), elaidyl alcohol (9E‐octadecen‐1‐ol), linoleyl alcohol (9Z, 12Z‐octadecadien‐1‐ol), elaidolinoleyl alcohol (9E, 12E‐octadecadien‐1‐ol), linolenyl alcohol (9Z, 12Z, 15Z‐octadecatrien‐1‐ol) and elaidolinolenyl alcohol (9E, 12E, 15E‐octadecatrien‐1‐ol). Equivalent conductivity and photon correlation spectroscopy confirmed that unsaturated long‐chain fatty alcohols form large and polydisperse aggregates in methanol. Critical micelle concentration (CMC) results showed that the degree of unsaturation and configuration of the double bonds in the fatty alcohol significantly influences aggregation. Aggregation of oleyl alcohol in a series of straight and branched medium‐chainlength (C3‐C8) alkanol solvents was studied. For shorter‐chained alkanols (C1‐C4), decreasing solvent dielectric constant decreases the CMC; however, for longer‐chained alkanols (C4‐C8), no significant effects occurred on the CMC. The effect of solubilized soybean oil on the viscosity of long‐chain fatty alcohol/methanol solutions was also analyzed. Relative viscosity results were consistent with those expected for microemulsions. Although preliminary in nature, these results generally support the notion that soybean oil is solubilized by incorporation into large soybean oil‐in‐fatty alcohol aggregates in methanol solvent, resembling a nonaqueous detergentless microemulsion.

Alterations to the catalytic properties of polyphenoloxidase in detergentless microemulsions and ternary water-organic solvent mixtures

The catalytic properties of mushroom polyphenoloxidase could be substantially altered by entrapment into hexane- and toluene-based microemulsions stabilized with isopropanol. The fast irreversible inactivation and drastic substrate inhibition of the enzyme were significantly reduced in detergentless microemulsions in comparison to conventional aqueous media. Similar changes in the catalytic behavior of polyphenoloxidase were observed in the normal ternary solutions of hexane-(toluene)-isopropanol-water, and in the H-bonded aggregates of isopropanol and water in toluene, but not in hexane.