Hydrophilic Organic Solvents Research Articles

Evaluation of the potential for use in biocatalysis of a lipase from a wild strain of Bacillus megaterium

We evaluated a lipase produced by Bacillus megaterium with respect to its potential for use in biocatalysis in organic solvents. After 72 h of fermentation the enzyme was precipitated directly from the culture broth with the addition of ammonium sulphate to 80% of saturation. It was then resuspended and dialysed. The preparation was shown to hydrolyse triacylglycerides, with further determinations of lipolytic activity being done in aqueous systems through the hydrolysis of p-nitrophenyl palmitate ( pNPP) at 37 °C. The temperature for maximum activity, based on product formation over the first minute of reaction, was 55 °C. The lipolytic activity was reasonably stable during incubation at temperatures of 40–70 °C, with a residual activity of 77% after 10 min incubation at 60 °C. Stability in organic solvents was studied by incubating the enzyme at 29 °C in butanol, toluene, hexane, isooctane and heptane and in various mixtures (25, 50, 80 and 100% (v/v)) of acetone, ethanol and isopropanol in water. Not only was it stable in butanol, hexane and toluene but it actually showed activation after being incubated in increasing percentages of isopropanol, ethanol and acetone, which is quite unusual for lipases. For the hydrolysis of pNPP at 37 °C in AOT (Dioctyl sodium sulfosuccinate)/heptane reversed micelles, the highest specific activities were obtained with W 0 ([H 2O]/[AOT]) values of 5 and 10, being 111 and 104 U mg −1, respectively. The R O/A (ratio of the reaction rates for the same reaction in organic medium and in aqueous medium) was 1.91 for the hydrolysis of pNPP, which is notable because R O/A values for lipases are typically much less than 1.0. The lipase of B. megaterium shows promise for application in biocatalysis due to its good stability at elevated temperatures and in hydrophobic and hydrophilic organic solvents.

Enzymatic transesterification of purine nucleoside having a low solubility in organic medium.

Enzymatic transesterification of guanosine having low solubility against organic solvent was examined. For the transesterification between guanosine and divinyl adipate catalyzed by alkaline protease from Bacillus (Bioprase), DMSO was added to DMF to increase the solublility of the nucleoside, and the conversion rate of guanosine to the vinyl guanosine ester was less than 30%. To overcome the reversible inactivation of enzyme by hydrophilic organic solvents, the reaction was carried out with 10% (v/v) water. The transesterification reaction was effectively catalyzed in DMF/DMSO in the presence of water and the conversion rate increased ca. 70% after 7 d reaction. The result shows that the water effect of Bioprase would be a useful method for the synthesis of low solublility nucleoside esters.

Penicillin Acylase Catalysed Synthesis of Ampicillin in Hydrophilic Organic Solvents

AbstractPenicillin acylase (EC 3.5.1.11) from Alcaligenes faecalis, immobilised as a cross‐linked enzyme aggregate (CLEA), catalysed the synthesis of ampicillin in water‐miscible organic solvents at low water concentrations. Below 4% water (v/v) no reaction was observed, showing the crucial role of water in maintaining the activity of penicillin acylase. The initial value of S/H was strongly affected by the nature of the solvent, but the effect of the water content was slight in the studied range of 4 to 15%. A reaction in acetonitrile containing 8% water afforded ampicillin in up to 86% yield.

Effect of water on the enzymatic synthesis of vinyl sugar ester in hydrophilic organic solvent

Full Paper: The transesterification of divinyl adipate with glucose in N,N-dimethylformamide (DMF) catalyzed by alkaline protease from Strptomyces sp. and Bacillus subtilis at various water contents were examined. The enzymatic reaction by the Bacillys protease was carried out effectively in the presence of more than 2% of water, and a maximum reaction rate was observed at a water content of 20%. On the other hand, the reaction by Streptomyces protease proceeded more effectively without the addition of water than in the presence of water. The results suggest that optimum water content is different, depending on the kind of enzyme protein used.

Stability of free and immobilised peroxidase in aqueous–organic solvents mixtures

The activity and stability of horseradish ( Amoracia rusticana) peroxidase (HRP) free in solution and immobilised onto silica microparticles was studied in the presence of organic co-solvents. The effect of several hydrophilic organic solvents, namely dimethyl sulfoxide, dimethylformamide, dioxan, acetonitrile and tetrahydrofuran, in the activity and stability of free HRP was studied. From the solvents tested, DMSO led to the highest activities and stabilities. After 2 h of incubation at 35°C, the remaining activity of the enzyme in the presence of 30% of each solvent was less than 30%, with exception of DMSO for which the enzyme remained fully active. In order to increase stability, HRP was covalently immobilised onto silica microparticles. The half-life of the enzyme in buffer at 50°C increased from 2 to 52 h when the enzyme was immobilised. The stability of both free and immobilised HRP was also studied at 50°C in aqueous mixtures of 3.5, 20, 35 and 50% (v/v) DMSO. Free HRP stability was not affected by the presence of 3.5 and 20% DMSO, but higher contents lead to a more pronounced deactivation. Immobilised HRP stability increased with DMSO content up to 20%, decreasing for higher contents. The enzyme half-life increased more than 300% when changing from buffer to 20% DMSO. The deactivation of free HRP was modelled using the simple exponential decay, and the deactivation of immobilised HRP was described by a two-step inactivation model.

Partitioning of Ethyl Acetate, Maltol, Glucose, and Fructose to Liquid Phases of the Carbon Dioxide + Water + 1-Propanol System

This investigation reports on a thermodynamic phenomenon that can be observed in aqueous solutions of hydrophilic organic solvents: A completely miscible binary liquid mixture of water and an organic solvent, e.g., 1-propanol, can reveal a liquid-phase split, when it is pressurized with a “near-critical” gas, i.e., a gas near its critical temperature. This phase split phenomenon is briefly explained and new experimental data are presented for the partitioning of some natural products (ethyl acetate, d-fructose, d-glucose, and maltol) on, at near-critical pressures, coexisting liquid phases of a three-phase vapor−liquid−liquid equilibrium in the ternary system carbon dioxide + water + 1-propanol. In addition to the experimental data, a semiempirical approach for the correlation of the partition coefficients is shortly outlined. As the partition coefficients significantly deviate from unity, this phase equilibrium phenomenon can be applied for a new mild high-pressure liquid−liquid extraction process for n...

Hydrolysis Behaviour of Lipase from Pseudomonas Cepacia Encapsulated in Silica Aerogels with Different Characteristics

Lipase from Pseudomonas Cepacia was encapsulated in silica aerogels dried by the supercritical method using CO2. Aerogels with a different balance of hydrophilic and hydrophobic functionalities were tested in a hydrolysis reaction performed in 1,4-dioxane. This solvent was chosen so that the organic substrate and the hydrolysis water were soluble in the reaction medium. The gels were expected to act somewhat like emulsion droplets, so as to provide an immediate solvent environment close to the enzyme, different from the general solvent environment in which the gel particles were dispersed and the substrates were soluble. For each type of gels, the porous texture was characterized by nitrogen adsorption isotherms. The results were analyzed in terms of gel network characteristics and their participation in the displacement of the kinetics and equilibrium. While the gel's chemistry and its preparation protocole are far from being optimized, it is shown that the gel composition can have an important influence on the biocatalyst activity. Depending on the characteristics of the gel, lipase-catalyzed hydrolysis can be performed in hydrophilic organic solvents which are known to be harmful to lipases.

Hydrolysis Behaviour of Lipase from Pseudomonas Cepacia Encapsulated in Silica Aerogels with Different Characteristics

Full Paper: Lipase from Pseudomonas Cepacia was encapsulated in silica aerogels dried by the supercritical method using CO 2 . Aerogels with a different balance of hydrophilic and hydrophobic functionalities were tested in a hydrolysis reaction performed in 1,4-dioxane. This solvent was chosen so that the organic substrate and the hydrolysis water were soluble in the reaction medium. The gels were expected to act somewhat like emulsion droplets, so as to provide an immediate solvent environ ment close to the enzyme, different from the general solvent environment in which the gel particles were dispersed and the substrates were soluble. For each type of gels, the porous texture was characterized by nitrogen adsorption isotherms. The results were analyzed in terms of gel network characteristics and their participation in the displacement of the kinetics and equilibrium. While the gel's chemistry and its preparation protocole are far from being optimized, it is shown that the gel composition can have an important influence on the biocatalyst activity Depending on the characteristics of the gel, lipase-catalyzed hydrolysis can be performed in hydrophilic organic solvents which are known to be harmful to lipases.

The applicability of subtilisin Carlsberg in peptide synthesis.

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous Bu(t)-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1' position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by subtilisin was achieved.

Screening of genes involved in isooctane tolerance in Saccharomyces cerevisiae by using mRNA differential display.

A Saccharomyces cerevisiae strain, KK-211, isolated by the long-term bioprocess of stereoselective reduction in isooctane, showed extremely high tolerance to the solvent, which is toxic to yeast cells, but, in comparison with its wild-type parent, DY-1, showed low tolerance to hydrophilic organic solvents, such as dimethyl sulfoxide and ethanol. In order to detect the isooctane tolerance-associated genes, mRNA differential display (DD) was employed using mRNAs isolated from strains DY-1 and KK-211 cultivated without isooctane, and from strain KK-211 cultivated with isooctane. Thirty genes were identified as being differentially expressed in these three types of cells and were classified into three groups according to their expression patterns. These patterns were further confirmed and quantified by Northern blot analysis. On the DD fingerprints, the expression of 14 genes, including MUQ1, PRY2, HAC1, AGT1, GAC1, and ICT1 (YLR099c) was induced, while the expression of the remaining 16 genes, including JEN1, PRY1, PRY3, and KRE1, was decreased, in strain KK-211 cultivated with isooctane. The genes represented by HAC1, PRY1, and ICT1 have been reported to be associated with cell stress, and AGT1 and GAC1 have been reported to be involved in the uptake of trehalose and the production of glycogen, respectively. MUQ1 and KRE1, encoding proteins associated with cell surface maintenance, were also detected. Based on these results, we concluded that alteration of expression levels of multiple genes, not of a single gene, might be the critical determinant for isooctane tolerance in strain KK-211.

A New High-Pressure Liquid-Liquid Extraction Process for Natural Products Recovery

A thermodynamic phenomenon which can be observed in aqueous solutions of hydrophilic organic solvents, i.e., a completely miscible binary liquid mixture of water and an organic solvent (e.g., ethanol + 1-propanol), reveals a liquid-liquid phase split when pressurized with a ‘near critical’ gas. Using methyl anthranilate, the authors report on a new high-pressure liquid-liquid extraction process.

A novel high‐pressure liquid–liquid extraction process for downstream processing in biotechnology: Extraction of cardiac glycosides

This investigation examines phase equilibrium phenomena that can be used to create two water-like solvents for liquid–liquid extraction in downstream processing in biotechnology: a completely miscible, binary liquid mixture of water and a hydrophilic organic solvent (e.g., an alcohol) reveals a liquid phase split, when it is pressurized with a “near-critical” gas (i.e., a substance which at ambient conditions is a gas, near its critical temperature). This phase split results in two hydrophilic liquid phases. Making use of this phenomenon in process development first requires research on the phase split phenomenon and, second, research on the feasibility of biomolecule extraction and separation. In this study, basic fluid phase equilibrium phenomena are briefly described. Then, experimental results are reported for the partitioning of small amounts of cardiac glycosides (digitoxin and digoxin) on coexisting liquid phases in the high-pressure, three-phase, vapor–liquid–liquid equilibrium of the ternary system of “near critical” CO2 + water + 1-propanol, at 313 K and 333 K. Finally, a process for extraction and separation of the aforementioned glycosides by means of the high-pressure phase equilibrium phenomenon is discussed. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 559–565, 2000.

Interaction of photosynthetic pigments with various organic solvents 2: Application of magnetic circular dichroism to bacteriochlorophyll a and light-harvesting complex 1

Magnetic circular dichroism (MCD) and absorption spectra of metal bacteriochlorin complexes have been measured on bacteriochlorophyll (BChl) a in various solvents and different forms of light-harvesting complexes 1 (LH1 complexes). In hydrophilic organic solvents, the MCD intensity of the Q y(0–0) transition of BChl a was sensitive to the wavelength of absorption maximum of Q x(0–0), and the ratio of MCD Q y(0–0) intensity to the dipole strength ( B/ D) was inversely proportional to the difference in energy between the Q x(0–0) and Q y(0–0). The similar correlation has been observed in metal chlorin derivatives as previously reported. The correlation depends on the coordination number of the Mg atom in BChl a and the molecules ligating to it. In a hydrophobic solvent such as carbon tetrachloride (CCl 4), however, the correlation did not hold because of the existence of aggregates. Hence, the correlation between the values of B/ D and the energy difference can be used to estimate the type and number of the molecules ligated to the Mg atom and to disclose the existence of aggregated pigments. We further apply the correlation to the LH 1 complex treated with n-octyl β- D-glucopyranoside.

Surfactant–protease complex as a novel biocatalyst for peptide synthesis in hydrophilic organic solvents☆

The peptide synthesis from N-acetyl- l-phenylalanine ethyl ester with alaninamide catalyzed by a surfactant-protease complex has been performed in anhydrous hydrophilic organic solvents. Proteases derived from various sources were converted to surfactant-coated complexes with a nonionic surfactant. The surfactant-subtilisin Carlsberg (STC) complex had a higher enzymatic activity than the other protease complexes and the initial reaction rate in tert-amyl alcohol was 26-fold that of STC lyophilized from an optimum aqueous buffer solution. Native STC hardly catalyzed the same reaction. The addition of water to the reaction medium activated the lyophilized STC, however, the reaction rate was much lower than that of the STC complex, and a hydrolysis reaction preferentially proceeded. The STC complex exhibited a high catalytic activity in hydrophilic organic solvents (e.g. tertiary alcohol). The addition of dimethylformamide as a cosolvent improved the solubility of amino acid amides and further activated the STC complex due to the water mimicking effect. When hydrophilic amino acid amides were employed as an acyl acceptor, the peptide formation proceeded efficiently compared to that using hydrophobic substrates. The surfactant-STC complex is a powerful biocatalyst for peptide synthesis because the STC complexes display a high catalytic activity in anhydrous hydrophilic organic solvents and did not require the excess amount of water. Thus the side (hydrolysis) reaction is effectively suppressed and the yield in the dipeptide formation is considerably high.

The applicability of subtilisincarlsberg in peptide synthesis

The synthesis of peptide bonds catalysed by subtilisin Carlsberg was studied in different hydrophilic organic solvents with variable H2O concentration. Z-Val-Trp-OMe and Z-Ala-Phe-OMe were used as acyl donors, and a series of amino acid derivatives, di- and tripeptides of the general structure Xaa-Gly, Gly-Xaa, Gly-Gly-Xaa (Xaa represents all natural L-amino acids except cysteine) and other peptides were used as nucleophiles. A comparative study of the enzymatic synthesis in aqueous DMF (50%, v/v) and acetonitrile containing 10% (v/v) of H2O demonstrated that the yields of peptide products were higher in most cases when acetonitrile with low H2O concentration was used. The acylation of weak nucleophiles was improved in organic solvents with very low H2O concentration (2%). The reactions in anhydrous But-OH proceeded with substantially lower velocity. Generally, the restricted nucleophile specificity of the enzyme for glycine and hydrophilic amino acid residues in P1′ position, as well as numerous side reactions, limit the utilization of subtilisin in peptide synthesis, especially in the case of the segment condensations. Contrary to the published data, we have proved that proline derivatives were not acylated in any media with the help of subtilisin Carlsberg. Effective ester hydrolysis of a protected nonapeptide corresponding to the N-terminal sequence of dicarba-eel-calcitonin catalysed by sublilisin was achieved. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

A novel high-pressure liquid-liquid extraction process for downstream processing in biotechnology: extraction of cardiac glycosides.

This investigation examines phase equilibrium phenomena that can be used to create two water-like solvents for liquid-liquid extraction in downstream processing in biotechnology: a completely miscible, binary liquid mixture of water and a hydrophilic organic solvent (e. g., an alcohol) reveals a liquid phase split, when it is pressurized with a "near-critical" gas (i.e., a substance which at ambient conditions is a gas, near its critical temperature). This phase split results in two hydrophilic liquid phases. Making use of this phenomenon in process development first requires research on the phase split phenomenon and, second, research on the feasibility of biomolecule extraction and separation. In this study, basic fluid phase equilibrium phenomena are briefly described. Then, experimental results are reported for the partitioning of small amounts of cardiac glycosides (digitoxin and digoxin) on coexisting liquid phases in the high-pressure, three-phase, vapor-liquid-liquid equilibrium of the ternary system of "near critical" CO(2) + water + 1-propanol, at 313 K and 333 K. Finally, a process for extraction and separation of the aforementioned glycosides by means of the high-pressure phase equilibrium phenomenon is discussed.

Sulfolobus Solfataricus β-Glycosidase-Catalysed Synthesis of Sugar-Alcohol Conjugates in the Presence of Organic Solvents

The thermostable β-D-glycosidase from the archaeon Sulfolobus solfataricus was used for the glucosylation of secondary alcohols at high organic solvent concentrations. Alkyl glucoside products were analysed by one- and two-dimensional NMR. Using a range of racemic secondary alcohols and various hydrophilic organic solvents, small but significant substrate- and solvent-dependent changes in the R/S ratios of alkyl glucoside products were detected. The anomeric specificity of the enzyme was apparently affected by butan-2-one. A possible mechanistic model for solvent-induced changes at the active site is presented.

有機媒体中での反応による層状構造をもったリン酸アルミニウムの合成とその特性

We have synthesized the pure single phase of aluminium tri-hydrogen bis (orthophosphate) monohydrate [AlH3(PO4)2·H2O] by using hydrophilic organic solvent, with high efficiency. It is a material with layered structure. The optimum condition to prepare it was as follows; the mixtures of 75wt% H3PO4 and Al(OH)3 (0.1mol) (molar ratio of H3PO4 to Al(OH)3=4.0) was refluxed at 94°C for 6 hours under stirring in 1-propanol (200cm3). Shape of products obtained was needle, and its length increased from 5μm to 80μm with increasing the reaction times. The maximal acid strength and amount of acid of solid surface were pKa=+1.5 and 1.81-2.13 (x10-2mmolg-1), respectively. AlH3(PO4)2·H2O changed to AlPO4 (berlinite, cristobalite-form) and Al(H2PO4)3 (B-form, C-form) by dehydration of monohydrate at 160°C.

The effects of hydrophilic organic solvents on CYP3A4 activity in human liver microsomes

The effects of hydrophilic organic solvents on CYP3A4 activity in human liver microsomes

High pressure multiphase equilibria in aqueous systems of carbon dioxide, a hydrophilic organic solvent and biomolecules

A binary mixture of water and a water-soluble organic solvent which at atmospheric pressure reveals no miscibility gap, can be forced to split into two liquid phases by pressurization with a `near-critical' gas. Since both coexisting liquid phases are water-rich, the liquid phase split might be used, for example, for the recovery of delicate, high value products by means of extraction in biotechnology. An overview is given on the phase behavior of ternary systems water–hydrophilic organic solvent–carbon dioxide, focusing on the high pressure three phase equilibrium LLV. Exemplary experimental results for such phase equilibria are presented, discussed and compared with predictions from an equation of state. Furthermore, new experimental results for the partitioning of vanillin and caffeine to the coexisting liquid phases in high pressure three-phase LLV equilibrium in water–acetone–carbon dioxide at 313 and 333 K are presented together with a simple method for correlating these results.