Water-miscible Organic Solvents Research Articles

Transphosphatidylation reaction of phosphatidylcholine to 4-methoxyphenol in water-immiscible organic solvents with immobilized phospholipase D

Immobilized phospholipase D (PLD) from Streptomyces sp. catalyzed the transfer reaction of the dipalmitoylphosphatidyl residue from 1,2-dipalmitoyl-3- sn-phosphatidylcholine (DPPC) to an aromatic hydroxy group on 4-methoxyphenol in water-immiscible organic solvents, to afford 1,2-dipalmitoyl-3- sn-phosphatidyl-4-methoxyphenol (DPP-PMP) with a 45% yield, accompanied by a trace amount of 1,2-dipalmitoyl-3- sn-phosphatidic acid sodium salt (DPPA-Na). The formation of DPP-PMP was affected by organic solvents used in the reaction. Benzene, toluene, and methylene chloride gave DPP-PMP with moderate yields but use of diethyl ether resulted in a low yield of DPP-PMP. In both ethyl acetate and water-miscible organic solvents, the transfer reaction did not take place. Immobilization of PLD was carried out by adding a 1 % volume of PLD solution to a suspension of a cation-exchange resin (Amberlite IRC-50, 5% w/v) in benzene with stirring and sonication. In a repeated batch reaction for DPP-PMP synthesis with immobilized PLD, after ten batch cycles the enzyme retained 74% of its initial activity.

A comparative study of the catalytic action of water-miscible organic solvents on the hydrolytic degradation of long-chain polyphosphates

The hydrolytic degradation of sodium polyphosphate (Na PP) catalysed by six water-miscible organic solvents—methanol, ethanol, n-propanol, t-butanol, acetone and dioxane—was studied. This was done principally to compare their catalytic actions and to have a fresh look at the factors responsible for it. The values of energy of activation ( E) obtained using 40% (v/v) of the solvents ranged from 12.1 to 15.3 kcal mol −1. Taking into account the values of dielectric constant (ϵ) of the solvent media at all temperatures at which the rate of degradation was studied, it was found that the lower the average value of ϵ, the lower was the value of E. The product of ϵ and the dipole moment, referred to as the electrostatic factor (EF), of the pure solvents also appeared to have a profound influence on the course of degradation. Dioxane, with a zero value of EF, was the most effective solvent in catalysing the hydrolytic degradation of Na PP.

Characterization of redox proteins from extreme thermophilic archaebacteria: studies on alcohol dehydrogenase and thioredoxins

Proteins isolated from extreme thermophilic microorganisms for their general stability to the common protein denaturants (heating, pH, organic solvents, urea, SDS, proteases) represent an important development in the potential exploitation of proteins and enzymes for practical purposes. Here we report studies on the thermoactivity and thermostability of an alcohol dehydrogenase purified from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus, in the absence and in the presence of water-miscible organic solvents, as well as studies on enzyme immobilization and co-enzyme recycling. Three thermostable thioredoxins isolated and purified from Bacillus acidocaldarius, Sulfolobus solfataricus and Pyrococcus furiosus, have been partially characterized.

Lignin Peroxidase Oxidation of Aromatic Compounds in Systems Containing Organic Solvents

Lignin peroxidase from Phanerochaete chrysosporium was used to study the oxidation of aromatic compounds, including polycyclic aromatic hydrocarbons and heterocyclic compounds, that are models of moieties of asphaltene molecules. The oxidations were done in systems containing water-miscible organic solvents, including methanol, isopropanol, N, N-dimethylformamide, acetonitrile, and tetrahydrofuran. Of the 20 aromatic compounds tested, 9 were oxidized by lignin peroxidase in the presence of hydrogen peroxide. These included anthracene, 1-, 2-, and 9-methylanthracenes, acenaphthene, fluoranthene, pyrene, carbazole, and dibenzothiophene. Of the compounds studied, lignin peroxidase was able to oxidize those with ionization potentials of <8 eV (measured by electron impact). The reaction products contain hydroxyl and keto groups. In one case, carbon-carbon bond cleavage, yielding anthraquinone from 9-methylanthracene, was detected. Kinetic constants and stability characteristics of lignin peroxidase were determined by using pyrene as the substrate in systems containing different amounts of organic solvent. Benzyl alkylation of lignin peroxidase improved its activity in a system containing water-miscible organic solvent but did not increase its resistance to inactivation at high solvent concentrations.

Investigation of a 17β-estradiol-monoclonal antiestradiol antibody binding mechanism using dilute solutions of organic solvents

Quantitative understanding of steroid hormone transport and receptor-mediated action requires knowledge of the bonding forces involved in each steroid-protein complex and the effects of a biological environment on these forces. An approach to these problems using dilute solutions of water-miscible organic solvents, with a range of polarity, dielectric and hydrogen bonding properties, was tested on an estradiol-antiestradiol antibody binding system on the basis that comparing the effects of the solvents would both permit the importance of hydrophobic and hydrogen bonding to be differentiated and give information on the effects of the environment on the reaction. The results were compared with thermodynamic measurements. All the solvents reduced the Gibbs free energy of binding as a function of their concentration in the medium. The decreases were virtually a monotonic function of their dielectric constant, indicating reduced hydrogen bonding. Analysis of the decreases in terms of the solvents' hydrogen bonding and polarity properties supported this. Thermodynamic measurement showed the binding reaction was enthalpy-driven with, overall, a slightly unfavorable entropy contribution. This again showed the hydrophobic effect was not the main bonding force. The most deleterious solvent, iso-propanol, not only decreased the enthalpic contribution to binding but rendered the entropic contribution more favorable. This approach still does not allow the relative importance of hydrogen bonding and van der Waals contacts in the actual binding to be differentiated but it does give indications on how a biological environment may affect a steroid-protein binding reaction in vivo.

Enzymatic esterification and transesterification of N-protected Glu or Asp and synthesis of sweetener aspartam analogues by a new glutamic acid specific endopeptidase

Esterification and transesterification reactions are carried out in water miscible organic solvents by using a new glutamic acid specific endopeptidase, isolated from Bacillus lichenifonnis. Sweetener aspartam analogues syntheses are also described.

Synthesis of L-tyrosine glyceryl ester catalyzed by α-chymotrypsin in water-miscible organic solvents: A possible sun-tan accelerator product

The synthesis of L-tyrosine glyceryl ester, from glycerol and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by α-chymotrypsin. Values of 60 % (v/v) for glycerol and 200 mM for L-tyrosine methyl ester were optimal for the transesterification reaction. Additionally to glycerol, several other water miscible cosolvents (acetonitrile, N,N'-dimetyl formamide and tetrahydrofurane) were tested in the reaction media, but their presence did not give an enhancement on the transesterification activity with respect to the glycerol/water medium. However, increasing the hydrophobicity of the cosolvent resulted in a reduction of the enzyme activity, the water:glycerol mixture being the best reaction media.

Effect of water-miscible organic solvents on the catalytic activity of cytochrome c

The effect of five water-miscible organic solvents (tetrahydrofuran, N, N-dimethylformamide, acetonitrile, 2-propanol, and methanol) on the oxidation of pinacyanol chloride (Quinaldine Blue) by horse heart cytochrome c was determined. Hydrogen peroxide was used as the oxidant, and a change in catalytic property of the dissolved protein was observed after a certain threshold concentration of the organic solvent had been reached. The maximum specific activity was correlated with the Dimroth-Reichardt parameter for the solvents, which is directly related to the free energy of the solvation process. The kinetic constants for the oxidation of pinacyanol chloride were determined in systems containing different proportions of tetrahydrofuran. The best catalytic efficiency ( k cat/ K M.app) was obtained in a system containing 50% tetrahydrofuran in phosphate buffer. In a mixture containing 90% tetrahydrofuran, cytochrome c showed 18% of its maximum activity. The inactivation of cytochrome c was mainly due to the presence of hydrogen peroxide, and a direct correlation was found between the inactivation constant and the concentration of hydrogen peroxide in the system. The chemical modifications and immobilization of cytochrome c were able to change its biocatalytic activity and stability in the organic solvent system. The kinetic constants and the inactivation of three other type c cytochromes, from Saccharomyces cerevisiae, Pseudomonas aeruginosa, and Desulfovibrio vulgaris Hildenborough in a system containing 90% tetrahydrofuran were compared with those of cytochrome c from horse heart. Cytochrome c 551 from P. aeruginosa showed the best stability against hydrogen peroxide and a higher catalytic efficiency than that of horse heart cytocrome c.

Borate ion-assisted stabilization of β-galactosidase from Aspergillus oryzae by polyhydroxy compounds in water-miscible organic solvents

The stability of β-galactosidase from Aspergillus oryzae in water-miscible organic solvents in different buffers at various pH values ranging from 4.6 to 8.0 was studied. The stability of the enzyme in all six organic solvents studied was dependent on pH and on the type of buffer ions present. At a given pH, destabilization by organic solvents was highest in sodium borate buffer. The destabilization of β-galactosidase by these solvents could be reversed by addition of sugars or polyhydroxy compounds exclusively in sodium borate, suggesting a role of borate ions in stabilization. A similar effect of addition of mannitol was observed on deactivation of β-galactosidase by N, N-dimethylformamide (DMF). Exclusively in sodium borate, at pH 8.0, the addition of mannitol (0.02 m) not only prevented the deactivation by DMF (8%, v/v) but increased the enzyme activity to the level at its optimum pH. Since β-galactosidase from Aspergillus oryzae is a glycoprotein, complexation of the borate ions to the carbohydrate part may result in change in protein conformation, which, without leading to denaturation or inactivation of the enzyme, may facilitate interaction of the organic solvents with the enzyme leading to its denaturation. Such a denaturation is probably prevented by addition of polyhydroxy compounds, which appear to compete favorably with the carbohydrate moiety of the protein in complexing with borate ions. This should result in the enzyme regaining its native conformation.

Enzymatic synthesis of [formula omitted] from [formula omitted] in the presence of organic cosolvent

Bacterial strain KBEL 101 isolated from soil was immobilized within polyacrylamide gel and used for the synthesis of d-p- hydroxyphenylglycine from dl-5-substituted hydantoin. The optimum reaction conditions for immobilized whole cell enzyme were pH 8.0 and temperature 30°C. Various water-miscible organic solvents were tested with respect to both the activity and the stability of whole cell enzyme, and 5% dimethylsulfoxide (DMSO) was chosen to be a proper solvent: hydantoinase activity increased twofold in the presence of 5% DMSO. From a practical standpoint, it is advantageous to obtain the highest conversion yield, and enzymatic synthesis of N- carbamoyl- d-p- hydroxyphenylglycine was carried out in repeated batch operation in the presence of 5% DMSO. The time required to reach 99% conversion yield was increased with increasing number of batch operations when batch operation was conducted four times.

Preparation and Characterization of 11-Molybdoarsenite(III) Complex

Abstract A green 11-molybdoarsenite(III) complex, which was formed in an Mo(VI)–AsO33−–HCl–CH3CN system at room temperature, was isolated as the tetrabutylammonium (n-Bu4N+) salt, being characterized by its voltammetric behavior and its IR and UV-visible spectra. The heteropolyanion undergoes two-step one-electron reductions in CH3CN and three-step two-electron reductions in acidified CH3CN. The electrochemical reduction produces mixed-valence blue species at the glassy carbon (GC) electrode. The formation conditions and chemical properties were compared with those of 12-molybdoarsenate(V) with the Keggin structure. The presence of water-miscible organic solvents like CH3CN and CH3COCH3 is absolutely necessary for the formation of the green heteropolyanion.

Cytochrome c as a biocatalyst for the oxidation of thiophenes and organosulfides

Cytochrome c from horse heart is able to catalyze the oxidation of some heterocyclic sulfur compounds and sulfides in a medium containing 10% of water-miscible organic solvent. The kinetic constants of the different substrates were determined and the oxidation products were analyzed. Cytochrome c oxidation of organosulfur compounds produced the respective sulfoxides. In addition to hydrogen peroxide, some peroxides are electron acceptors in the reaction. 3-Chloroperoxybenzoic acid showed the highest maximum oxidation rate. The activation energy and stability of cytochrome c were also determined, and the possibility of asphaltene modification using cytochrome c is discussed.

Lipase-catalyzed peptide synthesis using Z-amino acid esters as acyl donors in aqueous water-miscible organic solvents

The lipase(PPL)-catalyzed peptide synthesis(Z-Phe-Phe-NH2) in aqueous water-miscible organic cosolvents was studied using Z-Phe-OEt as an ester substrate and Phe-NH2 as a nucleophile. It was found that peptide yield increased with an increase of the concentration of DMF. At a higher concentration than 50%(v/v), however, peptide yield decreased rapidly. The optimal pH of an aqueous buffer and concentration of Phe-NH2 were 8 and 10-fold excess over Z-Phe-OEt, respectively. DMF, MeOH and DMSO were found to be effective cosolvents to promote peptide synthesis. The effect of reaction temperature was studied. It was found that both selectivity for peptide synthesis and the stability of PPL increased when the temperature was lowered. The selectivity reached about 90% at −10°C where the peptide synthesis became rather slow. The rate of peptide synthesis in aqueous-organic solvent mixture was good at room temperature. In a typical experiment (Z-Phe-OEt: 20 mM, Phe-NH2: 200 mM, 10°C, 50% DMF, PPL : 4 mgmL −1), Z-Phe-OEt was consumed almost completely within 6 h, yielding about 80% Z-Phe-Phe-NH2. The specificity of ester substrates toward PPL was also studied.

Thermostable mutants of kanamycin nucleotidyltransferase are also more stable to proteinase K, urea, detergents, and water-miscible organic solvents

A series of variants of kanamycin nucleotidyltransferase (KNTase), isolated previously on the basis of enhanced thermostability by cloning and selection for enzymatic activity in the thermophile Bacillus stearothermophilus, was used to systematically test the hypothesis that thermostable enzymes would also be more resistant to other forms of protein denaturation. The purified KNTases were treated with proteinase K or assayed at 37°C in the presence of urea, N-lauroylsarcosine, Triton X-100, tetrahydrofuran, ethanol, or dimethylformamide. With all these agents, the KNTases displayed increasing resistance to denaturation in the order: wild type, mutant TK9 (with a Thr130→Lys substitution), TK1 (Asp80→Tyr), and TK101 (both substitutions). This is the same order in which their thermostability increases, indicating that the structural mechanism(s) whereby the mutations yield enhanced resistance to heat denaturation also yield stabilization towards chemical forms of enzymatic inactivation. These results suggest that selection in thermophiles is a useful method to obtain enzyme variants with increased overall stability, even at nonthermophilic temperatures.

Soluble and immobilized enzymes in water-miscible organic solvents: glucoamylase and invertase

The efficiency of enzymes in organic solvents is determined by the catalytic parameters, which mostly are changed in comparison with those in aqueous solutions, as well as by reversible and irreversible denaturation of the enzyme protein. The interplay of these processes was studied on soluble and immobilized glucoamylase and invertase in mixtures of buffer and several water-miscible solvents such as methanol, glycerol, dimethylformamide, and dioxane. A comparison of the catalytic activity in 50 or 30% (v/v) solvents with the extent of irreversible inactivation during the time of assay showed that the solvents act on the catalytic parameters of the enzymes and their conformational stability in different ways. Irreversible inactivation is especially strong in dimethylformamide, whereas glycerol does not induce denaturation but strongly reduces the catalytic efficiency. Striking effects were observed in dioxane, where high stability to irreversible denaturation but strong reduction of catalytic power were observed. In all solvents K m values were more affected than V max values, suggesting that reversible denaturation plays an inferior role in activity losses. Similar effects of the individual solvents were also observed after immobilization of the enzymes by covalent binding to a macroporous aminomethyl polystyrene-divinylbenzene copolymer or a macroporous aminopropyl silica. In all cases, however, immobilized enzymes showed better long-term stability than the soluble enzymes.

Enzymatic Synthesis of Peptides with Hydrophilic Amino Acid in Water-Miscible Organic Solvent

Enzymatic Synthesis of Peptides with Hydrophilic Amino Acid in Water-Miscible Organic Solvent

Enzymatic Synthesis of Peptides with Hydrophilic Amino Acid in Water-Miscible Organic Solvent

Enzymatic Synthesis of Peptides with Hydrophilic Amino Acid in Water-Miscible Organic Solvent

Effect of organic solvents on enzymatic production of cyclodextrins from unliquefied corn starch in an attrition bioreactor

Cyclodextrins were produced from unliquified corn starch in the presence of water-miscible organic solvents using cyclodextrin glycosyltransferase (CGTase) in an attrition bioreactor. The production yield was significantly increased by isopropanol and tertiary butanol, and maximum enhancement was observed to be about 40% by 5% tertiary butanol. Increase in the production of cyclodextrins by organic solvents seems to be due to the fact that organic solvents decreased the product inhibition of CGTase by forming an inclusion complex with cyclodextrins.

How do organic solvents affect peroxidase structure and function?

The effect of organic solvents on horseradish peroxidase structure and function has been studied. Some, but not complete, enzyme denaturation occurs even in low volumes of water-miscible organic solvents (e.g., greater than 30% v/v dioxane, greater than 50% v/v methanol, and greater than 20% v/v acetonitrile) as determined by the decreased difference between the fluorescence of peroxidase's sole tryptophan residue and free L-tryptophan in solution. Absorbance and electron paramagnetic resonance spectroscopies indicate exposure of peroxidase's active site to the organic solvent. This reduces the local polarity in the enzyme's active site and results in stronger hydrogen bonding of phenolic substrates to the enzyme. In extreme cases (e.g., 95% v/v dioxane, 90% v/v acetonitrile, and ethyl and butyl acetate containing 2 and 1% v/v aqueous buffer, respectively), the transition state of the enzymic reaction is sufficiently perturbed so as to alter the magnitude of the Hammett rho value. This is most likely the result of the increased strength of hydrogen bonding between electron-donating alkoxyphenols (negative sigma values) and an electrophilic group in the enzyme's active site, thereby reducing catalytic efficiencies for such substrates relative to alkyl- and chlorophenols. Perhaps the most important effect of the organic solvent, however, is the significant ground-state stabilization of phenolic substrates in organic media as opposed to aqueous buffer. This stabilization can account for nearly 4 orders of magnitude in reduction of catalytic efficiency and is manifested in increased Km's. This study indicates that enzymes can maintain much of their native active-site structure in organic media and that the effect of solvent on substrate thermodynamics must be considered.

Bis(2-ethylhexyl)sulfoxide as an extractant for americium(III) from aqueous nitrate media

Solven extraction separation of americium(III) from dilute aqueous nitrate media into n-dodecane by bis(2-ethylhexyl)sulfoxide (BESO) has been investigated over a wide range of experimentgal conditioins. Very poor extractablity of Am(III), necessitated the use of calcium nitrate as the salting-out agent. Effects of certain variables such as acidity, extractant concentration, salting-out agent concentration, organic diluents on the metal extraction by BESO have been examined in detail. By increasing the concentration of BESO in organic phase or calcium nitrate in aqueous phase, nearly quantitative extraction of americium even from moderate acidity is accomplished. Slope analyses applied to Am(III) distribution experiments from acidic nitrate solutions indicate predominant formation of the risolvated organic phase complex, Am(NO3)3)·3BESO for which equilibrium constant is found to be, log Kx=1.99. Extraction behavior of Am(III) has also been evlauated in the presence of several water-miscible polar organic solvents to stuy their possible synergistic effects on its extraction. Extractability of americium increased 5 to 10-fold withi increasing conentration of some of these additives, with maximum enhancement being observed in the presence of acetone or acetonitrile. Recovery of BESO from loaded americium is easily obtained using dilute nitric acid as the strippant.