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1.
Developments and trends in enzyme catalysis in nonconventional media   总被引:8,自引:0,他引:8  
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2.
The performance of a crude extract lipase from Pseudomonas fluorescensin esterification was evaluated in microaqueous, biphasic and surfactant-enriched biphasic systems containing various amounts of water (from almost no water to pure water). The results showed a strong negative influence of the water content on the thermodynamic equilibrium of the reaction in biphasic systems. From a kinetic point of view, the enzyme was more efficient in systems involving a water/organic solvent interface (4 times in the biphasic system, 12 times in the surfactant-enriched biphasic system).  相似文献   

3.
The direct one-step synthesis of L-phenylalanine methyl ester in an organic-aqueous biphasic system using phenylalanine ammonia lyase (E.C.4.3.1.5, PAL) containing Rhodotorula glutinis yeast whole cells was reported earlier. We report here further optimization of this biotransformation using isolated PAL, when the lyophilized enzyme is treated with different water miscible and water immiscible organic solvents. Use of isolated PAL enzyme is advantageous in overcoming diffusion barriers encountered when using PAL containing R.glutinis whole cells, and resulted in increased product yield due to better interaction of enzyme with the substrate. Among the water miscible solvents, ethanol treated and methanol-treated enzymes supported maximum PAL forward and reverse activities; respectively. In the water immiscible solvents category, heptane-treated enzyme exhibited maximal activity for both PAL forward and reverse reactions. PAL activity obtained with enzyme specimens treated with methanol, ethanol, and heptane varied in the range of 91–99% of that observed in aqueous buffer medium for the forward reaction; and 89–95% for the reverse reaction. n-butanol,acetone, and benzene were found to have a inhibitory effect on PAL enzyme, in that, it resulted in only 31–33% activity of that obtained with aqueous solution. Raman spectroscopy was used to monitor amide I and II bands which are sensitive to changes in the secondary structure of proteins. No changes in structure could be detected from the analyses of AI and AII bands of PAL spectra. This data obtained for PAL, a tetramer, could be significant in predicting how solvent interactions affect the structure and function of multimeric proteins and enzymes in nonaqueous media.  相似文献   

4.
The kinetics of butyl butyrate synthesis by a lipase from Mucor miehei in different types of organic media were investigated. The three systems studied were a microaqueous medium containing enzyme in suspension in hexane, a water-hexane two-phase system, and reverse micelles. The synthesis of butyl butyrate was possible in all cases because of a favorable partition of the ester into the organic solvent. A sufficient stirring rate was necessary to achieve good reaction rates in the case of the liquid-liquid biphasic medium. The effect of water content was different according to the type of system used. The dependence of reaction rate and of conversion yield on enzyme and substrate concentrations was also investigated. From an applied point of view, the best performances were obtained with either microaqueous or liquid-liquid two-phase systems. The use of reverse micelles can be advocated only in particular conditions, such as low enzyme concentration, compatible with the specific constraints it involves.  相似文献   

5.
The degree of detergent insolubility of cell membranes is a useful parameter to test the strength of lipid–lipid interactions relative to lipid–detergent interactions. Thus, solubility studies could give insights about lipid–lipid interactions relevant in domain formation. In this work we perform a detailed study of the solubilization of four different erythrocyte membrane systems: intact human and bovine erythrocytes, and human and bovine erythrocytes depleted in cholesterol with methyl-β-cyclodextrin. Each system was incubated with different concentrations of the non-ionic detergent Triton X-100, and the insoluble fraction was characterized by determining cholesterol and phosphorus content. A distinct solubilization behavior was obtained for the four systems, which was quantified by a “detergent resistance parameter” obtained from the fit of the solubility curves. In order to correlate these findings with membrane structural parameters, we quantify the degree of acyl chain order/rigidity of the original membranes by EPR spectroscopy, finding that detergent resistance is higher when acyl chains are more rigid. Regarding compositional properties, we found a good correlation between detergent resistance parameters and the total amount of cholesterol plus sphingomyelin in the original membranes. Our results suggest that a high degree of acyl chain packing is the determinant membrane factor for resistance to the action of Triton X-100 in erythrocytes.  相似文献   

6.
Polycyclic aromatic hydrocarbons (PAHs) are toxic compounds presenting low water solubility and high hydrophobicity, which greatly hampers their natural biodegradation. The enzymatic degradation of a model compound, anthracene, was evaluated in presence of a miscible solvent for an increased solubility. Manganese peroxidase, a ligninolytic enzyme from white-rot fungi, was used as biocatalyst in a medium containing acetone. The kinetic parameters of the enzymatic degradation of anthracene, obtained from fed-batch experiments, were applied to model the operation of a continuous reactor. Kinetics comprised a Michaelis-Menten equation, modified with an autocatalytic term, assumed to the effect of quinones acting as electron carriers, and a logistic function related to enzyme activity. The continuous reactor has been operated for 108 h, attaining a 90% of anthracene degradation, which demonstrated the feasibility of the system for its application in the removal of poorly soluble compounds. The model of this reactor permitted to predict accurately anthracene degradation in different conditions, such as external addition of anthraquinone and different enzymatic activities.  相似文献   

7.
Biphasic reaction systems for enzyme catalysis are an elegant way to overcome limited solubility and stability of reactants and facilitate continuous processes. However, many synthetically useful enzymes are not stable in biphasic systems of water and organic solvent. The entrapment in polymer beads of polyvinyl alcohol has been shown to enable the stable operation of enzymes unstable in conventional biphasic reaction systems. We report the extension of this concept to continuous operation in a fluidised bed reactor. The enzyme benzaldehyde lyase was used for the continuous synthesis of enantiopure (R)-3,3'-furoin. The results show enhanced stability with half-life times under operation conditions of more than 100 h, as well as superior enzyme utilisation in terms of productivity. Furthermore, racemisation and oxidation of the product could be successfully prevented under the non-aqueous and inert reaction conditions.  相似文献   

8.
The three dimensional solubility parameters defined by Hansen are based on dispersion forces between structural units, interaction between polar groups and hydrogen bonding. For polar polymers such as poly(3-hydroxyalkanoates), P(3HA), this approach was used to obtain the three coordinates of a solubility parameter in terms of: a dispersion part, a polar part and a hydrogen bonding part. Thirty-eight different solvents for poly(3-hydroxybutyrate), PHB, which are mentioned in the literature are examined by this method and the theoretical predictions are compared with the experimental reports. Another overall comparison between PHA polymers provides their Hansen and Hildebrand parameters for side chain lengths up to C13. In this series a linear progression in calculated solubility parameters with side chain length was found. An Appendix provides information and data on calculation of the solubility parameters. While the solubility information is limited and only covers homopolymers, it should help to highlight some of the contradictions regarding PHB solubility. This semi-empirical approach is only valid for amorphous polymers hence crystallinity effects, which are important with PHB, as well as molecular weight effects still require analysis.  相似文献   

9.
This study reports a general strategy for the encapsulation of various enzymes in amphiphilic hollow carbonaceous microspheres (CMs). We found that enzymes could be spontaneously encapsulated in the interior cavity of the CMs via hydrophobic interactions. Due to strong hydrophobic interactions and robust confinement, leaching of the physically adsorbed enzymes is substantially restricted. As a novel immobilization matrix, the CMs display many significant advantages. They are capable of encapsulating a wide range of proteins/enzymes of different sizes, which can then be used in both aqueous and organic media and retain high activity, stability, and excellent reusability. Moreover, CMs could be considered as efficient microreactors that provide a favorable microaqueous environment for enzymes in organic systems. Therefore, this doubly effective and simple immobilization approach can be easily expanded to many other enzymes and has great potential in a variety of enzyme applications.  相似文献   

10.
Lu J  Nie K  Wang F  Tan T 《Bioresource technology》2008,99(14):6070-6074
The immobilized lipase Candida sp. 99-125 catalyzed methanolysis of glycerol trioleate was studied in twelve different solvents in order to deduce the solvent effect through an attempt to correlate the highest yield with such solvent properties as hydrophobicity (log P), dielectric constant (epsilon), and Hildebrand solubility parameter (delta). The results showed that the conversion of glycerol trioleate and yield of oleic acid methyl ester were quite dependent on the solvent. The catalyst lipase in various solvents also needed different optimum amount of water to keep its maximum activity, and generally this lipase in more hydrophobic solvents required more water. The correlation between the highest yield and log P value was found to be reasonable except deviation of data points of certain solvents, while no obvious correlation existed between the other two parameters, dielectric constant (epsilon) and Hildebrand solubility parameter (delta), and the enzyme activity. The study revealed that more hydrophobic solvents such as n-hexane or cyclohexane were more suitable solvents for Candida sp. 99-125 catalyzed transesterification of glycerol trioleate to oleic acid methyl ester.  相似文献   

11.
Hansen solubility parameters (HSPs), often used to predict the miscibility between two compounds, are an alternative tool in evaluating the ability of the solvent to interact via dispersion, dipole-dipole, and hydrogen bonding interactions. The aim of this paper is to find a simple way to calculate HSPs for complex mixtures of triglycerides (TAGs). HSPs were calculated using two approaches: the first assumes that the contributions to the dispersion, dipole-dipole, and hydrogen bonding interactions may be subdivided into larger functional moieties (i.e., fatty acids and fatty acid methyl esters) that are additive, while the second approach assumes that vegetable oils are comprised of mixtures of simple TAGs in the same mass fractions as the fatty acids. The HSPs obtained using the two approaches are compared to reference values determined using the “Hansen Solubility Parameters in Practice” software (HSPiP) considering the complex TAG profile for each vegetable oil.HSPs for vegetable oils, obtained with the HSPiP software, did not correspond well to the HSPs obtained from the group contribution approach, when using fatty acids, fatty acids + glycerol or fatty acid methyl esters. In contrast, the HSPs calculated for vegetable oils, assuming that all TAGs are simple and in the same mass fractions as the fatty acids, provide similar values to the HSPs obtained from the HSPiP software. Therefore, it is possible to calculate the HSPs for complex oils by simply knowing the fatty acid composition. Knowledge of HSPs may be used to rationalize the ability of certain low molecular weight molecules to develop organogels in vegetable oils as well as the crystallization of triglycerides.  相似文献   

12.
Five different ionic liquids (ILs) based on quaternary ammonium cations, with functional side chains ((3-hydroxypropyl)-trimethyl-, (3-cyanopropyl)-trimethyl-, butyl-trimethyl-, (5-cyanopentyl)-trimethyl- and hexyl-trimethyl-) associated with the same anion (bis(trifluoromethane)sulfonyl amide)), were synthesized, and their suitability for Candida antarctica lipase B (CALB)-catalyzed ester synthesis in IL/supercritical carbon dioxide (scCO(2)) biphasic systems was assayed. Catalytic efficiency of the system has been analyzed as a function of both enzyme properties and mass-transfer phenomena criteria. First, the suitability of these ILs as enzymic reaction media was tested for the kinetic resolution of rac-phenylethanol. All ILs were found to be suitable media for enzyme catalysis, the best catalytic parameter (5.3 U/mg specific activity, 94.9% selectivity) being obtained for the (5-cyanopentyl)-trimethylammonium. Second, enzyme stability in all of the ILs was studied at 50 degrees C over a period of 50 days, and data were analyzed by a two-step kinetic deactivation model. All of the ILs were shown to act as stabilizing agents with respect to hexane, producing an increase in the free energy of deactivation (to 25 kJ/mol protein) and an improvement in the half-life time of the enzyme (2000-fold), which agrees with the observed increased hydrophobicity of the cation alkyl side chain (measured by Hansen's solubility parameter, delta). By using two different CALB-IL systems with different hydrophobicity in the cation, continuous processes to synthesize six different short chain alkyl esters (butyl acetate, butyl propionate, butyl butyrate, hexyl propionate, hexyl butyrate, and octyl propionate) in scCO(2) at 10 MPa and 50 degrees C were carried out. Both rate-limiting parameters (synthetic activity and scCO(2)-ILs mass-transfer phenomena) were related with the delta-parameter of the ILs-alkyl chain and reagents.  相似文献   

13.
The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.  相似文献   

14.
Various factors affecting the catalytic activity of pure lipase of Pseudomonas fluorescens in microaqueous benzene were investigated with respect to lactonization of 15-hydroxypentadecanoic acid. Without deposition of the enzyme or of the enzyme plus activity enhancer (additive) on celite powder, the pure enzyme was very poorly dispersed in the microaqueous benzene, resulting in very low activity. The enzyme immobilized on celite powder exhibited the highest activity at a free water content of ca. 0.083%. When a sugar alcohol such as erythritol, arabitol, or sorbitol was added before lyophilization with approximate proportion of 3 g/g enzyme, marked increases in the enzyme activity were observed at a shifted optimal free water content, i.e., 0.04%. Inclusion of phosphotidylcholine resulted in a somewhat higher activity than in the system of enzyme plus celite only. Addition of lactose, bovine serum albumin, casein, dextran, polyvinyl alcohol, phosphate, or NaCl all caused a decrease in the enzyme activity. From the effects of the additives examined, it is deduced that the following three factors are required for a pure enzyme to exhibit its full activity in a water-immiscible organic solvent: (1) optimum moisture content, (2) disperser (support particles having enough surface area on which the enzyme is thinly deposited), and (3) activity enhancer (additive) at optimum concentration The importance of noting the purity of the enzyme preparation is emphasized when its catalysis in an organic solvent is investigated.  相似文献   

15.
Lipase catalysed stearoyl lactic acid preparation in non-aqueous media was treated as a model system to study the microaqueous phase containing hydrogen ions arising from dissociation of water soluble lactic acid in it. The thermodynamic factors operating at the microaqueous enzyme-water-solvent phase on the lipase in non-polar solvents were investigated in terms of the water of reaction which constitutes the microaqueous phase, partitioning of acid between water of the microaqueous phase and the organic solvent, dissolution and dissociation of the acid and the resultant number of H+ present in the microaqueous phase and the extent of esterification for a given amount of enzyme at various substrate concentrations. Using mass transfer equations, the theoretical number of H+ at the microaqueous phase were calculated and expressed as hydrogen ion numbers to generate plots which indicated various thermodynamic processes operating at the microaqueous phase to maintain this concentration to a safe minimum.  相似文献   

16.
A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.  相似文献   

17.
Phospholipase D (PLD)-mediated transphosphatidylation of phosphatidylcholine (PC) in a biphasic system was limited by the hydrolysis reaction. A biphasic system can produce a large amount of water. To solve this problem, a microaqueous water-immiscible organic solvent was used for the first time in the bioconversion of phosphatidylserine (PS). The transphosphatidylation among 40 µmol PC, 800 µmol L-serine, and 0.17 U/mL PLD in 2.133 mL of butyl acetate with 6.25% water (V/V) was conducted at a trans-phosphatidylation rate of 88% (mol/mol), and no hydrolytic reaction was observed. Compared to commonly used biphasic systems, this system shows a similar transphosphatidylation rate, whereas the undesirable hydrolysis of phospholipids was completely suppressed.  相似文献   

18.
On protein solubility in organic solvent   总被引:1,自引:0,他引:1  
Solubility of a model protein, hen egg-white lysozyme, was investigated in a wide range of neat nonaqueous solvents and binary mixtures thereof. All solvents that are protic, very hydrophilic, and polar readily dissolve more than 10 mg/mL of lysozyme (lyophilized from aqueous solution of pH 6.0). Only a marginal correlation was found between the lysozyme solubility in a non-aqueous solvent and the letter's dielectric constant or Hildebrand solubility parameter, and no correlation was observed with the dipole moment. Lysozyme dissolved in dimethyl sulfoxide (DMSO) could be precipitated by adding protein nondissolving co-solvents, although the enzyme had a tendency to form supersaturated solutions in such mixtures. The solubility of lysozyme, both in an individual solvent (1,5-pentanediol) and in binary solvent mixtures (DMSO/acetonitrile), markedly increased when the pH of the enzyme aqueous solution prior to lyophilization was moved away from the proteins's isoelectric point. (c) 1994 John Wiley & Sons, Inc.  相似文献   

19.
A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.  相似文献   

20.
Ternary solubility equilibria are studied for three chiral systems in various aqueous and nonaqueous solvents. The chosen systems were a pharmaceutical intermediate, threonine and mandelic acid. Measured solubility data are presented and the nature of the ternary solubility phase diagrams is described. On this basis possible procedures for a crystallization based enantioseparation are derived. Also, the impact of solubility equilibria on the resolution of racemates by liquid chromatography is analyzed and discussed for the systems under investigation. Finally, a hybrid approach coupling both separation techniques for an efficient chiral resolution is demonstrated by means of the fundamental solubility phase diagrams.  相似文献   

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