用磷酸二异辛酯/正辛醇反胶束从面包酵母粗提液中萃取谷胱甘肽

采用磷酸二异辛酯（P204）/正辛醇反胶束液从面包酵母粗提液中萃取还原型谷胱甘肽(GSH),研究了各种因素对萃取率的影响以及水相pH值和P204浓度与GSH萃取率关系的数学模型。正交实验确定,GSH的最佳萃取条件是水相pH值2.0,K+浓度0.1 mol•L-1,萃取时间15 min,P204浓度0.16 mol•L-1,GSH单次的萃取率为33.1%,2次萃取率达到49.7%。     

Extraction and re-extraction of phenylalanine by cationic reversed micelles in hollow fibre contactors

This work reports the extraction of phenylalanine with a reversed micellar system consisting of TOMAC/hexanol/n-heptane using hydrophobic hollow fibre modules. Extraction studies were performed under different hydrodynamic conditions and mass transfer correlations for the shell and tube sides were developed. The correlations were determined using a one-step calculation method and the results obtained are in agreement with the literature for the range of Reynolds numbers studied.Based on the obtained correlations and on the resistance in series model, a transport model was developed in which the phenylalanine concentration in the feed phase can be predicted during the experimental run. For the extraction process the model developed describes satisfactorily the evolution of phenylalanine concentration with time under different hydrodynamic conditions. The re-extraction process was found to be kinetically controlled due to the higher dynamic stability of reversed micelles when contacting a stripping phase with high ionic strength. The experimental results obtained were described using a kinetic model developed.Simultaneous extraction/stripping of phenylalanine was also accomplished using two hollow fibre modules in series, using different volume phase ratios. The mass transfer process was modelled and compared with the experimental results.     

Extraction by reversed micelles of triton N-42 for the spectrophotometric determination of cetyltrimethylammonium bromide

Micellar preconcentration of 1 : 2 associates of Bromophenol Blue with cetyltrimethylammonium bromide is proposed to improve the procedure for the spectrophotometric determination of cationic surfactants. The preconcentration procedure involves quantitative extraction by reversed micelles of Triton N-42 in decane followed by the decomposition of the micellar solution with chloroform. The loss of 10^–7–10^–5 M cetyltrimethylammonium bromide in 5–100-fold preconcentration was not supported by the added-found method (RSD = 3–5%). The determination limit for cetyltrimethylammonium bromide is 2 × 10^–7 M.Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 1, 2005, pp. 17–21.Original Russian Text Copyright © 2005 by Demidova, Bulavchenko.     

β-Xylosidase recovery by reversed micelles

β-Xylosidase, an enzyme produced by Penicillium janthinellum fungus, was prepurified by fractionated precipitation with ethanol and extracted by reversed micelles of N-benzyl-N-dodecyl-N-bis(2-hydroxyethyl) ammonium chloride (BDBAC) cationic surfactant. A 2^5-1 fractional factorial design was employed to evaluate the influence of the following factors on the enzyme extraction: pH (A), conductivity (B), surfactant concentration (C), cosolvent concentration (D) and temperature (E). A statistical analysis of the results revealed that, of the five variables studied, pH, surfactant concentration, and temperature had significant main effects (p<0.05) on the recovery of β-xylosidase (Y). However, the interactions between pH and temperature, conductivity and cosolvent concentration, conductivity and temperature, BDBAC concentration and temperature also had significant influences. A first-order model (R ²=0.98) expressed by the equation Y=7.73−5.55A−0.67B+3.49C−0.41D+5.26E+2.05AB−3.26AC+0.96AD−7.07AE−3.93BD−2.19BE+0.99CD+0.78CE was proposed to represent the enzyme recovery as a function of the effects that were really significant. This model predicts a recovery value of about 40%, which is similar to that obtained experimentally (39.5%).     

Determination of pH in reversed micelles

The pH in the reversed micellar system of di(ethylhexyl) sodium sulfosuccinate (Aerosol OT, AOT) / phosphate buffer solutions/octane was determined by a P-NMR technique, and pHs in the reversed micelles containing buffer solutions other than the phosphate buffer solution were measured by the spectrophotometric method with the aid of Phenol Red. pHs in reversed micelles were found to be substantially determined by the buffer capacity of buffer solutions solubilized into the systems. By means of both the methods, pKa of Phenol Red in the systems was found to be 7.7, which is almost consistent with that in water. Analysis of Na-NMR spectra indicates that the mobility of the sodium ion of AOT is independent of the molar ratio of water to AOT when the ratio is above 7 and is restricted strongly by the interaction with the sulfonate group of AOT. The relationship between pH and the mobility of the sodium ion was discussed on the basis of the data of Na-NMR spectra.     

Calorimetric study on the solubilization of some primary alcohols by reversed AOT micelles

A calorimetric method to evaluate, at the same time, the distribution constant and the standard enthalpy of transfer of a solute partitioned between organic phase and reversed micelles is proposed. The method was applied to the partition of methanol, 1-propanol and 1-pentanol between n-heptane and AOT reversed micelles containing water at 25°C. The results show that the distribution constant decreases as the alcohol alkyl chain length increases and that the solubilization site can change as the water content of reversed AOT micelles increases. In particular, at sufficiently high water content, methanol seems to be preferably solubilized in the aqueous pseudophase whereas 1-pentanol prefers always the palisade layer as site of solubilization.In part from Doctor in Biology thesis of F. Pinio, University of Palermo, Italy.     

Inhibitors of xylose reductase from the yeast pichia stipitis

Several compounds were examined for their inhibitory effects on xylose reductase from the yeastPichia stipitis NRC 2548. Mercuric chloride, cupric chloride, menadione sodium bisulfite, and sodium bisulfite inhibited enzyme activity in a sigmoidal dose-dependent manner, whereas quercetin and rutin were observed to have nonsigmoidal dose-response curves. Diphenylhydantoin, hydantoin, and valproic acid had no effect on xylose reductase activity. Mercuric chloride was the most potent inhibitor tested, with an IC₅₀ (the concentration that inhibited enzyme activity by 50%) of 4.7xl0^-6M. Three distinct inhibition patterns were observed amongst selected inhibitors. Mercuric chloride and quercetin were noncompetitive inhibitors of xylose reductase with respect to substrate and cofactor. Sodium bisulfite was an uncompetitive inhibitor with respect to substrate and cofactor, whereas menadione sodium bisulfite was a competitive inhibitor with respect to substrate, but noncompetitive to the cofactor.     

Conformational studies of the oligomers of L-lysine in reversed micelles

The effect of the chain length on the conformation of oligo-L-lysines (Lys-n, n= 9, 12 and 15) was examined in the reversed micelles of bis(2-ethylhexyl)sodium sulfosuccinate (AOT) in octane by the circular dichroism (CD) measurements. These oligomers seem to take a-structure in these systems. The structure-inducing effect of the reversed micelles is enhanced as the molar ratio of water to AOT (w₀=[H₂O]/[AOT]) becomes smaller. On the other hand, in the aqueous solutions the oligomers having 12 and 15 residues show the conformational transition from random coil to-helical structure by the addition of AOT, but the short oligomer of 9 residues does not show such a conformational transition.     

Exploitation of reversed micelles as membrane mimetic reagents

The aqueous micelle model and its relation to the various models proposed for aggregation of surfactants in nonaqueous solvents, and the controversy surrounding the applicability of the critical micelle concentration to these aggregates, have been discussed. The effect of the addition of water to, and the nature of acid-base interactions in, surfactants solutions in apolar solvents have been included in a review of the kinetics of the reactivity of esters and other organic sustrates, inoragatic reaction mechanisms, catalysis by solubilized enzymes and electron and proton transfer reactions in these media. Where possible, analogies have been made with membrance mediated processes.     

Photochemistry of a cyanine dye in reversed micelles

The effect of microenvironment on the existing state and spectral properties of a cyanine dye in different systems were investigated. Due to the space limitation and the polarity evolution of the water cell of reversed micelles, the optical behavior of the dye in reversed micelles was very different from in water and alcohol. The effect of surfactants with different charge on the interaction of a cyanine dye with AgCl nanoparticles in reversed micelles were also researched. The adsorption state of the dye on AgCl nanoparticles in reversed micelles was discussed.     

FT-IR investigation of the acetamide state in AOT reversed micelles

The state of acetamide nanoparticles encapsulated in the hydrophilic core of sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reversed micelles and dispersed in CCl ₄ has been investigated by Fourier transform infrared spectroscopy. The analysis of the vibrational spectra reveals even at the higher acetamide to AOT molar ratio some changes of the typical H-bonded structure of solid acetamide ascribable to their small size, confinement effects, and acetamide-AOT head group interactions. The stretching modes of acetamide CO and AOT sulfonate groups indicate unambiguously specific acetamide-AOT head group interactions.     

Calorimetric study of the alpha-tocopherol solubility in reversed AOT micelles

The experimental data of heat of mixing (Q) for heterogeneous system alpha-tocopherol/AOT/n-heptane with and without water at 25 degrees C are presented. The Q dependence on AOT (sodium bis (2-ethylhexyl) sulfosuccinate) concentration, and R parameter defined as R=[H2O]/[AOT] with flow calorimetric method were investigated. Using the D'Aprano model (which is formally identical to that used earlier by Magid et al.) the binding constant (K), the distribution constant of alpha-tocopherol (K distr) between hydrocarbon and the micellar phase, and the standard enthalpy of transfer (DeltaH tr 0) of alpha-tocopherol from the hydrocarbon to AOT reversed micelles were calculated. The solubility of alpha-tocopherol in AOT reversed micelles explored with the calorimetric technique was compared to the literature data obtained respectively with UV spectrophotometry for reversed micelles and by other techniques for the phospholipid bilayer.     

Environmentally responsive molecular baskets: unimolecular mimics of both micelles and reversed micelles

[structure: see text] When four facially amphiphilic cholate derivatives are attached to a tetraaminocalixarene scaffold, the resulting molecule responds to environmental changes by rotation of the cholate units. In polar solvents, the molecule adopts a micellelike conformation with the hydrophilic alpha-faces of the cholates pointing outward. In nonpolar solvents, it turns inside out, assuming a reversed micellelike conformation with the hydrophobic beta-faces pointing outward. Switching between the two conformations is driven by solvophobic interactions and is fully reversible.     

Changing flux of xylose metabolites by altering expression of xylose reductase and xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

We changed the fluxes of xylose metabolites in recombinant Saccharomyces cerevisiae by manipulating expression of Pichia stipitis genes (XYL1 and XYL2) coding for xylose reductase (XR) and xylitol dehydrogenase (XDH), respectively. XYL1 copy number was kept constant by integrating it into the chromosome. Copy numbers of XYL2 were varied either by integrating XYL2 into the chromosome or by transforming cells with XYL2 in a multicopy vector. Genes in all three constructs were under control of the strong constitutive glyceraldehyde-3-phosphate dehydrogenase promoter. Enzymatic activity of XR and XDH in the recombinant strains increased with the copy number of XYL1 and XYL2. XR activity was not detected in the parent but was present at a nearly constant level in all of the transformants. XDH activity increased 12-fold when XYL2 was on a multicopy vector compared with when it was present in an integrated single copy. Product formation during xylose fermentation was affected by XDH activity and by aeration in recombinant S. cerevisiae. Higher XDH activity and more aeration resulted in less xylitol and more xylulose accumulation during xylose fermentation. Secretion of xylulose by strains with multicopy XYL2 and elevated XDH supports the hypothesis that d-xylulokinase limits metabolic flux in recombinant S. cerevisiae.     

Increased xylose reductase activity in the xylose-fermenting yeastPichia stipitis by overexpression ofXYL1

Applied Biochemistry and Biotechnology - ThePichia stipitis xylose reductase gene (XYL1) was inserted into an autonomous plasmid thatP. stipitis maintains in multicopy. The plasmid pXOR with...     

Native and modified glucose oxidase in reversed micelles

The enzymatic activity of the native and modified glucose oxidase (GOx) from Aspergillus niger in the system of reversed micelles of Aerosol OT in octane was investigated. Two forms of the modified enzyme were studied: a hydrophobized form obtained by the attachment of palmitic chains to lysine amino groups by the reaction with palmitic acid ester of N-hydroxysuccinimide and a glycosylated (hydrophilized) form obtained by the attachment of the cellobiose moieties. The native glucose oxidase and its derivatives, while incorporated into micelles in a surfactant concentration range from 0.05 to 0.3 M, display an enzymatic activity, which is comparable with the activity in aqueous solution. The dependence of the enzymatic activity on hydration degree of surfactant (the molar ratio of water to surfactant, W₀) does not indicate the formation of qualitatively new associated forms of the enzyme subunits inside the micelles. The apparent size of Aerosol OT micelles obtained by dynamic light scattering gradually increases from 10±3 nm at low W₀ up to 25±5 nm at high W₀. Incorporation of the native and hydrophobized glucose oxidase into micelles does not affect their mean size. Kinetic analysis shows that the enzyme specificity is about an order of magnitude greater in the system of reversed micelles as compared with aqueous solution.     

Calorimetric study of the solubilization of cholesterol,retinol and retinal by reversed AOT micelles

Distribution constants and standard enthalpies of transfer of cholesterol, retinol and retinal partitioned between n-heptane and water containing reversed sodium bis(2-ethylhexyl) sulfosuccinate (AOT) micelles as a function of the molar concentration ratio (R=[water]/[AOT]) were evaluated by a calorimetric method. The results indicate that, in spite of the bulky hydrocarbon radical, these solubilizates behave like alcohols with a short alkyl chain. Moreover, cholesterol is always solubilized in the palisade layer of the reversed micelles whereas retinol and retinal are preferentially solubilized in the aqueous pseudophase. The influence of the enthalpic and the entropic contributions to the transfer of the solubilizates from n-heptane to reversed AOT micelles are also considered.     

Ribonucleotide activation by enzyme ribonucleotide reductase: understanding the role of the enzyme

This article focuses on the first step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This corresponds to the activation of the substrate. In this work a large model of the active site region involving 130 atoms was used instead of the minimal gas phase models used in previous works. The ONIOM method was employed to deal with such a large system. The results gave additional information, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme in this step. Enzyme-substrate interaction energies, specific transition state stabilization, and substrate steric strain energies were obtained. It was concluded that the transition state is stabilized in 4.0 kcal/mol by specific enzyme-substrate interactions. However, this stabilization is cancelled by the cost in conformational energy for the enzyme to adopt the transition state geometry; the overall result is that the enzyme machinery does not lead to a rate enhancement in this step. It was also found that the substrate binds to the active site with almost no steric strain, emphasizing the complementarity and specificity of the RNR active site for nucleotide binding. The main role of the enzyme at the very beginning of the catalytic cycle was concluded to be to impose stereospecifity upon substrate activation and to protect the enzyme radical from the solvent, rather than to be an reaction rate enhancement.