胰凝乳蛋白酶在反胶束体系中热稳定性的研究

研究了AOT／正辛烷反胶束体系中,表面活性剂AOT,助溶剂甘油和体 压力对胰凝乳蛋白酶热稳定性的影响,结果表明;常压下,40度时,体系中的甘油浓度分别为20％,30％,50％,60％（V／V）时,酶的活力分别为原来的10％,22％,59％,48％,说明在体系中加入甘油作为助溶剂可减少胰凝乳蛋白酶的运动,增强其稳定性,同时,发现体系压力的增加也能增强酶的稳定性,实验测出,AOT正辛烷反胶束体系萃取胰凝乳蛋白酶的最佳条件：R＝10,甘油浓度为50％,体系压力为50MPa,酶的萃取率为97％。     

Activity of lipoxygenase incorporated into reversed micelles of aerosol OT in octane

Soybean lipoxygenase (EC 1.13.11.12) incorporated into the reversed micelles of aerosol OT in octane has been studied for its catalytic properties. The enzyme is shown to preserve up to 10% activity as compared with the activity in the aqueous solution. In this case Km of lipoxygenase for linoleic acid increases from 10(-5) M to 5 X 10(-4) M. The activity of lipoxygenase is maximal, the aerosol OT concentration being 0.03 M and a degree of reversed micelle hydratation 40. Cationic detergents of the cetyltrimethyl ammonium bromide type are not good to form reversed micelles of lipoxygenase, since they inhibit the latter with IC50 = (4 divided by 6) x 10(-4) M. The lipoxygenase preparations in reversed micelles of aerosol OT in octane may be used to synthesize natural metabolites of polyunsaturated fatty acids, for instance of eicosanoids.     

Solubilization of rat kidney lysosomes in reversed micelles of aerosol OT in octane.

Intact lysosomes from rat kidneys were solubilized in the ternary system: surfactant (Aerosol OT)-buffer-organic solvent. According to data of laser light-scattering analysis and kinetic experiments with the lysosomal marker enzyme, N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30), the solubilization of lysosomes in this system resulted in the destruction of the lysosomes and the entrapping of their components in reversed micelles.     

Catalytic properties and stability of catalase in reversed micelles of aerosol OT in octane]

The catalytic function of catalase and its peroxidatic activity during tetramethylbenzidine (TMB) oxidation by cumene hydroperoxide were studied in reversed micelles of Aerosol OT (AOT) in octane relative to the [H2O]/[AOT] ratio and the initial catalase concentration. The optimum conditions permitting to retain the catalytic activity of the enzyme and its ability to induce peroxidation of TMB, were found. The catalytic function of the enzyme was shown to be dependent on its concentration in AOT micelles. The catalase stability monitored by the catalytic reaction and the decrease of the Soret band were analyzed. Both processes have two phases differing by the rate constants of the pseudo-first order. The catalase inserted into AOT micelles is characterized by the high stability as compared to other hemoproteins (cytochrome P-450, myoglobin, hemoglobin, peroxidase) under identical conditions.     

Sedimentation analysis of muscle glycogen phosphorylase b entrapped in hydrated reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscles have been studied in the system of hydrated reversed micelles of aerosol OT (AOT) in octane. Sedimentation studies show that the oligomeric state of the enzyme is controlled by the degree of hydration of micelles. Monomeric, dimeric, trimeric, tetrameric, hexameric, or octameric forms of the enzyme were observed depending on the degree of micelle hydration.     

Oligomeric state of the muscle glycogen phosphorylase b in the system of reversed micelles of aerosol OT in octane

The oligomeric state and formation of supramolecular structures of glycogen phosphorylase b from rabbit skeletal muscle was studied in the system of aerosol OT (AOT) reversed micelles in octane. The sedimentation experiments have shown that the enzyme oligomeric state depends on the degree of micelle hydration. The enzyme monomer, dimer, trimer, tetramer, hexamer, and octamer were observed, depending on the degree of hydration.     

Comparative study of the kinetics of the reduction of cytochrome c and the nitroxide radical in reversed aerosol OT micelles in octane

The comparison of the reduction kinetics of cytochrome c and nitroxide radical by ascorbate in reversed micelles of aerosol OT in octane was studied. The plot of the dependence of the reduction rate constant on the micelle hydratation is bell-shaped in the case of protein but shows the plateau form for the radical. The reaction rates decreases at high micelle concentrations. The equations have been drawn that connect the experimental rate constants with intramicellar biomolecular rate constant (km) for reagents unsolved in organic phase. In the case of strong hydratated micelles km for the radical reduction is practically equal to the rate constant in aqueous solution. For cytochrome c the ratio of these constants is less than 0.22, that may be explained by protein conformational changes detected by optical methods. For small micelle hydratation the dependence of the cytochrome reduction rate on ascorbate concentration is characterized by plateau. Under these conditions the limited stage of reaction is apparently the transition of the protein to the active conformation.     

Effectiveness of acylation of protein amino groups with fatty acid chlorides in a system of reversed micelles of aerosol OT in octane

The characteristics of water-soluble enzyme (alpha-chymotrypsin) modification with [3H] palmitoyl chloride in the reversed Aerosol OT micelles in octane were determined. The degree of enzyme modification depends on the molar ratio [palmitoyl chloride]/[protein]. The modification reaction is characterized by the wide pH-optimum range and proceeds with high speed.     

Hydrophobic formate dehydrogenase from Pseudomonas sp. 101 in the system of reverse micelles of aerosol OT in octane

NAD(+)-dependent formate dehydrogenase (FDH) was hydrophobized with palmitoyl chloride to give the samples with various modification degrees (2-10). The native and modified FDHs were comparatively studied in the system of reverse micelles of Aerosol OT in octane. Like the native, the modified enzyme displayed three maxima in the curve of dependence of its catalytic activity on the degree of surfactant hydration (the micelle size), which reflect the enzyme functioning in the form of a monomer, dimer, or octamer. The peak corresponding to the functioning of the FDH dimer was found to decrease along with an increase in the modification degree. Thus, the modified enzyme mainly functions in the form of monomer and octamer. The modified FDH displayed membranotropy and revealed the dependence of catalytic activity on surfactant concentration.     

Catalytic properties of the membrane enzyme prostaglandin H synthetase in a system of inverted micelles of aerosol OT in octane

Prostaglandin H synthetase solubilized in octane with the aid of hydrated reversed micelles of Aerosol OT (AOT) exhibits a catalytic activity dependent on hydration of the surface-active substance and its concentration. The maximum rate of the reaction is attained at the H2O/AOT molar ratio equal to 20 and amounts to a value close to the one observed in an aqueous solution. The inactivation rate of the enzyme in the course of the reaction does not depend on the water content in the system and is described by a Kin value commensurable with the Kin in an aqueous solution.     

Catalysis by hemoproteins and their structural organization in reversed micelles of surfactants in octane

The cytochromes $\text{P-450}$ LM-2 and $\text{b}{}_5$ from rabbit liver microsomes have been entrapped into reversed micelles of surfactants in octane. The optimum conditions providing for the maximum stability of the hemoproteins have been found: pH and concentration of the buffer solution, the glycerol addition, the surfactant concentration, the [H₂O]/[surfactant] ratio and, finally, the reversed micelles composition including aerosol OT and its mixture with Triton X-45, Tween 20 and cetyltrimethylammonium bromide (CTAB). The transformation kinetics of the hemoproteins solubilized by detergents has been studied by monitoring the absorbance of hemoproteins in the Soret band region. Significant changes in tryptophan fluorescence of cytochrome $\text{b}{}_5$ and in CD spectra of myoglobin in reversed micelles and their dependence on the [H₂O]/[aerosol OT] ratio have been shown. The three hemoproteins in reversed micelles have been found to exhibit high catalytic activity with respect to their reaction with cumene hydroperoxide. The kinetic and spectral data reveal the structural transformations of the proteins entrapped into the micelles due to the interactions of the lumenal surface of the micelles and the protein molecule surface.     

Mechanism of extraction of chymotrypsin into isooctane at very low concentrations of aerosol OT in the absence of reversed micelles

Chymotrypsin is easily extracted from an aqueous solution into isooctane containing the anionic surfactant aerosol OT (AOT). The concentration of AOT needed to efficiently extract 0.5 mg/mL CMT is as low as 1 mM and as low as 0.2 mM AOT was sufficient to extract the protein into isooctane. The extraction process was unaffected by 10% (v/v) ethyl acetate in the isooctane phase. Moreover, spectroscopic analysis by electron paramagnetic resonance indicated that CMT did not exist inside a discreet water pool of a reversed micelle. Calculations of the number of AOT molecules associated per extracted CMT molecule indicate that only ca. 30 surfactant molecules interact with the protein, a value too low for reversed micellar incorporation of the protein in isooctane. These studies suggested that reversed micelles do not need to be involved in the actual transfer of the protein from the aqueous to the organic phase and protein solubilization in the organic phase is possible in the absence of reversed micelles. Based on these findings, a new mechanism has been proposed herein for protein extraction via the phase transfer method involving ionic surfactants. The central theme of this mechanism is the formation of an electrostatic complex between CMT and AOT at the aqueous/organic interface between AOT and CMT, thereby leading to the formation of a hydrophobic species that partitions into the organic phase. Consistent with this mechanism, the efficiency of extraction is dependent on the interfacial mass transfer, the concentrations of CMT and AOT in the aqueous and organic phases, respectively; the ionic strength of the aqueous phase; and the presence of various cosolvents. (c) 1994 John Wiley & Sons, Inc.     

Transesterification of oil mixtures catalyzed by microencapsulated cutinase in reversed micelles

Recombinant cutinase from Fusarium solani pisi was used to catalyze the transesterification reaction between a mixture of triglycerides (oils) and methanol in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate (AOT) in isooctane for the purposes of producing biodiesel. The use of a bi-phase lipase-catalyzed system brings advantages in terms of catalyst re-use and the control of water activity in the medium and around the enzyme micro-environment. Small-scale batch studies were performed to study the influence of the initial enzyme and alcohol concentrations, and the substrates molar ratio. Conversions in excess of 75 were obtained with reaction times under 24?h, which makes this enzymatic process highly competitive when compared to similar lipase catalyzed reactions for biodiesel production using methanol.     

The lipase/lipoxygenase bienzyme system in AOT reversed micelles in octane

In this work it is shown that the bienzyme lipase/lipoxygenase system can function in reversed micelles of bis(2-ethyl)hexyl sulfosuccinate (AOT) in octane. As a lipase substrate, a fish fat preparation (fat of sea mammals) with a high content of polyunsaturated fatty acids was used. It was demonstrated that the bienzyme reaction proceeded in a stationary mode and had a rate-limiting step catalyzed by lipase. Under optimal conditions, the efficacy of functioning of the bienzyme system was by an order of magnitude higher than that in water. The lipase/lipoxygenase bienzyme system can be used as a new method of spectrophotometric determination of lipase activity. The English version of the paper.     

Regulation of the catalytic activity and supermolecular structure of penicillin acylase from Escherichia coli in an aerosol OT reversed micelle system in octane]

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles of Aerozol OT (AOT) in octane were studied. The catalytic activity dependence on the hydration degree, a parameter which determines the size of the micelle inner cavity, represents a curve with three optima, each corresponding to the enzyme functioning either in a dimer form (omega 0 = 23) or in the form of separate subunits--heavy, beta, and light, alpha, at omega 0 = 20 and 14, respectively. Reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis. Preparative isolation of penicillin acylase subunits, their catalytic activity being retained, was shown to be possible.     

Effect of hydration degree of aerosol OT reversed micelles and surfactant concentration in heptane on spectral and catalytic properties of catalase.

The influence of micelle hydration degree (w0) and AOT concentration on fluorescence, circular dichroism (CD), catalytic activity, and stability of catalase in Aerosol OT (AOT) reversed micelles in heptane was investigated. The quantitative parameters--differential fluorescence of catalase (DeltaI), protein molar ellipticity ([theta]lambda), initial rate of catalytic reaction, catalase efficiency (kcat/Km), and rate constant of enzyme inactivation (kin, sec-1)--decreased with increasing AOT concentration in micellar systems, reflecting the interaction of solubilized catalase with the AOT micellar aggregates in heptane. The dependences of all these parameters on increasing hydration degree of micelles (w0) were characterized by the appearance of maxima at w0 of 8, 15-18, and 26-30. These maxima are suggested to reflect three different states of catalase in the micellar system, distinguished by their conformations and catalytic activity, which is determined by the micellar microenvironment of the enzyme.     

Active centers of gamma-glutamyltransferase in the aerosol OT reverse micellar system in octane by an inhibitor analysis method]

Regulation of the supramolecular structure and catalytic activity of the heterodimeric enzyme gamma-glutamyltransferase in the system of Aerosol OT reversed micelles in octane was studied. Variation of the hydration degree causes a reversible dissociation of the enzyme to the light and heavy subunits, both possessing the catalytic activity. The subunits were separated on the preparative scale in the reversed micelle system using ultracentrifugation. The active centres of gamma-glutamyltransferase were studied using the enzyme's irreversible inhibitor AT-125 (L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). It is shown that the separation of the gamma-glutamyltransferase subunits results in "opening" of a new active centre in the heavy subunit, whereas in the enzyme's dimeric form this centre is masked and not accessible to the inhibitor's molecule. The kinetic and inhibitor analysis data indicate that the active centres in the light and heavy subunits are similar.     

Ice nucleation and crystallization in water-glycerol mixtures

Ice nucleation and crystallization processes play a very important role in research and, particularly, in crybiology. In this domain, water-glycerol mixtures are often used. The cryoprotective action of such solutions is not completely known but crystallization of water seems to play a dominant role. The aim of this work is to precisely define the devitrification conditions of such mixtures. Three different techniques are used in this study. Three temperature ranges have been distinguished in which nucleation, growth, and then Ostwald ripening are successively detected.