Action of Humicola lanuginosa lipase on long-chain lipid substrates: 2. Hydrolysis of diolein monolayers

The hydrolysis of 1,2-diolein (DO) monomolecular films by Humicola lanuginosa lipase (HLL) was studied by simultaneous measuring the decrease in the film area and the changes in the surface potential in the “zero-order trough” at constant surface pressure and in the presence of β-cyclodextrin (β-CD). The decrease with time in the film area reflects both the reduction in the area per molecule due to the transformation of substrate DO molecules into the products molecules of monoolein (MO) and oleic acid (OA) and the desorption of the soluble inclusion complexes β-CD–MO and β-CD–OA. The surface potential data were interpreted as an accumulation at the interface of negatively charged products of OA and insoluble β-CD–DO complexes. In the proposed kinetic model, the product solubilization rates in the presence of β-CD and the flux supplied progressively by the moving barrier from the reservoir to the reaction compartment in order to keep the constant surface pressure were taken into account. The surface concentrations of MO and OA transiently present at the interface were determined. The values of the global kinetic constant Q_m′ of hydrolysis of DO to MO were obtained. Comparison with the values of the global kinetic constant of hydrolysis of monoglyceride MO to OA shows that the rates of hydrolysis of diglyceride and monoglyceride by HLL are of the same order of magnitude.     

A kinetic study of the formation of beta-cyclodextrin complexes with monomolecular films of fatty acids and glycerides spread at the air/water interface

The kinetics of formation of inclusion complexes between beta-cyclodextrin and monolayers of one-, two- and three-chained lipid molecules, namely, oleic acid (OA), monoolein (MO), diolein (DO) and triolein (TO), was investigated at various pH using three independent dynamic methods. The formation and solubilization of soluble inclusion beta-CD/OA and beta-CD/MO complexes was detected by measuring the decrease of the surface area and surface pressure of the OA and MO monolayers in the presence of beta-CD within a wide range of concentrations. A third approach, describing the dilatational properties of the monolayers, influenced by the formation and solubilization of the complexes, was developed. Using the three above-mentioned independent methods, the rate constants of formation (k1) and dissociation (k2) of beta-CD/OA and beta-CD/MO, were determined. We observed that solubilization flux i s for OA monolayer increases with pH and at pH 11 reached a value, which is closed to the diffusion flux iD and the process thus becomes diffusion controlled. For MO monolayer no significant effects of pH was observed above pH 6. The surface pressure (Deltapi)--area per molecule (A) and surface potential (DeltaV)--area per molecule (A) isotherms and rheological properties of DO and TO monolayers were measured in the presence or absence of beta-CD. DO and TO form water-insoluble complexes with beta-CD, as visualized by AFM images.     

Interfacial characteristics of β-casein spread films at the air–water interface

Casein is well known to be a good protein emulsifier and β-casein is the major component of casein and commercial sodium caseinate. This work studies the behaviour of β-casein at the interface. The interfacial characteristics (structure and stability) of β-casein spread films have been examined at the air–water interface in a Langmuir-type film balance, as a function of temperature (5–40°C) and aqueous phase pH (pH 5 and 7). From surface pressure–area isotherms (π–A isotherms) as a function of temperature we can draw a phase diagram. β-Casein spread films present two structures and the collapse phase. That is, there is a critical surface pressure and a surface concentration at which the film properties change significantly. This transition depends on the temperature and the aqueous phase pH. The film structure was observed to be more condensed and β-casein interfacial density was higher at pH 5. β-Casein films were stable at surface pressures lower than equilibrium surface pressure. In fact, no hysteresis was observed in π–A isotherms after continuous compression-expansion cycles or over time. The relative area relaxation at constant surface pressure (10 or 20 mN m⁻¹) and the surface pressure relaxation at constant area near the monolayer collapse, can be fitted by two exponential equations. The characteristic relaxation times in β-casein films can be associated with conformation–organization changes, hydrophilic group hydration and/or surface rheology, as a function of pH.     

Enzymatic hydrolysis reaction of phospholipids in monolayers

The hydrolysis reaction of , and , -dipalmitoylphosphatidylcholine (DPPC) catalized by bee venom phospholipase A₂ was studied in spreading monolayer at the water/air interface. DPPC and the hydrolysis products, palmitic acid and -lysophosphatidylcholine, palmitoyl were characterized at the interface by means of surface pressure, surface potential and ellipsometric measurements. Furthermore, mixed monolayers of reagents and products were investigated to ascertain their miscibility. The results show that the hydrolysis reaction can be followed by the decrease of surface pressure with time on subphases containing β-cyclodextrin, a well-known complexing agent of many amphiphilic compounds. The order of the reaction, the kinetic constant and other kinetic parameters are deduced.     

Savinase proteolysis of insulin Langmuir monolayers studied by surface pressure and surface potential measurements accompanied by atomic force microscopy (AFM) imaging

The mechanism of the enzymatic action of Savinase on an insulin substrate organized in a monolayer at the air-water interface was studied. We followed two steps experimental approach classical surface pressure and surface potential measurements in combination with atomic force microscopy imaging. Utilizing the barostat surface balance, the hydrolysis kinetic was followed by measuring simultaneously the decrease in the surface area and the change of the surface potential versus time. The decrease in the surface area is a result of the random scission of the peptide bonds of polypeptide chain, progressively appearance of amino acid residues, and their solubilization in the aqueous subphase. The interpretation of the surface potential data was based on the contribution of the dipole moments of the intact and broken peptide groups which remain at the interface during the proteolysis. An appropriate kinetic model for the Savinase action was applied, and the global kinetic constant was obtained. The application of the AFM revealed the state of the insulin monolayers before and after the Savinase action. The comparison of the topography of the films and the roughness analysis showed that insulin Langmuir-Blodgett (LB) films transferred before the enzyme action were flat, while at the end of hydrolysis, roughness of films has increased and the appearance of 3D structures was observed.     

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.     

Layered assemblies composed of sulfonated cyclodextrin and poly(allylamine)

Layered assemblies containing cyclodextrin (CD) have been prepared by layer-by-layer deposition of poly(allylamine) and sulfonated α-CD or β-CD on a solid surface. A quartz crystal microbalance study revealed that α-CD and β-CD form multilayers in the films upon each deposition. The sulfonated-α-CD-containing film bound methyl orange (MO) by forming an inclusion complex, resulting in the isolation of MO as a monomer in the film, whereas MO formed aggregates in the sulfonated-β-CD and CD-free films.     

Kinetics and layer organization during the polymerization of coniferyl alcohol at the air-water interface

The initial stage of the polymerization of coniferyl alcohol catalyzed by a peroxydase was studied at the air-water interface. The properties of the monolayers were investigated at constant area and at constant surface pressure by surface pressure, surface potential and ellipticity measurements. On the basis of the results obtained at constant surface area, it is proposed that the formation of a 2D layer occurs up to the inflection point of the surface pressure-area isotherm, and that for larger surface pressures a 3D structure is formed during the polymerization. If the barostat is set at the inflection point (the surface pressure is constant while the surface area increases), the 2D organization of the monolayer is retained. A kinetic model describing the adsorption of the reaction products in a 2D layer is proposed. The kinetic constants were determined from the surface potential, ellipsometry, surface pressure and surface area data.     

Reorganization of lipid nanocapsules at air-water interface. I. Kinetics of surface film formation

The size, the electrical properties and the behaviour at air-water interface of lipid nanocapsules (LNC) with various compositions were investigated. Two populations of LNC are presented in the suspension after the preparation: with (LNC II) and without (LNC I) phospholipid molecules. After the spreading at air-water interface, a rapid disaggregation of LNC I, located in the vicinity of interface, occurs leading to formation of surface film. The phospholipid molecules stabilize the structure of nanocapsules and LNC II are more stable at the interface in comparison with LNC I. The formation of a surface film was followed after by measuring the evolution of the surface pressure, relative surface area change and surface potential. A kinetic approach describing the various processes during the surface film formation was proposed. The corresponding kinetic constants were estimated.     

Studies of an inclusion complex of a redox-active barbiturate with β-cyclodextrin

The effects of β-cyclodextrin (β-CD) on the spectral properties and electrochemical behaviour of barbitone sodium were studied using electrochemical and optical techniques. The apparent changes in the UV-visible absorption spectrum and fluorescence quenching of the barbiturate upon addition of β-CD afford clear evidence of the inclusion complex formation of β-CD in aqueous solutions. The redox acitivity of barbitone sodium in both the absence and presence of β-CD was assessed by an electrochemical method in 0.05 M potassium nitrate-nitric acid (pH 1.8) solution. While the complexation reaction remains is equilibrium, the apparent formation constant of the inclusion complex could be obtained by either spectral or electrochemical methods. Quantitative evaluation of the data gave 326 l mol⁻¹ under equilibrium conditions at 20°C. Possible conclusions are discussed on the basis of environmental changes around the barbiturate molecules on inclusion.     

Surface forces and the stability of colloids and disperse systems

There are discussed two factors controlling the thickness h of a free film of liquid between two bubbles of radius R pressed against one another, the mechanical properties of the film and the temperature. The role of the viscosity, η, is analyzed on the basis of the formula h = 2.64 R(ηu/σ)^2/3 (σ surface tension, u thereceding velocity of the film perimeter), which is the simple consequence of the formula for the thickness of the film left behind the receding wetting perimeter (Derjaguin, 1943). A similar formula is also used for the analysis of the role of yield value of the film. The temperature does not change the thickness of the black free films (100A.) in contrast to the thicker films governed by electric repulsion. The jumpwise thinning of free films is studied by microfilming.

The method of crossed polarized metal wires is described, which permits measurement of the potential barrier preventing metallic contact in liquid media. The measurements of this barrier in water solutions of electrolytes as a function of potential of the wires are interpreted on the basis of our theory of colloid stability. The existence of repulsive forces (disjoining pressure) at high electrolyte concentrations independent of the potential proves their nonelectrostatic origin. These measurements were also used to calculate Hamaker's constant of molecular attraction and to determine the potential of zero charge of some metals.     

Synthesis and characterization of an ultrathin polyion complex membrane containing β-cyclodextrin for separation of organic isomers

An ultrathin polyion complex (PIC) layer containing β-cyclodextrin (β-CD) was formed on the surface of a charged base membrane. Thus, a positively charged copolymer containing β-CD was synthesized by the radical copolymerization of β-CD monomer and allylamine, and was used to modify the surface of a Nafion membrane containing negatively charged fixed ions. Electron spectroscopy for chemical analysis (ESCA) confirmed the presence of the copolymer on the surface of the Nafion membrane due to the formation of a PIC layer. The ability of the membrane to separate organic isomer mixtures was tested by the pervaporation technique, using butanol isomer mixtures as the feed model. The membrane exhibited a good selectivity toward butanol isomers, indicating the effectiveness of β-CD as a selective fixed carrier for the isomers.     

Cyclodextrin-enhanced fluorescence and photochemically-induced fluorescence determination of five aromatic pesticides in water

The effect of β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) aqueous solutions upon the fluorescence and photochemically-induced fluorescence (PIF) properties of five pesticides, including coumatetralyl, pirimiphos-methyl, chlorpyriphos, deltamethrin and fenvalerate was investigated. A 1:1 stoichiometry was found for the β-CD and HP-β-CD complexes formed with all compounds. Binding constant values, ranging between about 90 and 830 M⁻¹ were calculated using the iterative nonlinear least-squares regression approach. Cyclodextrin-enhanced fluorescence and PIF methods were developed for the determination of these pesticides with linear dynamic ranges over two orders of magnitude, and limits of detection (LOD) between 0.2 and 54 ng ml⁻¹ according to the compound. Application to the analysis of tap water and river water samples yielded satisfactory recoveries (88–116%). The method seems to be suitable for environmental water analysis.     

Sample stacking in laboratory-on-a-chip devices

In this study, enantioseparations of five phenothiazines, including promethazine, ethopropazine, trimeprazine, methotrimeprazine, and thioridazine, in cyclodextrin (CD)-modified capillary zone electrophoresis were investigated using a phosphate buffer (40 mM) at pH 3.0. We focussed on the separation of phenothiazines with the use of CDs at low concentrations. Three different CDs, including β-CD, hydroxypropyl-β-CD (HP-β-CD) and γ-CD, were chosen as chiral selectors. The results indicate that effective enantioseparation of phenothiazines, except for methotrimeprazine, is simultaneously achievable with addition of γ-CD at a concentration of 2.5–6.0 mM. The enantiomers of ethopropazine and trimeprazine are effectively separated with addition of HP-β-CD at low concentrations, in the range 0.4–6.0 mM, whereas those of promethazine and trimeprazine are baseline resolved with β-CD at much lower concentrations (0.02–3.0 mM) than with HP-β-CD. The results also confirm that the separation window is greatly enlarged at low CD concentrations. Moreover, the drastic variations of the electrophoretic mobility of phenothiazines as a function of CD concentration reveal that phenothiazines interact very strongly with CDs in the order γ-CD相似文献   

Enzymatic hydrolysis by cutinase of PEG-co PLA copolymers spread monolayers

The mechanism of the hydrolysis by cutinase and the progressive fragmentation of lactic chains in diblock copolymers of PLA with various sizes attached to PEG were studied in a 2D monolayers model system. The hydrolysis kinetics was followed by measuring simultaneously the decrease of the surface area and evolution of the surface potential with time at barostatic conditions. The decrease of the surface area is due to the solubilization of the copolymers as well as of their hydrolytic products: detached PEG blocks and small soluble PLA fragments. The evolution of the surface potential detects the transient interfacial accumulation of charged insoluble PLA fragments. A kinetic model describing the enzymatic hydrolysis was developed and the values for the global hydrolytic kinetic constant were obtained without any fitting parameter. It was found that the global kinetic constant no practically depend on the length of the lactic and the presence of polyethylene–glycol chains.     

Preparation and enantiomer separation behavior of selectively methylated β-cyclodextrin-bonded stationary phases for high performance chromatography

Native and three selectively methylated β-cyclodextrin (β-CD)-bonded stationary phases without an unreacted spacer arm for liquid chromatography were prepared, where heptakis(2-O-methyl)-β-CD, heptakis(3-O-methyl)-β-CD and heptakis(2,3-di-O-methyl)-β-CD were used as the methylated β-CDs. The enantiomer separation abilities of the resulting β-CD stationary phases for 12 pairs of dansylamino acid enantiomers and six pairs of N-3,5-dinitrobenzoyl amino acid methyl esters as model solutes were investigated. The effects of pH and methanol content of the mobile phase on the retention and resolution were examined to optimize the mobile phase conditions. The optimum resolution for the dansylamino acids was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 4/6) on the β-CD stationary phase. Heptakis(3-O-methyl)- and heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases showed little enantiomer separation abilities for the dansylamino acids. The heptakis(2-O-methyl)-β-CD-bonded stationary phase exhibited no enantioselectivities for those solutes.

For the N-3,5-dinitrobenzoyl amino acid methyl esters, the optimum resolution was achieved using a mobile phase consisting of 1.0% triethylammonium acetate buffer (pH 5.0)–methanol (v/v 9/1) on a heptakis(2-O-methyl)-β-CD stationary phase. The heptakis(2,3-di-O-methyl)-β-CD-bonded stationary phases exhibited no enantioselectivities for the N-3,5-dinitrobenzoyl amino acid methyl esters. β-CD and heptakis(3-O-methyl)-β-CD-bonded stationary phases had no enantiomer separation abilities for those solutes except for the N-3,5-dinitrobenzoyl phenylalanine methyl ester.     

Modified cyclodextrin polymers as selective ion carriers for Pb(II) separation across plasticized membranes

In the present work the hydrophobic β-cyclodextrin (β-CD) polymers have been used as macrocyclic ion carriers for separation of Pb(II), Zn(II), and Cu(II) ions from dilute aqueous solutions by transport across polymer inclusion membranes. The β-CD polymers were prepared by cross-linking of β-CD with 2-(1-docosenyl)-succinic anhydride derivatives in anhydrous N,N-dimethylformamide in the presence of NaH. The metal ions were transported from aqueous solutions containing heavy metal ions through plasticizer triacetate membranes with dimer and polymer β-CD derivatives into distilled water. The selectivity of lead(II) over other metal ions in the transport through polymer inclusion membrane was very high, especially for dimer cyclodextrin carrier. In the case of competitive transport of Pb(II), Cu(II), and Zn(II) ions through plasticized immobilized membranes the selectivity of process is controlled via formation of ion pairs of β-CD hydroxyl groups with metal cations. The polymer and dimer of β-CD linked by 2-(1-docosenyl)-derivative used as ionic carriers for competitive transport of metal ions show preferential selectivity order: Pb(II)  Cu(II) > Zn(II). Application of ion carriers mixtures (β-CD polymers and palmitic acid) causes the increase of Pb(II) maximal removal from dilute aqueous solution. The weight-average molecular weight (M_W) and the chemical structure of the β-CD polymers were determined using high-performance size exclusion chromatography with refractive index detector, and ¹H NMR spectroscopy.     

Effect of the acyl group and the dodecylsulfonate chain on the inclusion of pyrene inside the cavity of β-cyclodextrin

The solubilization of pyrene in aqueous solution of β-cyclodextrin (β-CD) or its derivatives such as β-CD-hexanoyl, β-CD-benzoyl and β-CD-dodecylsulfonate was investigated by spectrophotometry. Linear and non-linear regression methods were used to estimate the association constants (K₁). A 1:1 stoichiometric ratio and different effects of the hexanoyl, benzoyl and dodecylsulfonate groups on the association constant were observed for the binary inclusion complex between pyrene and β-CD. The formation constant was shown to decrease when β-CD was modified by a dodecylsulfonate chain. The value of K₁ was 190 ± 10 L mol⁻¹ for the [pyrene/β-CD] complex and 145 L mol⁻¹ for the [pyrene/β-CD-dodecylsulfonate] complex. Partitioning of the pyrene molecules between the dodecylsulfonate chains and cyclodextrin cavities can explain the decrease in the association constant value. In the cases of β-CD-hexanoyl and β-CD-benzoyl derivatives, no association constants were detected. Results suggest that the high hydrophobicity of the hexanoyl and benzoyl groups prevents the inclusion of pyrene molecules inside the cyclodextrin cavity.     

Influence of Poly(ethylenimine) on the Monolayer of Oleic Acid at the Air/Water Interface

The effect of poly(ethylenimine) (PEI) dissolved in water on the surface pressure-area isotherm of oleic acid (OA) at the air/water interface was investigated. On a concentrated PEI solution, the isotherm of the OA monolayers exhibited a noticeable difference as a function of subphase pH. PEI caused the collapse pressure of the OA monolayer to increase up to 45 mN/m, due to a stronger acid-base-type interaction occurring between the amine group of the PEI and the carboxyl group of OA; on a pure water subphase, the collapse pressure was;28 mN/m. On the other hand, owing to a stronger OA-PEI interaction, the OA monolayers favored a liquid-expanded state more on the PEI-containing water subphase than on the pure water. From the QCM measurement, each OA molecule appeared to interact, on average, with 4.3-5.8 ethylenimine repeating units at basic pHs. We also found that OA multilayers could be assembled on a hydrophilic substrate by a Z-type Langmuir-Blodgett (LB) deposition in a PEI-containing subphase at basic pHs. The ATR-IR spectral data revealed that, in a Z-type LB film, the headgroup of OA was mostly present as carboxylate, interacting in an ionic state with the protonated amine groups of PEI. In acidic conditions, neither a Y-type nor a Z-type deposition was really accomplished. Nonetheless, the ATR-IR spectral data suggested that OA molecules should exist in a monomeric state in a LB film assembled at acidic pHs without PEI while they would form intermolecular hydrogen bridges and/or dimers in the presence of PEI. Copyright 2000 Academic Press.     

Polyreactions in ordered systems: Polymerization of octadecyl methacrylate in monolayers at the gas–water interface

The ultraviolet-initiated polymerization of octadecyl methacrylate (ODMA, octadecyl 2-methyl-2-propenoate) as a monomolecular layer at the gas-water interface was studied. The polymerization was carried out at 27°C at the nitrogen-water interface; air inhibits the polymerization. At 27°C the ODMA monolayer exhibits three different states which were characterized by surface pressure-area diagrams and by surface potential measurements. The ODMA monolayer was polymerized under constant surface pressure in the range between 0 and 10 dyne/cm. The polymerization was followed by recording the contraction of the film. The conversion was determined by comparison of the area per monomer unit during polymerization with the area of mixed monolayers formed from ODMA and poly-ODMA as well as by infrared spectroscopy. The polymerization in the condensed state of the monolayer (at high surface pressure) is considerably faster than in the expanded state (at low surface pressure). As the polymer has a condensing effect on the monomer in the mixed monolayer, autocatalysis is suggested at low surface pressures. The resulting polymer was studied by x-ray diffraction. The structure of the freshly collapsed film is determined by the conformation of the polymer at the interface; the data after crystallizing the same sample from the melt point to higher tacticity of the poly-ODMA prepared in monolayers than that of poly-ODMA prepared by normal radical polymerization in solution.