Aristotelis Xenakis

Bioorganic reactions in microemulsions: the case of lipases

Water-in-oil microemulsions, or reverse micelles, are being evaluated as a reaction medium for a variety of enzymatic reactions. These systems have many potential biotechnological applications. Important examples are the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. This review illustrates the biotechnological applications of microemulsions as media for bioorganic reactions. The principal focus is on lipase catalyzed processes.

Microemulsion-based organogels as matrices for lipase immobilization

Organogels based on water-in-oil microemulsions can be formed using various natural polymers such as gelatin, agar or cellulose derivatives. Enzymes entrapped in the water core of the microemulsion can keep their activity and enhance their stability within the gel matrix. The importance of the microemulsion based organogels (MBGs) leans on their numerous potential biotechnological applications. An important example is the use of various lipase microemulsion systems for hydrolytic or synthetic reactions. In this review, several MBGs are being evaluated as immobilization matrices for various enzymes. The main subject focuses on the parameters that affect the use of MBGs as media for bioorganic reactions using lipases as catalysts.

Biocompatible microemulsions based on limonene: formulation, structure, and applications.

The preparation of biocompatible (w/o) microemulsions based on R-(+)-limonene, water, and a mixture of lecithin and either 1-propanol or 1,2-propanediol as emulsifiers was considered. The choice of the compositions of the microemulsions used was based on the pseudo-ternary phase diagrams of the four-component system determined at 30 degrees C for different weight ratios of the components. When 1-propanol was considered as co-surfactant, the area of the microemulsion zone was remarkably increased. Interfacial properties and the dynamic structure of the emulsifiers monolayer were studied by electron paramagnetic resonance (EPR) spectroscopy using the spin-labeling technique. The rigidity and polarity of the interface were affected by the nature of the alcohol used as co-surfactant. When 1-propanol was used, the emulsifiers interface was much more flexible, indicating a less tight packing of lecithin molecules than in the case of 1,2-propanediol. In addition, the membranes polarity was decreased when the diol was added as co-surfactant in the microemulsion system. To evaluate the size of the dispersed aqueous domains as a function of water content and other additives concentration, dynamic light scattering (DLS) measurements were carried out. Radii in the range from 60 to 180 nm were observed when 1-propanol was used as co-surfactant, and the water content varied from 0 to 12% w/w. Electrical conductivity measurements of R-(+)-limonene/lecithin/1-propanol/water microemulsions with increasing weight fractions of water indicated the appearance of a percolation threshold at water content above 4% w/w. Lipase from Rhizomucor miehei was solubilized in the aqueous domains of the biocompatible microemulsions, and the esterification of octanoic, dodecanoic, and hexadecanoic acids with the short-chained alcohols used as co-surfactants for the formulation of microemulsions was studied. The enzyme efficiency was affected by the chain length of the carboxylic acids and the nature of the alcohol. In the case of 1-propanol, a preference for the long-chain carboxylic acids was observed. On the contrary, when 1,2-propanediol was used formulation of the corresponding esters was not observed. This behavior could be possibly attributed to either the specificity of the lipase toward the alcohol employed for the esterification of the acids or the structural changes induced in the system when 1-propanol was replaced by 1,2-propanediol.

Characterization of a 13-lipoxygenase from virgin olive oil and oil bodies of olive endosperms

Olive oil is one of the oldest known vegetable oils, and it is almost unique in that it can be consumed without any refining treatment. One of its most important quality problems is oxidative rancidity due to the oxygenation of polyenoic fatty acids and formation of compounds that derive from these fatty acid hydroperoxides. Beside autoxidation, lipoxygenases (LOXs) were suggested to be involved in this process. Here we show, that approximately 1.6% of all linoleic acid (LA) molecules within olive oil samples had been converted into LOX-derived (13S,9Z,11E)-13-hydroperoxy-9,11-octadecadienoic acid (13-HPODE) as determined by 1H NMR- and HPLC analysis. LOX activity tests indicated the occurrence of an active 13-LOX exhibiting a pH optimum between pH 5.5 and 6.0. Furthermore, this enzyme preferentially metabolized free fatty acids. In order to elucidate the origin of this LOX, we analyzed olive endosperms for LOX forms. Chromatography of total protein extracts of the tissue showed LOX activity almost exclusively associated with a high molecular mass fraction. Light microscopic inspection, as well as the calculated phosphate, neutral lipid, and protein content of this fraction, suggested that this fraction may contain oil bodies and that LOX activity was associated with their membrane. This LOX activity had a pH optimum of 6.0. Activity assays at various temperatures indicated a significant catalytic efficiency of the enzyme up to 55°C. HPLC analysis of LA oxygenation products within the lipid fraction and of activity tests of isolated oil bodies showed that the LOX present in mature olive endosperm oil bodies was, as the enzyme from olive oil, a linoleate 13-LOX preferentially active on free LA. We suggest, that this oil body LOX from olive endosperm, is the one detected originally in olive oil and may survive at least in part olive oil production. Charakterisierung einer 13-Lipoxygenase aus kalt geprestem Olivenol und Olkorpern des Endosperms von Oliven Olivenol ist eines der altesten bekannten Pflanzenole, und es ist darin einzigartig, das es ohne Reinigung direkt konsumiert werden kann. Eines der wichtigsten Qualitatsprobleme ist, das es ranzig wird durch die Oxidation von Polyenfettsauren und der Bildung von Verbindungen, die aus diesen Fettsaurehydroperoxiden entstehen konnen. Neben der Autoxidation wurde die Beteiligung einer Lipoxygenase (LOX) vorgeschlagen. Hier zeigen wir mittels 1H-NMR- und HPLC-Analysen, das Olivenol ca. 1.6% (13S,9Z,11E)-13-Hydroperoxy-9,11-octadecadiensaure (13-HPODE) enthalt, welche durch eine LOX-katalysierte Umsetzung von Linolsaure entsteht. Durch LOX-Aktivitatstests konnte das pH-Optimum dieser 13-LOX fur pH 5,5 bis 6,0 bestimmt werden. Daruber hinaus zeigte sich eine Praferenz des Enzyms fur freie Fettsauren. Zur Analyse der Herkunft dieser LOX wurde das Endosperm von Oliven auf das Vorkommen von LOX-Formen untersucht. Zunachst wurde durch chromatographische Auftrennung von Proteinextrakten dieses Gewebes gezeigt, das LOX-Aktivitat nahezu ausschlieslich mit einer hochmolekularen Fraktion assoziiert ist. Lichtmikroskopische Analysen dieser Fraktion in Kombination mit der Bestimmung des Phosphat-, Lipid- und Proteingehalts, deuteten auf eine Assoziation der LOX-Aktivitat mit der Membran von Olkorpern hin. Das pH-Optimum dieser LOX-Aktivitat wurde fur 6,0 bestimmt, und Aktivitatsbestimmungen bei verschiedenen Temperaturen zeigten eine hohe Stabilitat des Enzyms bis zu einer Temperatur von 55°C. Auch diese LOX-Aktivitat konnte durch HPLC-Analysen der Produkte, analog zu der aus Olivenol, als eine 13-LOX mit Praferenz fur freie Fettsauren identifiziert werden. Wir schlagen daher vor, das es sich bei der Olkorper-LOX des Endosperms von Oliven und der LOX aus Olivenol um dasselbe Enzym handelt und seine katalytische Aktivitat beim Herstellungsprozes von Olivenol zumindest teilweise erhalten bleibt.

Biocatalysis using microemulsion-based polymer gels containing lipase

Abstract Natural gelling agents such as gelatin, agar and κ-carrageenan have been tested for the formation of lecithin microemulsion-based gels as well as hydrogels (without surfactant and oil). The results presented in this work provide information concerning the utility of these solid gels as lipase immobilization matrices. It was found that lipase from Pseudomonas cepacia keeps its catalytic function after entrapment in the gels, catalyzing the esterification reaction of propanol with lauric acid in various hydrocarbons at room temperature. Various parameters which affect the lipase catalytic behavior such as the nature and the concentration of the gelling agent, as well as the concentration of the biocatalyst and the mole ratio of the substrates have been examined. High yields (80%) were obtained with agar and κ-carrageenan organogels in isooctane. The remaining lipase activity, in repeated syntheses was found to depend on the nature of the biopolymer used for the formation of the organogels. Gelatin and agar microemulsion-based gels showed the highest operational stability.

Characterization of cephalexin loaded nonionic microemulsions.

Water/propylene glycol/sucrose laurate/ethoxylated mono-di-glyceride/isopropyl myristate/peppermint oil U-type microemulsions were used to solubilize cephalexin. Microemulsion dilution and interfacial factors contributing to the cephalexin solubilization were evaluated. Cephalexin solubilization capacity increases with the increase in the aqueous phase volume fraction (φ) up to 0.4 then decreases. Electrical conductivity of drug loaded and drug free microemulsions increases with φ. The hydrodynamic radius measured by dynamic light scattering of the oil-in-water loaded microemulsions decreases with temperature. The microemulsions were characterized by the volumetric parameters, density, excess volume, ultrasonic velocity and isentropic compressibility. The microemulsion densities increase with φ up to 0.8 then decrease. The excess volume decreases with φ up to 0.8 then stabilizes. Ultrasonic velocities increase with the increase in φ while isentropic compressibility decreases. Analysis of the volumetric parameters enabled the characterization of structural transition along the microemulsion phase region. The presence of water-in-oil, bicontinuous and oil-in-water microemulsions, at aqueous phase volume fractions below 0.2, between 0.3 and 0.7 and above 0.8, respectively were found. Interfacial properties and dynamic structure of the monolayer for drug loaded and drug free microemulsions, were studied by electron paramagnetic resonance spectroscopy employing the nitroxide spin probe 5-doxylstearic acid. The rigidity of the interface was affected by the water content and also the presence of cephalexin.

A comparison of different strategies for lipase-catalyzed synthesis of partial glycerides

SummaryFour different approaches for the synthesis of monolaurylglycerol (MLG) by non specificPseudomonas cepacia lipase in a crude and purified form have been studied: a. The direct esterification of glycerol by lauric acid in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsion systems; b. the transesterification of glycerol by vinyl laurate in the presence or not of any solvent; c. solid-phase glycerolysis of trilaurin; and, d. transesterification of protected glycerol, 1,2-O-isopropylidene glycerol, by vinyl laurate, in the presence or not of any solvent. It was found that in the two latter cases (d and c) the formation of pure MLG was occurred, while in the first two cases (a and b) apart of MLG the formation of DLG was also observed.

Activity and Stability Studies Of Mucor miehei Lipase Immobilized in Novel Microemulsion-based Organogels

Lipase from Mucor miehei was immobilized in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT) as well as lecithin water-in-oil (w/o) microemulsion-based organogels (MBGs) formulated with biopolymers such as agar and hydroxypropylmethyl cellulose (HPMC), respectively. These lipase-containing MBGs prove to be novel solid-phase catalysts for use in organic media. Using these organogels at 25°C, various esterification reactions in non-polar solvents as well as in solvent free systems were possible. Apparent lipase activity was influenced to some extent by the nature and the concentration of biopolymers used. Lipase stability in such MBGs is much higher than that observed in w/o microemulsions. MBGs containing lipase functioned effectively in repeated batch syntheses of fatty esters. Kinetic studies have shown that ester synthesis catalyzed by immobilized lipase occurs via the Ping-Pong bi-bi mechanism in which only inhibition by excess of alcohol has been identified. Values of all kinetic parameters were determined.

Enantiomeric selectivity of a lipase from Penicillium simplicissimum in the esterification of menthol in microemulsions

SummaryLipase from Penicillium simplicissimum catalyzes the stereospecific esterification of menthol with fatty acids. The studies on the specificity of this new lipase were carried out using (+), (-) and racemic menthol with water soluble enzyme entrapped in microemulsion systems stabilized with sodium(bis-2-ethylhexyl)sulfosuccinate (AOT) as surfactant, in isooctane. Microemulsions appear to be an effective and fast system for racemic resolution of alcohols.

Hydrophilic matrices as carriers in felodipine solid dispersion systems

Hydrophilic matrices or hydrocolloids are polymers which swell on contact with aqueous solutions and dissolve slowly from the surface forming a gel mass. Several studies have been carried out in the past few years on the use of hydro-colloids in controlled release formulations. The present study used three modified celluloses, carboxymethyl cellulose sodium, hydroxyethyl cellulose (HEC), and hydroxypro-pylmethyl cellulose (HPMC) in systems using the dihydropyridine felodipine, which is slightly soluble in water, as the active ingredient. This study was concerned with solid dispersions, which were prepared following the dissolution method using a common solvent. The drug—polymer interactions were studied using differential scanning calorimetry and IR techniques, as well as high-performance liquid Chromatographic purity after storage in strength conditions. Neither significant interactions nor degradation of the active ingredient was observed after storage at 40 °C for 3 months. In addition, felodipine release from the solid dispersion systems was studied and the factors influencing release, such as the drug—polymer ratio, interactions, and polymer properties were investigated. HPMC was observed to promote a more significant retard and a more linear release of the active ingredient than HEC. Finally, the natural mixtures presented a larger variation and high relative standard deviation values.