Extractive Synthesis of Ethyl‐Oleate Using Alginate Gel Co‐Entrapped Yeast Cells and Lipase Enzyme

To synthesize ethyl‐oleate ester, a complex Ca‐alginate gel co‐entrapped system was prepared. The gel beads contained two kinds of biocatalysts (living yeast cells and a lipase enzyme) and various amounts of glucose (100–400 g/L). These alginate beads dispersed directly in pure oleic acid. To follow the bioconversion of the cell growth, the glucose uptake of yeast cells, the concentration of ethanol inside the gel beads and the ethyl‐oleate concentration in oleic acid phase was monitored. The glucose was quantitatively taken up by yeast cells during 24–72 h, depending on the concentration of glucose. After this 24–72‐hour period, the glucose uptake was stopped. In accordance with changes in glucose concentration, the concentration of ethanol and ethyl‐oleate increased rapidly during the first day of fermentation and thereafter slowed down. It is supposed that the inhibitory effect of produced ethanol would be resolved by co‐immobilization of lipase in the same gel particles. Using lipase, one is able to transform ethanol to ethyl‐oleate, which is soluble in oleic acid. According to the data obtained a minimum of 4 U/mL lipase is required to increase ethyl‐oleate production significantly. Summing up it can be concluded that by means of this system a maximum yield of ethanol and ethyl‐oleate was achieved when gel beads containing 100 g/L glucose and 4 U/mL lipase enzyme were used.     

Lipase-Catalyzed Synthesis and Hydrolysis of Cholesterol Oleate in Aot/Isooctane Microemulsions

We have examined a lipase-catalyzed bidirectional ester synthesis/hydrolysis reaction in a water-in-oil microemulsion system. The reactants were cholesterol (alcohol), oleic acid (acid) and cholesterol oleate (ester), and the solvent system consisted of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water. The reactions were assayed by using [³H]oleic acid, [³H]cholesterol, or [³H]cholesterol oleate for the synthesis and hydrolysis reactions, respectively (separate incubations). The lipase that we used derived from Candida cylindracea, and was used at a concentration of 0.1mg/ml microemulsion. The reactions were performed at 22°C as the reactions proceeded more slowly at higher temperatures. With the initial reactant concentrations set to 10 mM cholesterol, 1 min oleic acid, and 1 mM cholesterol oleate, it was observed that the optimal [H₂O]/[AOT] ratio was at about 9 both for the esterification reaction and for the hydrolysis reaction (after 24 h). The hydrolysis reaction was slower than the synthesis reaction at all [H₂O]/[AOT] ratios studied (0-20), but the difference in reaction yield for the synthesis and the hydrolysis reactions became smaller as the reaction time increased (up to 11 days). When the reaction yield was followed as a time function, it was observed that about 80% of the oleic acid was esterified within 3 days of reaction ([H₂O]/[AOT] ratio of 6), whereas the corresponding value of 80% hydrolysis of cholesterol oleate was reached within 11 days. The results of the present study indicate that by choosing optimal reactant concentrations and reaction conditions, it is at least in part possible to determine the direction of the lipase-catalyzed synthesis/hydrolysis reaction.     

Acid Carboxypeptidase-like Activity Contaminating Proctase B

Ester synthesis by the purified lipase from Pseudomonas fragi 22.39 B was investigated. The lipase could synthesize esters from oleic acid and primary or secondary alcohols, but it did not react with tertiary alcohols. Also, the enzyme could use the fatty acids with straight carbon chains as substrates. The activity was enhanced by increasing the carbon number of the fatty acid, but this is not the case for alcohol. The lipase synthesized glycerides from glycerol and oleic acid. 1(3)-Monoolein and 1,3-diolein were the main products and triolein was minor. Synthesis of monoester such as butyl oleate was scarcely affected by the water content in the reaction mixture, while that of glyceride of oleic acid was much affected.     

Computational approach to solvent-free synthesis of ethyl oleate using Candida rugosa and Candida antarctica B Lipases. I. Interfacial activation and substrate (ethanol, oleic acid) adsorption

This paper presents the results of a MM2 study of the adsorption of oleic acid and ethanol/water in the tunnel and active-site models of lipases from Candida rugosa and Candida antarctica B. The role of an interface polar/no polar in the opening of C. rugosa lipase's lid is also addressed, discussed and analyzed at the level of the conformational changes needed to achieve the lipase open form. The adsorption of oleic acid and alcohols considering C. antarctica B, a lipase not interfacially activated, is also presented. In this case, the tunnel is shorter than in case of C. rugosa lipase. Two different pockets can be visualized at the active site-tunnel model of C. antarctica B lipase: one for the acyl group and another for the alcohol. Wrong location of alcohol and oleic acid severely hinders reaction because it hinders the H-transfer to histidine, a key step in the reaction mechanism. Right location of alcohol decreases the possibility of alcohol inhibition. In the case of C. rugosa, no restrictions for ethanol/water location are found. For that lipase, a second adsorption site for oleic acid (outside the tunnel) is presented. This site is the exit tunnel of the ester product when oleic acid is adsorbed in the tunnel. Experimental results of our own that correlate with this study are presented.     

Growth and shape transformations of giant phospholipid vesicles upon interaction with an aqueous oleic acid suspension

The interaction of two types of vesicle systems was investigated: micrometer-sized, giant unilamellar vesicles (GUVs) formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and submicrometer-sized, large unilamellar vesicles (LUVs) formed from oleic acid and oleate, both in a buffered aqueous solution (pH 8.8). Individual POPC GUVs were transferred with a micropipette into a suspension of oleic acid/oleate LUVs, and the shape changes of the GUVs were monitored using optical microscopy. The behavior of POPC GUVs upon transfer into a 0.8 mM suspension of oleic acid, in which oleic acid/oleate forms vesicular bilayer structures, was qualitatively different from the behavior upon transfer into a 0.3 mM suspension of oleic acid/oleate, in which oleic acid/oleate is predominantly present in the form of monomers and possibly non-vesicular aggregates. In both cases, changes in vesicle morphology were observed within tens of seconds after the transfer. After an initial increase of the vesicle cross-section, the vesicle started to evaginate, spawning dozens of satellite vesicles connected to the mother vesicle with narrow necks or tethers. In 60% of the cases of transfer into a 0.8 mM oleic acid suspension, the evagination process reversed and proceeded to the point where the membrane formed invaginations. In some of these cases, several consecutive transitions between invaginated and evaginated shapes were observed. In the remaining 40% of the cases of transfer into the 0.8 mM oleic acid suspension and in all cases of vesicle transfer into the 0.3 mM oleic acid suspension, no invaginations nor subsequent evaginations were observed. An interpretation of the observed vesicle shape transformation on the basis of the bilayer-couple model is proposed, which takes into account uptake of oleic acid/oleate molecules by the POPC vesicles, oleic acid flip-flop processes and transient pore formation.     

Erratum to: Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40?°C, geraniol to oleic acid molar ratio of 5:1, 150?rpm and 10?wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5?wt%) and temperature of 50?°C afforded nearly complete reaction conversion after 1?h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

Ultrasonication enhanced hydrolytic activity of lipase in water/isooctane two-phase systems

The hydrolysis of olive oil catalyzed by Chromobacterium viscosum lipase (EC 3.1.1.3) in a water/isooctane two-phase system was carried out both under ultrasound and conventional stirring. The maximum activity of lipase in the ultrasonicated system was 1.75 times higher than that in the stirred system. The lipase activity was dependent on ultrasonic power and volume ratio of isooctane to water. The optimum reaction temperature in both systems was around 25°C. The stability of lipase at 25°C in the ultrasonicated system decreased more rapidly than that in the stirred system. In the presence of exogenous oleic acid, however the half-life of lipase in the ultrasonicated system was improved to a value, which was respectively half and twice of that in stirred systems with and without oleic acid. The maximum reaction rate (V_max) was increased by ultrasonication whereas the Michaelis constant (K_m) remained unaltered.     

Solvent-free geranyl oleate production by enzymatic esterification

This study reports the maximization of geranyl oleate production by esterification of geraniol and oleic acid in a solvent-free system using a commercial lipase as catalyst. The operating conditions that maximized geranyl oleate production were determined to be 40 °C, geraniol to oleic acid molar ratio of 5:1, 150 rpm and 10 wt% of enzyme, with a resulting reaction conversion of about 93%. After determining the best reaction parameters, a kinetic study was performed and the results obtained in this step allow to conclude that an excess of alcohol (alcohol to acid molar ratio of 5:1), relatively low enzyme concentration (5 wt%) and temperature of 50 °C afforded nearly complete reaction conversion after 1 h of reaction. New experimental data on enzymatic esterification of geraniol and oleic acid for geranyl oleate production are reported in this work, showing a promising perspective of the technique to overcome the inconvenience of the chemical-catalyzed route.     

First lipase-catalysed synthesis of fatty carbonate esters

Summary Ethyl carbonate reacts with fatty acids and alcohols in the presence of an immobilized lipase. The biocatalyst converts oleic acid into ethyl oleate almost quantitatively. From octadecenol, the main product is ethyl octadecenyl carbonate. These experiments show that lipase is capable of catalytic activity in carbonic series.     

Ethyl oleate synthesis using Candida rugosa lipase in a solvent-free system. Role of hydrophobic interactions

The solvent-free esterification reaction of a commercial oleic acid and ethanol was selected as the test reaction for Candida rugosa lipase immobilized on polypropylene (PP) at 318 K (initial molar ratio 1:1). Adding of water from 0 to 30 wt. % (in gram per gram of fatty acid x 100) and the pretreatment of Candida rugosa lipase with polyethylenglycol (PEG), octane, and acetone increases the conversion to ethyl esters. The role of hydrophobic interactions of the lipase with PP and PEG was studied using molecular mechanics (MM2) for calculation of steric energies and the parametrized model (PM3) for calculation of enthalpy changes upon interaction. The nonpolar lateral groups of amino acids interact strongly with PP, whereas polar groups interact more strongly with PEG. Both interactions stabilize the open, active conformation of the lipase from Candida rugosa. Activities ranged from 5 x 10(-5) to 2.0 x 10(-4) mol ethyl oleate/h/mg enzyme, depending on reaction conditions. Steric energy changes vary between +30 and -10 kcal/mol, whereas the enthalpy changes ranged from +10 to -10 kcal/mol.     

Visualization by freeze fracture, in vitro and in vivo, of the products of fat digestion

The technique of freeze fracture was used to visualize triglyceride (TG) hydrolysis and the production of lipolytic products (LPs) in vitro and in vivo in the presence of bile salts (BS). Three systems were investigated: pure lipolytic products (oleic acid and monoolein) in the presence of a pure bile salt (taurodeoxycholate (TDC)), lipolytic products produced from TG by pancreatic lipase in the presence of a variety of bile salts, and lipolytic products produced in the intestine of the killifish, Fundulus heteroclitus, after fat feeding. In vitro, lamellae (4-5 nm thick with 0-8-nm water spacings) appeared on the surface of TG droplets in all preparations with LP/BS molar ratios of 1.5 or greater and spherical vesicles (diameter range, 20-130 nm) were produced from these lamellae. With model killifish bile (taurocholate-cholate 1:1) at LP/BS ratios between 1.5 and 4, homogeneous vesicles or particles (mean diameter, 23.8 nm) were produced by lipase at pH 6.9. In vivo, lamellar product phases also occurred after fat feeding. The smallest visible LP/BS structures by freeze fracture electron microscopy were approximately 20 nm globular particles. Large disc-shaped micelles either were not present or were below the resolution limit of the replica (approximately 10 nm). The dominant aggregated lipolytic product phase was composed of multiple layers of rough-textured lamellae. No evidence of cubic structure was seen. These results show that lamellar and vesicular lipolytic product phases can be intermediates in intestinal fat digestion. However, no evidence for the direct endocytotic absorption of these product phases by the intestinal microvillus membrane was found.     

Enzymatic synthesis of cetyl oleate in a solvent-free medium using microwave irradiation and physicochemical evaluation

Abstract

A cosmetic ester, cetyl oleate was synthesized using microwave irradiated system. The esterification reaction was carried using Candida antarctica lipase B in a solvent-free media. The influence of various reaction parameters was studied, and the efficiency of Fermase CALB^TM10000 was compared with other enzymes. Equilibrium conversion of 97.5% was obtained within 20?min at 60?°C temperature, 1:2 oleic acid to cetyl alcohol molar ratio and 4% w/w dose of lipase. A comparative study showed that microwave irradiation is a much more efficient method than ultrasound irradiation and conventional heating. Fermase CALB^TM10000 was reusable over 6 enzymatic cycles as its stability improved under microwave system. Physicochemical parameters of cetyl oleate were tested in order to analyze its suitability for further cosmetic use.     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6 h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40-60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

CLEAs of Candida antarctica lipase B (CALB) with a bovine serum albumin (BSA) cofeeder core: Study of their catalytic activity

Highly active CALB cross-linked enzyme aggregates (CLEAs) were synthesized using a layered methodology based on the synthesis of a cross-linked protein cofeeder core over which an external layer of lipase was later cross-linked. The layered CALB CLEAs were characterized in terms of their catalytic activity in three different test reactions: esterification of oleic acid and ethanol in absence of solvents, esterification of oleic acid and heptanol in organic medium, and hydrolysis of triolein in emulsioned medium. The impact of the cross-linker/protein mass ratio on CLEAs activity, and its evolution with storage time were evaluated in the solventless synthesis of ethyloleate. The amount of cross-linker used showed to be a key parameter for the evolution of the catalytic activity of CLEAs during storage. Under the best conditions found, hyperactivated CALB CLEAs with up to 188% of recovered activity in ethyl oleate synthesis were obtained. In terms of hydrolytic activity mature layered CALB CLEAs showed a retained activity of 68%. The assay of dried mature layered CALB CLEAs in heptyl oleate synthesis showed catalytic activities much higher than the one exhibited by free CALB, reaching 1 h-fatty acid conversions of 14% and 2%, respectively. The high catalytic activity shown by layered CALB CLEAs, suggests that they are an interesting alternative specially for the catalysis of fatty acid esterifications in both organic and solventless medium.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Lysozyme effect on oleic acid/oleate vesicles

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.     

Pancreatic cholesterol esterases. 2. Purification and characterization of human pancreatic fatty acid ethyl ester synthase

Human pancreatic fatty acid ethyl ester synthase has been isolated and purified 1200-fold to homogeneity, and its activities, binding properties, and N-terminal amino acid sequence indicate that it is a member of the lipase family. This 52-kDa monomeric protein is present at 0.6-1.2 mg/g of pancreas, and it catalyzes the synthesis and hydrolysis of ethyl oleate at rates of 2400 nmol mg-1 h-1 and 30 nmol mg-1 h-1, respectively. Kinetic analyses reveal a pronounced substrate specificity for unsaturated octadecanoic fatty acids, with ethyl ester synthetic rates of 2400 nmol mg-1 h-1 (linoleic), 2400 nmol mg-1 h-1 (oleic), 400 nmol mg-1 h-1 (arachidonic), 300 nmol mg-1 h-1 (palmitic), and 100 nmol mg-1 h-1 (stearic). Like cholesterol esterase, the enzyme binds to immobilized heparin, and this property was critical for its purification to homogeneity. Its N-terminal amino acid sequence is virtually identical with that reported for human triglyceride lipase, NH2-X-Glu-Val-Cys-5Tyr-Glu-Arg-Leu-Gly-10Cys-Phe-Ser-Asp- Asp-15Ser-Pro-Trp-Ser-Gly-20Ile, and it differs by only four residues from that reported for porcine pancreatic lipase. The synthase purified here also cleaves triglycerides, hydrolyzing triolein at a rate of 30 nmol mg-1 h-1, and this activity is stimulated by colipase and inhibited by sodium chloride. Conversely, commercially available porcine triglyceride lipase exhibits fatty acid ethyl ester synthase activity (1530 nmol mg-1 h-1) and hydrolyzes triolein at a rate of 23 nmol mg-1 h-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemo-enzymatic epoxidation of oleic acid and methyl oleate in solvent-free medium

Chemo-enzymatic epoxidation of oleic acid (OA) and its methyl ester has been performed using hydrogen peroxide and immobilized lipase from Candida antarctica (Novozym® 435). The purpose of the study was to characterize the reaction under solvent-free conditions. The reaction temperature had a significant impact on epoxidation of OA. At lower temperatures, the substrate conversion was hindered by the formation of solid epoxystearic acid product. Nearly 90% conversion of OA to the epoxide product was obtained after 6?h at 50°C. Longer reaction times at 40°C and above resulted in by-product formation and eventually lowered the product yield. In contrast, the reaction with methyl oleate (MO) was less influenced by temperature. Almost complete epoxidation was achieved at 40–60°C; the higher the temperature the shorter was the reaction time. The main epoxidation product obtained was epoxystearic acid methyl ester (EME), and the remaining was epoxystearic acid (EA) formed by the hydrolytic action of the lipase. Recycling of the lipase for epoxidation of MO at 50°C indicated that the immobilized enzyme was prone to activity loss.     

Regulation of fatty acid 18O exchange catalyzed by pancreatic carboxylester lipase. 1. Mechanism and kinetic properties.

The exchange of 18O between H2O and long-chain free fatty acids is catalyzed by pancreatic carboxylester lipase (EC 1.1.1.13). For palmitic, oleic, and arachidonic acid in aqueous suspension and for 13,16-cis,cis-docosadienoic acid (DA) in monomolecular films, carboxyl oxygens were completely exchanged with water oxygens of the bulk aqueous phase. With enzyme at either substrate or catalytic concentrations in the argon-buffer interface, the exchange of DA oxygens obeyed a random sequential mechanism, i.e., 18O,18O-DA in equilibrium with 18O,16O-DA in equilibrium with 16O,16O-DA. This indicates that the dissociation of the enzyme-DA complex is much faster than the rate-limiting step in the overall exchange reaction. Kinetic analysis of 18O exchange showed a first-order dependence on surface enzyme and DA concentrations, i.e., the reaction was limited by the acylation rate. The values of kcat/Km, 0.118 cm2 pmol-1 s-1, for the exchange reaction was comparable to that for methyl oleate hydrolysis and 5-fold higher than that for cholesteryl oleate hydrolysis in monolayers [Bhat, S., & Brockman, H. L. (1982) Biochemistry 21, 1547]. Thus, fatty acids are good "substrates" for carboxylester lipase. With substrate levels of carboxylester lipase in the interfacial phase, the acylation rate constant kcat/Km was 200-fold lower than that obtained with catalytic levels of enzyme. This suggests a possible restriction of substrate diffusion in the protein-covered substrate monolayer.     

Lysozyme Effect on Oleic Acid/Oleate Vesicles

We have analysed by means of turbidimetric, dynamic light scattering (DLS), and fluorimetric techniques the effect of lysozyme on negatively charged oleic acid/oleate vesicles. The addition of lysozyme brings about a decrease in optical density of the vesicle population, which finally results in a size distribution of oleate vesicles shifted toward smaller mean diameters. On the contrary, (a) when phosphatidylserine vesicles were used, lysozyme induces an increase of turbidity and a shift toward larger vesicle sizes; and (b) the addition of histone H1 or poly-L-lysine produces an aggregative behavior both in oleate and in phosphatidylserine vesicles. Experiments carried out with calcein-containing vesicles indicate that the observed changes in the lysozyme/oleate system occur with partial leakage of the vesicle content. All this is taken to suggest that the interaction between lysozyme and oleate vesicles is of quite specific nature, and certainly not just due to electrostatic interactions.