Lipase catalyzed esterification of lactic acid

Reactions between lactic acid and alcohols or carboxylic acids catalyzed by lipase from Candida antarctica were evaluated with hexane as solvent. Lactic acid was a good acyl donor and esters of both primary and secondary alcohols were effectively synthesized. No interfering dimer formation due to lactic acid acting as both nucleophile and acyl donor was observed. In agreement with this, no esterification occurred between lactic acid and carboxylic acids.     

Papain catalyzed esterification in polar organic solvents

Summary A wide range of N-substituted amino acids were converted to their corresponding esters by Sephadex G-50 immobilized papain in 95% methanol or other alcohols.     

Lipase catalyzed esterification of glycidol in organic solvents

We studied the resolution of racemic glycidol through esterification with butyric acid catalyzed by porcine pancreatic lipase in organic media. A screening of seven solvents (log P values between 0.49 and 3.0, P being the n-octanol-water partition coefficient of the solvent) showed that neither log P nor the logarithm of the molar solubility of water in the solvent provides good correlations between enantioselectivity and the properties of the organic media. Chloroform was one of the best solvents as regards the enantiomeric purity (e. p.) of the ester produced. In this solvent, the optimum temperature for the reaction was determined to be 35 degrees C. The enzyme exhibited maximum activity at a water content of 13 +/- 2% (w/w). The enantiomeric purity obtained was 83 +/- 2% of (S)-glycidyl butyrate and did not depend on the alcohol concentration or the enzyme water content for values of these parameters up to 200 mM and 25% (w/w), respectively. The reaction was found to follow a BiBi mechanism. (c) 1993 John Wiley & Sons, Inc.     

NMR on-line monitoring of esterification catalyzed by cutinase

A nuclear magnetic resonance (NMR) method has been developed to monitor on-line lipase-catalyzed esterification reactions without the need to sample the reaction medium. The technique, through (1)H NMR, measures the concentrations of alcohol, ester, hydroxylic hydrogens in the organic phase, and hydroxylic hydrogens in the aqueous phase, if any. Also, the chemical shift evolution of the two types of hydroxylic hydrogens has been followed, providing information on water content of the organic phase and on the appearance of a distinct aqueous phase. As far as (13)C NMR is concerned, it has been possible to measure, first the acid and the ester concentrations in the carbonyl region, and second, the alcohol and the ester concentrations in the methylene region. All (1)H and (13)C results are in agreement with one another. Furthermore, NMR allows for the choice of detection zone. Preliminary studies on the solid phase proved the presence of much more water in the solid phase than in the organic phase, and also gave evidence of the existence of two types of esters, one in the organic phase, mainly associated with the acid, and the other one not associated with the acid, most probably entrapped within the solid enzyme.     

Enzyme catalyzed electricity-driven water softening system

Hardness in water, which is caused by divalent cations such as calcium and magnesium ions, presents a major water quality problem. Because hard water must be softened before use in residential applications, there is great interest in the saltless water softening process because, unlike ion exchange softeners, it does not introduce additional ions into water. In this study, a saltless hardness removal driven by bioelectrochemical energy produced through enzymatic oxidation of glucose was proposed and investigated. Glucose dehydrogenase was coated on a carbon electrode to catalyze glucose oxidation in the presence of NAD⁺ as a cofactor/mediator and methylene green as an electrocatalyst. The results showed that electricity generation stimulated hardness removal compared with non-electricity conditions. The enzymatic water softener worked upon a 6 h batch operation per day for eight days, and achieved an average hardness removal of 46% at a high initial concentration of 800 mg/L as CaCO₃. More hardness was removed at a lower initial concentration. For instance, at 200 mg/L as CaCO₃ the enzymatic water softener removed 76.4 ± 4.6% of total hardness. The presence of magnesium ions decreased hardness removal because of its larger hydrated radius than calcium ions. The enzymatic water softener removed 70–80% of total hardness from three actual hard water samples. These results demonstrated a proof-of-concept that enzyme catalyzed electricity generation can be used to soften hard water.     

Kinetic study of lipase catalyzed esterification reactions in water-in-oil microemulsions

The kinetics of the esterification of lauric acid by (-)menthol, catalyzed by Penicillium simplicissimum lipase, was studied in water/bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsions. Due to their low water content, microemulsions assist in reversing the direction of lipase activity, favoring synthetic reactions. The kinetics of this synthesis follows a Ping-Pong Bi--Bi mechanism. The values of all apparent kinetic parameters were determined. The theoretical model for the expression of enzymic activity in reverse micelles, proposed by Verhaert et al. (Verhaert, R., Hilhorst, R., Vermüe, M., Schaafsma, T. J., Veeger, C. 1990. Eur. J. Biochem. 187: 59-72) was extended to express the lipase activity in an esterification reaction involving two hydrophobic substrates in microemulsion systems. The model takes into account the partitioning of the substrates between the various phases and allows the calculation of the intrinsic kinetic constants. The experimental results showing the dependence of the initial velocity on the hydration ratio, W(o) = [H(2)O]/[AOT], of the reverse micelles, were in accordance with the theoretically predicted pattern. (c) 1993 John Wiley & Sons, Inc.     

Enzymes catalyzed esterification of N-protected amino acids with secondary alcohols

Secondary alcohols were converted to their corresponding esters with N-Cbz-L-amino acids by Celite-immobilized protease or lipase in organic solvents at pH 7.5. The esterification of 2-butanol and 2-phenylethanol were achieved up to 71% yields. The optimal reaction condition for Aspergillus oryzae protease (AOP) catalyzed synthesis of N-Cbz-L-aspartyl- sec-butyl ester (97.6 de) was at pH 7.5 and a duration of 96 hours.     

Kinetics of amino acid esterification catalyzed by hydrophobic derivatives of alpha-chymotrypsin

Hexyl-alpha-chymotrypsin, a hydrophobic derivative of the enzyme, is explored for the proteinase-catalyzed ester synthesis reaction with N-acetyl-L-tyrosine and ethanol. To guarantee the preservation of the enzyme activity and to allow for the extraction of the product in the organic phase, a biphasic system was used. The Vm increased for the modified enzyme. This phenomenon was linked to the modification of the protein as demonstrated by its chemical denaturation with sodium dodecyl sulfate.     

Microbial catalyzed esterification of primary and secondary alcohols in organic solvent

Summary Microbial esterification of primary and secondary short chain alcohols with butyric acid in organic solvent has been studied. A screening for 2-octylbutyrate hydrolysis between microorganisms belonging to different genera allowed the selection of 12 microbial strains able to hydrolyze this substrate. The potential of these microorganisms in catalyzing ester formation was checked for various 1- and 2-alkylbutyrate derivatives:Rhizopus delemar,Rhizopus oryzae andSarcina lutea promoted both 1- and 2-alkylbutyrate synthesis with almost complete molar conversion of the primary alcohols, whileAspergillus niger andYarrowia lipolytica only catalyzed 1-alkanol esterification.     

Enzyme studies on the esterification of vitamin D in rat tissues

1. The mechanism of vitamin D esterification in the rat was studied with liver, small-intestinal mucosa, pancreatic juice and blood plasma as enzyme sources and [1-(3)H]cholecalciferol, [U-(14)C]ergocalciferol and [4-(14)C]cholesterol as substrates. 2. No esterification of vitamin D could be detected with liver preparations nor with homogenates or acetone-dried powder extracts of intestinal mucosa. 3. Pancreatic juice esterified [1-(3)H]cholecalciferol with oleic acid, and specificity studies indicated that a cholesterol-esterifying enzyme was using vitamin D as substrate. 4. Plasma cholesterol-esterifying enzyme also esterified vitamin D. 5. The specificity of the esterification reaction is discussed in relation to (a) the molecular structure of the substrates and (b) their availability, in a micellar solution, to the enzyme. 6. It is concluded that cholesterol-esterifying enzymes esterify vitamin D in vivo during absorption from the small intestine and while it is transported in blood.     

Whole cell catalyzed esterification of fatty acids to biodiesel using Aspergillus sp.

Abstract

Powdered biomass of Aspergillus sp. (RBD01) was used to carry out esterification of long chain fatty acids for biodiesel production. Dry biomass of Aspergillus sp. at 20% (w/w) with respect to the fatty acid was able to completely esterify oleic acid to ethyl ester at 35°C, in 36 h, with step wise addition of alcohol. Similar conditions also resulted in yield of 64.5% and 58% of ethyl ester from stearic acid and palmitic acid, respectively. However, the presence of methanol resulted in 87.5%, 71% and 41% of methyl ester from oleic, palmitic and stearic acid. Furthermore, it was found that the efficiency of biomass decreased by only 10% with its repeated use up to four cycles.     

Enantioselective esterification of 2-methylbutyric acid catalyzed via lipase immobilized in microemulsion-based organogels

Chromobacterium viscosum lipase (c.v. lipase) was immobilized in microemulsion-based órganogels and successfully utilized for the enantioselective esterification of (+/?)-2-methylbutynic acid to preferentially form ethyl-(+)-2-methylbutyrate. The reaction time course and enantioselectivity obtained with the organogel—lipase system was compared and contrasted to that achieved in a reversed micellar solution system that contained lipase solubilized in its inner water core as well as that in which powdered lipases were directly dispersed in an organic solvent. The unique properties and potential benefits of the organogel system are discussed. © 1994 Wiley-Liss, Inc.     

Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity

We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide. (c) 1994 John Wiley & Sons, Inc.     

Optimization of the reaction medium for esterification catalyzed by A lipase from Pseudomonas fluorescens

The performance of a crude extract lipase from Pseudomonas fluorescensin esterification was evaluated in microaqueous, biphasic and surfactant-enriched biphasic systems containing various amounts of water (from almost no water to pure water). The results showed a strong negative influence of the water content on the thermodynamic equilibrium of the reaction in biphasic systems. From a kinetic point of view, the enzyme was more efficient in systems involving a water/organic solvent interface (4 times in the biphasic system, 12 times in the surfactant-enriched biphasic system).     

Enzyme catalyzed transesterification of waste cooking oil with dimethyl carbonate

The detrimental effects of waste cooking oil on sewer system attracted attention toward its proper management and reusing this waste oil for making biodiesel provides commercial and environmental advantage. In the present study, biodiesel has been successfully produced from waste cooking oil and dimethyl carbonate by transesterification, instead of the conventional alcohol. In this optimization study, the effect of various reaction conditions such as solvent, time and temperature, molar ratio of DMC to oil, enzyme loading and reusability, on the yield of fatty acid methyl ester (FAME) has been studied. The Maximum conversion of FAMEs achieved was 77.87% under optimum conditions (solvent free system, reaction time of 24 h, 60 °C, molar ratio of DMC to oil 6:1, catalyst amount 10% Novozym 435 (based on the oil weight)). Moreover, there was no obvious loss in the conversion after lipases were reused for 6 batches under optimized conditions.     

Magnetic nanoparticles supported ionic liquids for lipase immobilization: Enzyme activity in catalyzing esterification

Candida rugosa lipase was immobilized on magnetic nanoparticles supported ionic liquids having different cation chain length (C₁, C₄ and C₈) and anions (Cl⁻, BF₄⁻ and PF₆⁻). Magnetic nanoparticles supported ionic liquids were obtained by covalent bonding of ionic liquids–silane on magnetic silica nanoparticles. The particles are superparamagnetic with diameter of about 55 nm. Large amount of lipase (63.89 mg/(100 mg carrier)) was loaded on the support through ionic adsorption. Activity of the immobilized lipase was examined by the catalysis of esterification between oleic acid and butanol. The activity of bound lipase was 118.3% compared to that of the native lipase. Immobilized lipase maintained 60% of its initial activity even when the temperature was up to 80 °C. In addition, immobilized lipase retained 60% of its initial activity after 8 repeated batches reaction, while no activity was detected after 6 cycles for the free enzyme.     

The nature of fatty acid modifies the equilibrium position in the esterification catalyzed by lipase

The equilibrium position in lipase mediated esterification of various fatty acids and butanol was studied. The influence of the chain length and the presence of unsaturations in the fatty acids on the equilibrium position was measured and predicted. To predict equilibrium position the program TREP extended (TREPEX) based on the UNIFAC group contribution method was used. Using an equilibrium constant of 35, calculated on the basis of thermodynamic activities, the equilibrium position between butanol and saturated and/or unsaturated fatty acids with different chain lengths can be predicted. The ester mole fraction at equilibrium increases with the fatty acid chain length, and for fatty acids with the same carbon number, the highest values are found for unsaturated fatty acids. For reaction systems containing two saturated fatty acids, a slightly higher mole fraction is obtained for the fatty acid with the higher chain length, while for mixtures consisting of saturated and unsaturated fatty acids, the mole fractions of the unsaturated esters are lower than those of the saturated ones, regardless the chain length of the fatty acid. These experimental results are in good agreement with the calculations with TREPEX.     

Lipase catalyzed production of monoacylglycerols by the esterification of fish oil fatty acids with glycerol

In this study, we attempted the efficient production of monoacylglycerols (MAG) via the lipase-catalyzed esterification of glycerol with fatty acids obtained from sardine oil. The reaction factors that influenced MAG synthesis were the glycerol to fatty acid mole ratio, amount of enzyme, organic solvent, temperature, and the type of lipase used. Porcine pancreas lipase was selected to catalyze this reaction. The optimum conditions we determined for MAG synthesis were a glycerol to fatty acid mole ratio of 1∶6, 100 mg/mL of lipase, and 30°C in dioxane. Under these conditions, the MAG content was 68% (w/w) after 72 h of reaction. The MAGs synthesized via the lipase-catalyzed esterification of glycerol with fatty acids included monomyristin, monopamiltin, and monoolein, as identified by GCMS.