Lipase-catalyzed synthesis and properties of estolides and their esters

Eight lipases were screened for their ability to synthesize estolides from a mixture that contained lesquerolic (14-hydroxy-11-eicosenoic) acid and octadecenoic acid. With the exception ofAspergillus niger lipase, all 1,3-specific enzymes (fromRhizopus arrhizus andRhizomucor miehei lipases) were unable to synthesize estolides.Candida rugosa andGeotrichum lipases catalyzed estolide formation at >40% yield, with >80% of the estolide formed being monoestolide from one lesquerolic and one octadecenoic acyl group:Pseudomonas sp. lipase synthesized estolides at 62% yield, but the product mixture contained significant amounts of monoestolide with two lesquerolic acyl groups as well as diestolide. ImmobilizedR. miehei lipase was chosen to catalyze the esterification of mono-and polyestolide, derived synthetically from oleic acid, with fatty alcohols or α,ω-diols. Yields were >95% for fatty alcohol reactions and >60% for diol reactions. In addition, the estolide linkage remained intact through the course of the esterification process. Esterification of estolides improved the estolide’s properties—for example, lower viscosity and higher viscosity index—but slightly raised the melting point. Estolides and, particularly, estolide esters may be suitable as lubricants or lubricant additives.     

Enzymatic alcoholysis reaction of soy phospholipids

Lipase-catalyzed alcoholysis of soy phospholipids was investigated to simultaneously make lysophospholipids and fatty acid esters of individual alcohols. Alcoholysis was carried out by stirring a mixture of soy phospholipids and individual alcohols in equimolar proportions with 10% (by weight of reactants) Mucor miehei lipase at 55°C for 24 h. The products were isolated by column chromatography after removal of the lipase. Lysophospholipids (in 69–78% molar yield) were obtained from soy phospholipids, and the yield of esters of various alcohols also conformed nearly with theoretical yields.     

Optimization of lipase-catalyzed sorbitol monoester synthesis in organic medium

The influence of acid/polyol molar ratio and reaction time on lipase-catalyzed esterification of oleic acid (OA) and sorbitol was studied to determine optimal conditions for monoester synthesis. A simple mathematical model was developed to determine relationships between various parameters of technical and/or economical importance. A direct relationship. independent of OA initial concentration and reaction time, was shown between the percentage of monoester in total esters, monoester concentration (the maximum was 25–30 mM for 70–80% monoester), and sorbitol conversion rate. A high sorbitol conversion was always associated with a low percentage of monoester in total ester. No absolute optimum could be found, so that compromises should be chosen, with the help of the results presented herein, depending on the constraints on the process. Two possible optima are proposed. In both examples, monoester (25–30 mM) is 80% pure. In the first case, productivity is maximized (15 mmol·L⁻¹·h⁻¹), but OA and sorbitol conversions are only 40 and 60%, respectively. In the second case, a high OA conversion (>99%) is favored at the expense of monoester productivity (1.8 mmol·L⁻¹·h⁻¹), 60% of the sorbitol being converted. It was shown that, although sorbitol monoester has better surface properties than diester, the addition of 20% diester did not modify the interfacial activity of monoester and slightly increased its surface activity.     

Enzymatic Synthesis of Glucose- and Xylose Laurate Esters Using Different Acyl Donors,Higher Substrate Concentrations,and Membrane Assisted Solvent Recovery

Glucose- and xylose laurate esters are enzymatically synthesized using equimolar substrate concentrations in 2-methyl-2-butanol, comparing free lauric acid with methyl- and vinyl-laurate as acyl donors. All reactions result in ≥70% acyl donor conversions after 72 h but the activated donors are also partially hydrolyzed to lauric acid, highlighting the difficulty in controlling water presence in this particular reaction system. The esterification of xylose generates a complex product profile, with several regioisomers of monoesters and diesters. The esterification of glucose is quite selective, forming mainly the 6-O monoester (≥96%) with a small presence of two diester isomers (4%). Increasing substrate concentration up to 800 millimoles kg⁻¹ results in lower conversion values (down to 58%) but shows that the reaction proceeds successfully even in the presence of high amounts of insoluble glucose. However, the reaction is less selective and the proportion of diester increases, becoming up to 46% (molar fraction) of the final product. Solvent recovery after esterification can be achieved by organic solvent nanofiltration through a polymeric membrane able to retain ≥80% of all reaction substrates and products. Practical Applications: The use of high substrate concentrations during the enzymatic synthesis of sugar ester biosurfactants leads to product titers that are more industrially appealing, without the need to find a solvent that can solubilize all initial substrate. The sustainability of the enzymatic conversion at mild temperatures can be enhanced by recycling of the reaction solvent through organic solvent nanofiltration, an energy efficient alternative to other traditional methods like distillation.     

Enzymatic synthesis of monosaccharide fatty acid esters and their comparison with conventional products

5-O-Acyl-1,2-O-isopropylidene-D-xylofuranose and 6-O-acyl1,2∶3,4-di-O-isopropylidene-D-galactopyranose were enzymatically prepared from the corresponding monosaccharide acetals and commercial (crude) fatty acid mixtures. Subsequent acid-catalyzed hydrolysis of the isopropylidene group(s) gave monosaccharide esters with overall yields of 59–88%, where the monoester content was at least 80% (galactose oleate) and typically 90% for the other preparations. In contrast to sugar fatty acid esters prepared by conventional, high-temperature (trans)esterification, the enzymatically obtained monosaccharide esters contained no appreciable quantities of undersirable side products, and the only contaminants were monosaccharides and fatty acids.     

Substrate preferences for lipase-mediated acyl-exchange reactions with butteroil are concentration-dependent

Substrate preferences for pancreatic lipase-mediated acyl-exchange reactions with butteroil were concentration-dependent for the series of acyl donors and alcohol acceptors evaluated. For acidolysis reactions, the initial reaction rates and percent reaction yields after 18 h at 50 μmol acyl donor per gram substrate mixture were similar forn-fatty acids and their methyl and glycerol esters. At 400–500 μmol g⁻¹ (and greater), order of initial reaction rates and percent reaction yield was fatty acid glycerol esters > fatty acid methyl esters > fatty acids. At concentrations above 300–500 μmol g⁻¹, reaction inhibition was observed for fatty acid substrates, and inhibition took place at lower concentrations for the shorter-chainlength fatty acids of those evaluated (5–17 carbons). Inhibition was primarily attributed to acidification of the microaqueous environment of the lipase. Desorption of water by the fatty acid substrate may be a secondary mode of inhibition. The concentration dependence of initial reaction rates and percent reaction yield was similar for then-alcohol substrates evaluated (2–15 carbons) for alcoholysis reactions with butteroil. Optimum alcohol concentration was 375–500 μmol g⁻¹ (except for butanol, which was 1 mmol g⁻¹, above which reaction inhibition was observed. Inhibition was attributed to desorption of water from the enzyme by the alcohol substrate. Relative reactivity of classes of alcohols for this reaction system was primary alcohols > secondary alcohols > tertiary alcohols. Generally, alcoholysis reactions were faster than acidolysis reactions, and triacylglycerols were the best substrates for acidolysis reactions with butteroil at high levels (up to 2 mmol g⁻¹) of acyl donor substrate.     

Production of propylene glycol fatty acid monoesters by lipase-catalyzed reactions in organic solvents

Fatty acid monoesters of propylene glycol (1,2-propanediol) are good water-in-oil emulsifiers. These esters were synthesized enzymatically to overcome the problems associated with chemical processes. APseudomonas lipase was added to reaction mixtures containing propylene glycol and various acyl donors (fatty acids, fatty acid ethyl esters, fatty acid anhydrides and triglycerides) in organic solvents, and the mixtures were shaken at 30°C. The products were analyzed by gas chromatography. The yield of monoesters was affected by the acyl donors, organic solvents, temperature, water content, pH memory and reaction time. The anhydrous (lyophilized) enzyme and fatty acid anhydrides were best for monoester production. The optimum pH ranges were 4–5 and 8–10. The yields of propylene glycol monolaurate, monomyristate, monopalmitate, monostearate and monooleate with 50 mM fatty acid anhydrides as acyl donors were 97.2, 79.6, 83.7, 89.7 and 93.4 mM, respectively; those with 50 mM fatty acids as acyl donors were 37.3, 28.7, 28.7, 35.3 and 36.2 mM, respectively. The yields of propylene glycol monopalmitate, monostearate and monooleate with 50 mM triglycerides as acyl donors were 87.4, 65.1 and 83.2 mM, respectively.     

Chain-breaking fused heterocyclic antioxidants: Antioxidant activities of 9H-xanthene-2,7-diols and α-tocopherol upon liposomal membranes

We evaluated the antioxidant activities of 9H-xanthene-2,7-diols and α-tocopherol (α-Toc) upon the oxidation of soybean phosphatidylcholine liposomal membranes, induced by 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) and 2,2′-azobis(2,4-dimethylvaleronitrile (AMVN). The stoichiometric factors of 9H-xanthene-2,7-diols, initiated with water-soluble AAPH and lipid-soluble AMVN, were 1.9–2.7 and 1.2–1.8-fold greater than those of α-Toc, respectively. The consumption profile of the antioxidant confirmed that 9H-xanthene-2,7-diol was completely consumed within the induction period (t _inh) and that the 9H-xanthene-2,7-diol oxidation product was formed. When all oxidation product was depleted, t _inh was terminated, and rapid oxidation occurred. These results suggested that the antioxidant activities of 9H-xanthene-2,7-diol depend not only on the initial hydrogen abstraction from 9H-xanthene-2,7-diol but also on a second hydrogen abstraction from the residual phenolic OH group of the oxidation product. Ascorbic acid (AsA) could not scavenge the radicals by itself in the lipid bilayer. However, when 9H-xanthene-2,7-diol was located in the lipid bilayer, the addition of AsA into the aqueous phase prolonged t _inh and reduced the rate of decay of 9H-xanthene-2,7-diol.     

Performance and physicochemical properties of α-sulfo fatty acid methyl esters

The detergency properties of α-sulfonated fatty acid methyl esters (α-SFMe) were evaluated and compared to those of conventional anionic surfactants by using a model heavy-duty detergent formulation. Several physicochemical properties of surfactants were measured to investigate the effective factors on detergency. α-SFMe showed good detergency performance under various washing conditions. These results were considered to correlate well with the good adsorption behavior on oils and dispersing capabilities for particles, as well as with the good solubilization capacity. Solubilization behavior of α-SFMe is discussed in relation to micelle properties. It is suggested that α-SFMe can form suitable micelles for solubilizing polar oils advantageously, due to its bulky hydrophilic group.     

Lipase-catalyzed synthesis of hydroxy stearates and their properties

Hydroxy fatty acid (HFA) esters of long-chain alcohols, such as hydroxy stearates, have potential applications from lubricants to cosmetics. These esters were synthesized enzymatically to overcome the problems associated with chemical processes. An immobilized lipase, Rhizomucor miehei, was employed as catalyst in the esterification reaction between hydroxy-stearic acid as a source of HFA and monohydric fatty alcohols (C₈–C₁₈). The yields of esters were in the range of 82–90% by conducting the reactions at 65±2°C, 2–5 mm Hg pressure, and 10% lipase concentration. The products were analyzed by infrared spectroscopy, and some of their analytical characteristics were determined.     

9H-xanthene-2,7-diols as antioxidants for autoxidation of linoleic acid

The antioxidant activities of 9H-xanthene-2,7-diols and α-tocopherol were studied during the oxidation of linoleic acid in a homogeneous solution and in an aqueous micelle dispersion. The antioxidant activities of 9H-xanthene-2,7-diols for both systems were 1.0–2.4 times greater relative to α-tocopherol. In addition, the 1,3,4,5,6,8-hexamethylxanthene-2,7-diol showed less cytotoxicity toward human fibroblasts than did 2,6-di-t-butyl-4-methylphenol.     

α-Sulfonated fatty acid esters: I. Structural effects of sodium α-sulfonated fatty acid higher alcohol esters on surface-active properties and emulsification ability

The surface-active properties and emulsification ability of sodium α-sulfonated fatty acid esters, C_mH_2m+1CH-(SO₃Na)COOC_nH_2n+1, were studied as a function of the hydrophobic alkyl chainlength in the fatty acid (m=8−16) and the alcohol (n=8−18). As a result, it was discovered that sodium α-sulfonated fatty acid esters have a structural effect on the Krafft point different from that of amphiphiles with short alkyl chains. Moreover, some of the α-sulfonated fatty acid esters have quite low interfacial tensions, as well as non-foaming properties, which depend upon the total (m+n) number of carbon atoms in the alkyl chains.     

Increased rate of lipase-catalyzed saccharide-fatty acid esterification by control of reaction medium

Through proper selection of initial conditions and control of the reaction medium composition, a productivity rate over 10-fold higher than that previously reported was achieved for lipase-catalyzed fructose-oleic acid esterification. From a screening process, tert-butanol (t-BuOH) was selected as the most effective solvent for cosolubilizing fructose and oleic acid during the initial stage of the reaction. A t-BuOH concentration of 0.35–0.55 w/w produced the highest rate and extent of reaction at 60°C. Neither water addition nor removal applied to initial reaction materials improved the rate. Since both fructose-oleic acid mono- and diester promoted higher fructose solubility than either oleic acid or oleic acid/t-BuOH mixtures, t-BuOH was not needed during the latter stage of the reaction. Also, the presence of t-BuOH hindered the removal of water by free evaporation. Thus, complete removal of t-BuOH during the middle-to-latter stage of reaction was found to enhance the reaction rate. In addition, the introduction of fructose to the reactor in small batchwise increments accelerated the reaction. The monoester to diester ratio decreased during the initial and middle stages of the reaction owing to the disappearance of t-BuOH, but increased slightly during the later stages presumably because of the ester formation.     

Identification of α-parinaric acid in the seed oil ofSebastiana brasiliensis sprengel (Euphorbiaceae)

Besides some usual fatty acids, the seed oil ofSabastiana brasiliensis (Euphorbiaceae) contains up to 39% (estimated by ultraviolet spectroscopy) of α-parinaric acid (cis, trans, trans, cis-9, 11, 13, 15-octadecatetraenoic acid). The fatty acids were analyzed by gas chromatography and gas chromatography/mass spectrometry as their methyl esters. The structure of α-parinaric acid was proven by a combination of chemical and spectroscopic methods, conducted with the crude oil, the methyl ester mixture, and the isolated fatty acid methyl ester. Complete assignment of the¹H and¹³C nuclear magnetic resonance (NMR) shifts of α-parinaric acid was carried out by two-dimensional NMR experiments Presented in part at the 21st world Congress and Exhibition of the International Society for Fat Research (ISF), October 1–6, 1995, The Hague, The Netherlands.     

Inhibitory Effect of Conjugated α-Linolenic Acid from Bifidobacteria of Intestinal Origin on SW480 Cancer Cells

In this study, we assessed the ability of six strains of bifidobacteria (previously shown by us to possess the ability to convert linoleic acid to c9, t11-conjugated linoleic acid (CLA) to grow in the presence of α-linolenic acid and to generate conjugated isomers of the fatty acid substrate during fermentation for 42 h. The six strains of bifidobacteria were grown in modified MRS (mMRS) containing α-linolenic acid for 42 h at 37 °C, after which the fatty acid composition of the growth medium was assessed by gas liquid chromatography (GLC). Indeed, following fermentation of one of the strains, namely Bifidobacterium breve NCIMB 702258, in the presence of 0.41 mg/ml α-linolenic acid, 79.1% was converted to the conjugated isomer, C18:3 c9, t11, c15 conjugated α-linolenic acid (CALA). To examine the inhibitory effect of the fermented oils produced, SW480 colon cancer cells were cultured in the presence of the extracted fermented oil (10–50 μg/ml) for 5 days. The data indicate an inhibitory effect on cell growth (p ≤ 0.001) of CALA, with cell numbers reduced by 85% at a concentration of 180 μM, compared with a reduction of only 50% with α-linolenic acid (p ≤ 0.01).     

Enzymatic fractionation of fatty acids: Enrichment of γ-linolenic acid and docosahexaenoic acid by selective esterification catalyzed by lipases

Immobilized lipase preparations from seedlings of rape (Brassica napus L.) andMucor miehei (lipozyme) used as biocatalysts in esterification and hydrolysis reactions discriminate strongly against γ-linolenic and docosahexaenoic acids/acyl moieties. Utilizing this property, γ-linolenic acid contained in fatty acids of evening primrose oil has been enriched seven to nine-fold, from 9.5 to almost 85% by selective esterification of the other fatty acids with butanol. Similarly, docosahexaenoic acid of cod liver oil has been enriched four to five-fold, from 9.4 to 45% by selective esterification of fatty acids (other than docosahexaenoic acid) with butanol. As long as the reaction is stopped before reaching equilibrium, very little of either γ-linolenic acid or docosahexaenoic acid are converted to butyl esters, which results in high yields of these acids in the unesterified fatty acid fraction.     

How is chemical interesterification initiated: Nucleophilic substitution or α-proton abstraction?

Esters of carboxylic acids including 2-methylhexanoic, 2-methylbutyric, 2,2-dimethyl-4-pentenoic, palmitic, and oleic acids were tested as substrates in methoxide-catalyzed interesterification and transesterification. The aliphatic acid esters participated in the ester-ester interchange upon addition of catalytic sodium methoxide. Their isopropyl esters also produced methyl esters on heating with sodium methoxide. The esters of α-methyl-substituted acids did not participate in the ester-ester interchange. Their isopropyl esters did not react with methoxide to produce methyl esters. However, upon addition of methanol with sodium methoxide, their methyl esters were produced. These results indicate that the first step in interesterification is possibly that methoxide abstracts the α-hydrogen of an ester to form a carbanion. Interesterification is then likely completed via a Claisen condensation mechanism involving the β-keto ester anion as the active intermediate. The β-keto ester anion contains positively charged ketone and acyl carbons that are active sites for nucleophilic attack by anions such as methoxide and glycerinate, which would produce a methyl ester or rearrange acyls randomly. On the other hand, transesterification is a nucleophilic substitution by methoxide at the acyl carbon in the presence of methanol.     

Analysis of the dark-colored impurities in sulfonated fatty acid methyl ester

A fractionally distilled C₁₄−C₁₆ fatty acid methyl ester, derived from palm oil, was sulfonated with gaseous SO₃ in a falling film reactor to form an α-sulfo fatty acid methyl ester (α-SF; unbleached and unneutralized form). The included dark-colored impurities were then separated from α-SF as a diethyl ether-insoluble matter. After purification by thin-layer chromatography, the colored species were analyzed by ion-exchange chromatography, gel-permeation chromatography, and nuclear magnetic resonance spectrometry. These data suggested that the colored species were polysulfonated compounds with conjugated double bonds. Minor components in the raw fatty acid methyl ester, found by gas chromatography/mass spectrometry, were spiked into the purified methyl palmitate and then sulfonated. The unsaturated methyl ester and hydroxy ester showed the worst color results. The methyl oleate and methyl 12-hydroxystearate were then sulfonated and analyzed. Deep black products were obtained, which showed the same properties as the colored species in α-SF. It was concluded that low levels of unsaturated fatty acid methyl esters and hydroxy esters in the fatty acid methyl ester are the main causes of the coloring.     

Oxidation kinetics of β-carotene in oleic acid solvent with addition of an antioxidant, α-tocopherol

Oxidation experiments with β-carotene were performed in oleic acid solvent with addition of an antioxidant, α-tocopherol. A kinetic model was proposed based on a reaction mechanism consisting of the oxidation of β-carotene, oleic acid, and α-tocopherol; the antioxidation reactions of β-carotene and oleic acid by α-tocopherol; the cross-reaction of β-carotene and oleic acid; and the radical-exchange reaction of β-carotene and α-tocopherol. The model quantitatively described the oxidation behavior of β-carotene over a wide range of temperatures, oxygen compositions, and initial antioxidant concentrations. The model simulated well the time over which β-carotene was almost totally consumed under practical storage conditions at room temperature in air.     

Transesterification of cocoa butter by fungal lipases: Effect of solvent on 1,3-specificity

Transesterification and alcoholysis reactions catalyzed by immobilized lipases fromMucor miehei andHumicola lanuginosa in hexane gave fatty acid esters that did not reflect the expected 1,3-specificity of the enzymes, due to competing acyl migrations in the partial glyceride products. However, both lipases were 1,3-specific in reactions when diethyl ether was used as a solvent, and this provided a convenient analytical methodology in combination with gas chromatography and nuclear magnetic resonance spectroscopy for the determination of fatty acid distribution within triglycerides.