Enzymatic esterification of glycerol I. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols

Regioisomerically pure 1,3-sn-diacylglycerols are conveniently prepared in high yields (>80%) and in large quantities by enzymatic esterification of glycerol in the presence of various 1,3-selective lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) and a variety of different acyl donors like free fatty acids, fatty acid alkyl esters and vinyl esters. All reactions are carried out in aprotic organic solvents of low water content, namelyn-hexane, diethyl ether or tBuOMe. The creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction media by the adsorption of glycerol onto a solid support prior to use was essential for the success of these transformations. The effects of reaction conditions and the regioselectivities of the lipases on the product yields are described in detail.     

Fatty acid vinyl esters as acylating agents: A new method for the enzymatic synthesis of monoacylglycerols

Lipase-catalyzed synthesis of monoacylglycerols (MAG) was performed by transesterification reactions between fatty acid vinyl esters and either glycerol (1) or 1,2-O-isopropylidene-rac-glycerol (2), without solvents or in the presence ofn-pentane. Vinyl decanoate, vinyl laurate, vinyl stearate and vinyl palmitate have been converted to the corresponding monoacylglycerols. As expected for the reaction with1, a mixture of mono-, di- and triacylglycerols was synthesized. The highest concentrations of MAG were achieved with vinyl stearate (30% 2-MAG and 15% 1-MAG). The reactions of fatty acid vinyl esters with the protected glycerol (2) led to the corresponding protected 3-monoacylglycerols with 100% conversion after short reaction times. The subsequent cleavage of these acetonides was performed by four different methods. The fastest cleavage was found with trifluoroacetic acid as catalyst, whereas the highest concentration of MAG (100%) was obtained for the boric acid-catalyzed hydrolysis of the acetonides.     

Lipase-catalyzed incorporation of n−3 polyunsaturated fatty acids into vegetable oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.     

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.     

Enzymatic synthesis of acetylated glucose fatty acid esters in organic solvent

Two immobilized lipases fromCandida antarctica (SP 382) andC. cylindraceae, nowrugosa (2001), catalyzed the synthesis of novel acetylated glucose fatty acid esters with glucose pentaacetate (GP) and Trisun 80 (80% oleic) vegetable oil or methyl oleate as substrates in organic solvents. The relative yield was between 6.4–52%, and the incorporation of oleic acid onto the glucose was between 31–100%. In addition, these enzymes were able to catalyze the synthesis of glucose fatty acid esters with free glucose as the sugar substrate. The highest oleic acid incorporation (100%) was obtained in benzene with SP 382 lipase and Trisun 80 as the acyl donor. With methyl oleate as the acyl donor, greater incorporation was obtained in benzene (90.5%) compared to 75% in isooctane. The 2001 lipase was better in benzene/pyridine (2∶1 vol/vol) 74%) and chloroform (61%) compared to benzene and isooctane. However, with free glucose and Trisun 80 as substrates, both enzymes gave acceptable levels of oleic acid incorporation (82–100%) in benzene, benzene/pyridine and pyridine. The best conditions for the ester interchange reaction reported are: lipase (10% by weight of substrate); incubation time 48 h; molar ratio of Trisun/GP 1∶2; 3 mL solvent and 3% added water. These glucose esters have potential applications as emulsifiers in food, cosmetics and pharmaceutical formulations.     

Enzymatic modification of trilinolein: Incorporation of n-3 polyunsaturated fatty acids

Two immobilized lipases, IM60 fromMucor miehei and SP435 fromCandida antarctica, were used as biocatalysts for the modification of trilinolein with n-3 polyunsaturated fatty acids (PUFA), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), by using their ethyl esters as acyl donors (EEPA and EDHA, respectively). Transesterification (ester-ester interchange) reactions were carried out in organic solvent. The products were analyzed according to their equivalent carbon number and polarity by reverse-phase high-performance liquid chromatography, and the fatty acid profiles were determined by gas-liquid chromatography. Modified triacylglycerol products contained 1 or 2 molecules of n-3 PUFA. With EEPA as the acyl donor, the total EPA product yields with IM60 and SP435 as biocatalysts were 79.6 and 81.4%, respectively. However, with EDHA as the acyl donor and IM60 and SP435 as biocatalysts, the total DHA product yields were 70.5 and 79.7%, respectively. Effects of reaction parameters, such as type of solvent, enzyme load, time course, and molar ratio of substrates on the n-3 PUFA incorporation, were followed with SP435 as the biocatalyst. High yields were obtained, even in the absence of organic solvent. These lipids do hold promise for specialty nutrition and other therapeutic uses.     

Reactive extraction of oilseeds with dialkyl carbonates

Using dialkyl carbonates as reagents for lipase‐catalyzed transesterification, the reaction is driven by the evolvement of carbon dioxide as the co‐product and thus no longer an equilibrium reaction. Therefore this transesterification method is faster and quantitative conversions can be obtained. Short‐chain dialkyl carbonates, like other short‐chain esters, are also suitable solvents for seed oil extraction. Thus, extraction and transesterification can be combined in a single reaction. This reaction, called reactive extraction, was carried out in a standard Soxhlet apparatus with rapeseed, linseed and calendula seed as the raw materials and with dimethyl and diethyl carbonate as extraction solvent and transesterification reagent at the same time. Fatty acid methyl esters and ethyl esters respectively were obtained with higher yields than those achieved by conventional two step extraction / transesterification. In the case of linseed fatty acid esters and especially calendula seed fatty acid esters, the iodine values of the products obtained by one‐pot‐two‐step reactive extraction were significantly higher.     

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.     

Lipase-catalyzed enantioselective acidolysis of chiral 2-methylalkanoates

Lipase fromCandida cylindracea catalyzes the acidolysis between racemic 2-methylalkanoates and fatty acids in heptane with a preference for the (S)-configurated esters. Velocity and enantioselectivity of the acyl transfer are strongly influenced by the structures of the initial substrates. The potential of this strategy for kinetic resolution of the enantiomers of chiral 2-methylalkanoates is demonstrated. Limitations of the procedure due to the reversible character of the reaction are discussed.     

Enzymatic removal of 3‐monochloro‐1,2‐propanediol (3‐MCPD) and its esters from oils

3‐Monochloro‐1,2‐propanediol (3‐MCPD) is a contaminant in processed food well known for about 30 years. More recently, this compound has observed attendance due to its occurrence as fatty acid esters in edible oils and products derived from them. In this study, the first enzymatic approach to remove 3‐MCPD and its esters from aqueous and biphasic systems by converting it into glycerol is described. First, 3‐MCPD was converted in an aqueous system by an enzyme cascade consisting of a halohydrin dehalogenase from Arthrobacter sp. AD2 and an epoxide hydrolase from Agrobacterium radiobacter AD1 with complete conversion to glycerol. Next, it could also be shown, that the corresponding oleic acid monoester of 3‐monochloropropanediol‐1‐monooleic‐ester (3‐MCPD‐ester) was converted in a biphasic system in the presence of an edible oil by Candida antarctica lipase A to yield free 3‐MCPD and the corresponding fatty acid. Hence, also 3‐MCPD‐esters can be converted by an enzyme cascade into the harmless product glycerol. Practical applications: Since several reports have been recently published on the contamination of foods with 3‐MCPD and its fatty acid esters, there is a great demand to remove these compounds and an urgency to find useful methods for this. In this contribution, we present an easy enzymatic way to remove 3‐MCPD and its esters from the reaction media (i.e., plant oil) by converting it to the nontoxic glycerol. The method requires neither high temperature nor organic solvents.     

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.     

Impact of Fatty Acid Structure on CALB‐Catalyzed Esterification of Glucose

Enzymatic synthesis of fatty acid glucose esters from different fatty acyl donors are performed via enzymatic catalysis in the presence of Candida antarctica lipase B (CALB), using acetonitrile as the solvent. The acyl donor nature (fatty acid or fatty acid vinyl ester) and structure are varied. Lower reaction rates and lower conversions are obtained with fatty acids in comparison to their corresponding vinyl esters. Moreover, the acyl donor with the longest chain length gives the highest conversions. The presence of unsaturation on the acyl donor chain is also shown to be detrimental to the conversion. Practical Applications: The practical applications of the present work are related to the production of gluco‐esters that could be used as nonionic surfactants as detergents, cosmetics and food emulsifiers, emollients or conservatives, respectively. In this study, it is shown that in order to get high production yields, each reaction parameter has to be tuned properly.     

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99–92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4–C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12–C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.     

Lipase-catalyzed synthesis of structured low-calorie triacylglycerols

Because of their unique fatty acid specificities and regioselectivities, lipases have been found to be effective catalysts for the synthesis of structured lipids that have a predetermined composition and distribution of fatty acyl groups on the glycerol backbone. The prospective plant-derived lipase found in the exudate of Carica papaya is known for its shortchain acyl group specificity, 1,3-glycerol regioselectivity, and sn-3 stereoselectivity. Carica papaya latex (CPL) was therefore examined for its potential ability to synthesize structured lowcalorie short- and long-chain triacylglycerols (SLCT). In this paper, we describe the utility of CPL in the lipase-catalyzed interesterification reaction of triacetin and hydrogenated soybean oil. Normal-phase high-performance liquid chromatography, combined with mass spectrometry, was used to distinguish the structured SLCT synthesized using the lipase from the corresponding SLCT produced by chemical synthesis.     

Enzymatic fat hydrolysis and synthesis

The hydrolysis of tallow, coconut oil and olive oil, by lipase fromCandida rugosa, was studied. The reaction approximates a firstorder kinetics model. Its rate is unaffected by temperature in the range of 26–46 C. Olive oil is more rapidly hydrolyzed compared to tallow and coconut oil. Hydrolysis is adversely affected by hydrocarbon solvents and a nonionic surfactant. Since amounts of fatty acids produced are almost directly proportional to the logarithms of reaction time and enzyme concentration, this relationship provides a simple means of determining these parameters for a desired extent of hydrolysis. All three substrates can be hydrolyzed, almost quantitatively, within 72 hr. Lipase fromAspergillus niger performs similarly. The lipase fromRhizopus arrhizus gives a slow hydrolysis rate because of its specificity for the acyl groups attached to the α-hydroxyl groups of glycerol. Esterification of glycerol with fatty acid was studied with the lipase fromC. rugosa andA. niger. All expected five glycerides are formed at an early stage of the reaction. Removal of water and use of excess fatty acid reverse the reaction towards esterification. However, esterification beyond a 70% triglyceride content is slow.     

Alcoholysis of palm oil mid-fraction by lipase from Rhizopus rhizopodiformis

A mycelial lipase from Rhizopus rhizopodiformis was prepared in fragment form. The lipase was examined to catalyze the alcoholysis of palm oil mid-fraction (PMF) in organic solvents. High percentage conversions of PMF to alkyl esters were achieved when methanol or propanol was used as acyl acceptor. Of the two most prevalent fatty acids in PMF, palmitic acid seemed to be preferred over oleic acid in the formation of methyl and propyl esters. The optimal ratio of oil to methanol in the alcoholysis reaction is 1 to 2 moles. The lipase exhibited high alcoholysis activities in nonpolar solvents (log P>2), such as hexane, benzene, toluene, and heptane. The enzyme showed exceptionally high thermostability.     

Gradients of n-heptane and acetonitrile in silver-ion high-performance liquid chromatography analyses of cis and trans bonds in lipids

Cis and trans isomers of fatty acid methyl esters, fatty alcohols, and triacylglycerols were analyzed with a silverion high-performance liquid chromatography system. Gradients of n-heptane and acetonitrile were used to elute molecules with up to nine cis double bonds. The analyses were as fast and reliable and had a resolution similar to that of the best published analyses. However, published analyses were performed with chlorinated solvents, and these solvents are carcinogenic and mutagenic. The solvents we used, heptane and acetonitrile, are less dangerous to the analyst.     

Utilization of biodiesel and glycerol for the synthesis of epoxidized acyl glycerides and its application as a plasticizer

In many regions, even if waste vegetable oil is used as raw material, the profits of biodiesel (fatty acid methyl ester, FAME) enterprises still remain restricted, and they are facing environmental problems caused by the by-product glycerol. Hence, value-added utilization of FAME and glycerol is a promising alternative to improve the competitiveness of biodiesel enterprises in the economy and environmental protection. In this paper, FAME and glycerol were utilized to prepare epoxidized acyl glycerides by transesterification and epoxidation. In the transesterification, acyl glycerides were prepared by reacting FAME and glycerol catalyzed by K₂CO₃. The contents of monoacyl glyceride, diacyl glyceride, and triacyl glyceride in the acyl glycerides were about 50%, 40%, and 10%, and the optimal conditions were a temperature of 200°C and the molar ratio of FAME to glycerol of 1:1. In the epoxidation, selectivity of epoxides was significantly affected by monoacyl glyceride content, and a treatment method was developed to control the monoacyl glyceride content <5%. In addition, the plasticity of epoxidized acyl glycerides to partially substitute dioctyl phthalate in polyvinyl chloride was studied, the overall mechanical properties, thermal stability, and extraction resistance were improved. In this work, FAME and glycerol were utilized to prepare epoxidized acyl glycerides with higher value-added, which provide a potential solution for improving the market competitiveness of biodiesel enterprises.     

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.     

Quality control of rapeseed oil methyl esters by determination of acyl conversion

A simple procedure for the evaluation of vegetable oil conversion to methyl esters of fatty acids has been developed. These methyl esters, prepared by the transesterification of vegetable oil with methanol, are used as alternative fuel for diesel engines. A method of gas-liquid chromatography (GLC) on packed columns is used to determine the conversion of acyls bound in acylglycerols to methyl esters. This procedure is based on comparison of the peak areas of methyl esters in fuel samples before and after reaction with an effective transesterification reagent, which will transform unreacted acylglycerols to methyl esters. A correlation between the bound glycerol content, determined by the thin-layer chromatography/flame-ionization detector method, and the acyl conversion, determined by GLC, is given. In acyl conversions to methyl esters over 96.0%, the bound glycerol content is less than 0.25% by weight.