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.     

An Improved Method for Synthesis of <Emphasis Type="Italic">N</Emphasis>-stearoyl and <Emphasis Type="Italic">N</Emphasis>-palmitoylethanolamine

Certain N-acylethanolamines interact with cannabinoid receptors and have anorexic and neuroprotective effects. Traditional methods for the synthesis of N-acylethanolamines use fatty acid chlorides, fatty acid methyl esters, free fatty acids and triacylglycerols as acyl donors to react with ethanolamine. In the present study, we investigated the feasibility of using fatty acid vinyl esters as the acyl donor to synthesize N-stearoyl and N-palmitoylethanolamine. Theoretically, the use of fatty acid vinyl esters should lead to an irreversible reaction because the volatile acetaldehyde by-product is easily removed. Four reaction conditions, i.e. catalyst concentration, substrate ratio, temperature, and time were evaluated. The reaction performed at mild temperatures and with an excess amount of ethanolamine which acted as both reactant and solvent resulted in the formation of high purity N-stearoyl and N-palmitoylethanolamine. When 20 mmol ethanolamine was reacted with 1 mmol vinyl stearate at 80 °C for 1 h with 1% sodium methoxide as catalyst, N-stearoylethanolamine with 96% purity was obtained after the removal of excess ethanolamine without further purification, while N-palmitoylethanolamine with 98% purity was obtained by reacting with the same substrate ratio at 60 °C for 1.5 h with 3% catalyst. Complete conversion of vinyl stearate and palmitate was achieved.     

Enzymatic esterification of glycerol II. Lipase-catalyzed synthesis of regioisomerically pure 1(3)-rac-monoacylglycerols

Regioisomerically pure 1(3)-rac-monoacylglycerols are conveniently prepared in high yields (>75%) and in multigram quantities by enzymatic esterification of glycerol in the presence of various lipases(Chromobacterium viscosum, Rhizopus delemar, Rhizomucor miehei) with a variety of different acyl donors, such as free fatty acids, fatty acid alkyl esters, vinyl esters and triacylglycerols, as well as natural fats and oils. All reactions are carried out in aprotic organic solvents with low water content, namelyn-hexane, diethyl ether, tBuOMe or mixtures of these solvents. Essential for the success of these transformations were the following two factors. First, the creation of an artificial interphase between the solvent-immiscible hydrophilic glycerol and the hydrophobic reaction medium by its adsorption onto a solid support. Second, a facile system for the separation of the desired monoacylglycerol from the reaction mixture, coupled with the continuous recycling of acyl donor and undesirable by-products.     

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.     

Lipase-Catalyzed Solid Phase Synthesis of Sugar Esters

Lipase-catalyzed synthesis of sugar fatty acid esters was performed in a heterogeneous reaction system in the presence of an organic solvent serving as adjuvant. Although the sugar is almost insoluble in such a system, high conversions to the corresponding sugar esters were achieved, due to crystallization of the product. Acylation occurred regioselectively at the primary hydroxyl group and subsequent diacylation was observed only in the case of caprylic acid (2–5%). Best conditions were found for solvents having low log P values and low product solubility such as acetone, using immobilized lipase from Candida antarctica (CAL-B, Novo SP435) and fatty acids with chain lengths from C₁₂ to C₈ as acyl donors. The esterification of β-D(+)-glucose with stearic acid resulted in up to 100% conversion after 48 hours equal to a productivity of 0.4 mmol sugar ester per gram lipase and hour.     

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 kinetic resolution of secondary alcohols by esterification with FA under vacuum

Kinetic resolution of some chiral secondary alcohols [2-octanol, 1-phenylethanol, and 1-(2-naphthyl)ethanol] with high enantioselectivity (E>300) was achieved by direct esterification with FFA catalyzed by immobilized Candida antarctica B lipase. The reaction equilibrium was shifted toward the synthetic side by the removal of the water formed under vacuum. Esterification of rac-2-octanol at an alcohol/FFA molar ratio of 2∶1 was used as a model reaction. The chain length of FFA and their structure influenced the reaction rate but did not have a measureable effect on E. The best acyl donor was decanoic acid: >47% conversion at 4 h with virtually perfect E. Temperature did not affect E in the range studied (15–45°C), but temperatures at the higher end afforded improved reaction rates. The reaction rates and E were compared for three different acyl donors. The initial reaction rate increased in the following order: ethyl laurate < lauric acid < vinyl acetate. E was high (E>300) for all acyl donors. Racemic 1-phenylethanol and 1-(2-naphthyl)ethanol were also resolved by direct esterification with decanoic acid in short times (3 and 4 h, respectively) with E>300 and excellent conversions. Preparative-scale kinetic resolution of 2-octanol was also performed.     

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.     

Enzymatic Synthesis of Tyrosol-Based Phenolipids: Characterization and Effect of Alkyl Chain Unsaturation on the Antioxidant Activities in Bulk Oil and Oil-in-Water Emulsion

Oxidative stability of lipids is one of the most important parameters affecting their quality. Lipase‐catalyzed lipophilic tyrosyl esters with an equivalent carbon alkyl chain but different degrees of unsaturation (C18:0 to C18:4n3) were prepared, characterized, and used as antioxidants. Free fatty acids and fatty acid ethyl esters (substrate molar ratio tyrosol: acyl donor, 1:10) were used as acyl donors and immobilized lipase from Candida antarctica was the biocatalyst (10 %). The phenolipids were isolated and characterized using ESI–MS, ¹H‐NMR, and ¹³C‐NMR. Peroxide value (PV) and para‐anisidine value (p‐AV) were measured to evaluate their antioxidant activities in bulk oil structured lipid (SL) and in an oil‐in‐water emulsion (SL‐based infant formula). No significant difference was found in yield and reaction time between the two types of acyl donors. However, as the unsaturation of the fatty acids increased the reaction time also increased. In SL, tyrosyl esters exhibited lower antioxidant activity than tyrosol whereas the addition of an alkyl chain enhanced the antioxidant efficiency of tyrosol in infant formula. Tyrosyl oleate was the most efficient antioxidant in the emulsion system followed by tyrosyl stearate and tyrosyl linoleate. These results suggest that the synthesized phenolipids may be used as potential antioxidants in lipid‐based products.     

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.     

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.     

Wax ester-synthesizing activity of lipases

The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, was ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium.     

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.     

Simultaneous derivatization of acyl and S-alkyl moieties of acyl thioesters by using trimethylsulfonium hydroxide for gas chromatographic analysis

The reaction of long-chain acyl thioesters, viz. lauric acid dodecyl thioester, 1,8-di-S-palmitoyl octanedithiol, and tristearoyl α-monothioglycerol, with trimethylsulfonium hydroxide in the presence of methanol leads to simultaneous derivatization of acyl moieties and S-alkyl moieties of acyl thioesters to the corresponding fatty acid methyl esters and methyl alkylsulfides, respectively. These derivatized products were analyzed by gas chromatographic technique.     

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 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.     

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.     

Epoxy fatty acid ester plasticizers. Preparation and properties

Summary The preparation of epoxy fatty acid ester plasticizers has been reviewed. These plasticizers can be prepared by epoxidation reactions, utilizing a preformed peracid or anin-situ technique. A newin-situ epoxidation process, utilizing hydrogen peroxide and acetic acid in the presence of strong acid catalyst, has been described. Excellent conversions to epoxide have been achieved with a series of monounsaturated and polyunsaturated esters. Epoxy fatty acid esters are excellent plasticizers for vinyl resins. Their performance in a series of additional epoxy plasticizers of vegetable and marine origin is described. Novel epoxy plasticizers based upon mixed acetylated mono- and di-glyceride have been prepared and are found to have excellent plasticizer performance. Presented at the 28th fall meeting, American Oil Chemists’ Society, October 1954, Minneapolis, Minn.     

Lipase-catalyzed reactions involving thia fatty acids and ester derivatives

Enzymatic hydrolysis of synthetic methyl 5-, 9-, and 12-thiastearates in aqueous media withCandida cyclindracea or porcine pancreatic lipases gave the corresponding fatty acids in 70–100% yield. Hydrolysis of the 3- and 4-positional isomers gave only 15–25% of the free thia fatty acids, suggesting discrimination against these isomers by lipases. No lipolysis was achieved with methyl 2-thialaurate under a range of reaction conditions. Esterification of the 3-, 4-, 5-, 9-, and 12-thiastearic acids withn-butanol inn-hexane using Lipozyme (immobilizedRhizomucor miehei) as the biocatalyst gave the corresponding butyl esters in 80–95% yield. Interesterification (acyl exchange) of triolein with methyl 9-thiastearate in the presence of Lipozyme showed the incorporation of 9-thiastearoyl chain at only one of the α-positions of triolein. In the case of methyl 2-thialaurate, no lipase-catalyzed acyl exchange reaction was possible. This study showed that the position of the sulfur atom in thia fatty esters affects the lipase-catalyzed hydrolysis and interesterification reactions.     

The synthesis,properties, and applications of ascorbyl esters

Ascorbic acid is a naturally occurring antioxidant. Nevertheless, its primary applications as an antioxidant in the life science, food and pharmaceutical industries are limited because of its hydrophilic nature. Alternatively, ascorbyl acid esters are potential surfactants and antioxidants. Chemical methods for the synthesis of ascorbyl esters lead to the formation of side products and simultaneous decomposition of ascorbic acid due to harsh reaction conditions. In contrast, lipases are used as regioselective and mild catalysts for the synthesis of ascorbyl esters. So far, various acyl donors namely, fatty acids, fatty acid alkyl esters, fatty acid vinyl esters and triacylglycerols have been explored for the synthesis of ascorbyl fatty esters. Other compounds such as L‐methyl lactate, bixin, phenyl butyric acid are also used as acyl donors. This article is focused on the recent developments of lipase‐catalyzed synthesis of ascorbyl esters, their antioxidant properties and applications.