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.     

Enzymatic esterification of θ-3 fatty acid concentrates from seal blubber oil with glycerol

Enzymatic synthesis of acyglycerols directly from glycerol and an θ-3 fatty acid concentrate, prepared from seal blubber oil, in organic solvents was studied. Seven lipases were used as biocatalysts for esterification, Lipase LP-401-AS from Chromobacterium viscosum showed the highest activity for esterification. Effects of reaction parameters, namely, temperature, time course, type of solvent, water content, amount of glycerol, enzyme load and solvent volume, were followed with lipase IP-401-AS as the biocatalyst of choice. Optimal reaction conditions were established, and the maximal degree of acylglycerol synthesis reached was 94.3%. The concentrations of monoacylglycerols, diacylglycerols, and triacylglycerols were 13.8, 43.1, and 37.4%, respectively. Therefore, acylglycerols containing predominantly θ-3 fatty acid concentrates may be easily synthesized directly via their reaction with glycerol.     

Esterification of glycerol with conjugated linoleic acid and long-chain fatty acids from fish oil

Free fatty acids from fish oil were prepared by saponification of menhaden oil. The resulting mixture of fatty acids contained ca. 15% eicosapentaenoic acid (EPA) and 10% docosahexaenoic acid (DHA), together with other saturated and monounsaturated fatty acids. Four commercial lipases (PS from Pseudomonas cepacia, G from Penicillium camemberti, L2 from Candida antarctica fraction B, and L9 from Mucor miehei) were tested for their ability to catalyze the esterification of glycerol with a mixture of free fatty acids derived from saponified menhaden oil, to which 20% (w/w) conjugated linoleic acid had been added. The mixtures were incubated at 40°C for 48h. The ultimate extent of the esterification reaction (60%) was similar for three of the four lipases studied. Lipase PS produced triacylglycerols at the fastest rate. Lipase G differed from the other three lipases in terms of effecting a much slower reaction rate. In addition, the rate of incorporation of omega-3 fatty acids when mediated by lipase G was slower than the rates of incorporation of other fatty acids present in the reaction mixture. With respect to fatty acid specificities, lipases PS and L9 showed appreciable discrimination against esterification of EPA and DHA, respectively, while lipase L2 exhibited similar activity for all fatty acids present in the reaction mixture. The positional distribution of the various fatty acids between the sn-1,3 and sn-2 positions on the glycerol backbone was also determined.     

Characterisation of acylglycerol synthesis in aqueous foams by lipases

Low water systems have become important for organic synthetic reactions catalysed by enzymes. Esterification reactions could be catalysed by lipase in multiphasic reaction media, such as foams, containing excess water. The esterification of glycerol with oleic acid catalysed by lipase in aqueous foams has been characterised. More than 80% of the oleic acid was converted to glycerides within 10 h. Preliminary characterisation of the reaction with respect to time, pH and temperature indicate that acylglycerol synthesis in foam media as similar to hydrolysis by the same lipase. The observed high K_m of glycerol for the esterification reaction may be due to poor surface active properties of glycerol. The unique advantages of foam as a medium to conduct lipase reactions are discussed. © 1999 Society of Chemical Industry     

Castor bean lipase as a biocatalyst in the esterification of fatty acids to glycerol

Castor bean lipase was investigated as biocatalyst in the esterification of fatty acids to glycerol. For this purpose, pressed seeds were pretreated with phosphate-citrate buffer solution at optimal preincubation time and pH and used as a lipase source in esterification of fatty acids with glycerol. The effect of process parameters in the esterification, i.e., molar ratios of reactants, temperature, water content of glycerol and concentration of lipase, were determined by using pretreated castor bean.     

Enzymatic Production of Fatty Acid Methyl Esters by Hydrolysis of Acid Oil Followed by Esterification

Acid oil, a by-product of vegetable oil refining, was enzymatically converted to fatty acid methyl esters (FAME). Acid oil contained free fatty acids (FFA), acylglycerols, and lipophilic compounds. First, acylglycerols (11 wt%) were hydrolyzed at 30 °C by 20 units Candida rugosa lipase/g-mixture with 40 wt% water. The resulting oil layer containing 92 wt% FFA was used for the next reaction, methyl esterification of FFA to FAME by immobilized Candida antarctica lipase. A mixture of 66 wt% oil layer and 34 wt% methanol (5 mol for FFA) were shaken at 30 °C with 1.0 wt% lipase. The degree of esterification reached 96% after 24 h. The resulting reaction mixture was then dehydrated and subjected to the second esterification that was conducted with 2.2 wt% methanol (5 mol for residual FFA) and 1.0 wt% immobilized lipase. The degree of esterification of residual FFA reached 44%. The degree increased successfully to 72% (total degree of esterification 99%) by conducting the reaction in the presence of 10 wt% glycerol, because water in the oil layer was attracted to the glycerol layer. Over 98% of total esterification was maintained, even though the first and the second esterification reactions were repeated every 24 h for 40 days. The enzymatic process comprising hydrolysis and methyl esterification produced an oil containing 91 wt% FAME, 1 wt% FFA, 1 wt% acylglycerols, and 7 wt% lipophilic compounds.     

Simple, high-efficiency synthesis of fatty acid methyl esters from soapstock

We report a simple method that efficiently esterifies the fatty acids in soapstock, an inexpensive, lipid-rich by-product of edible oil production. The process involves (i) alkaline hydrolysis of all lipid-linked fatty acid ester bonds and (ii) acid-catalyzed esterification of the resulting fatty acid sodium salts. Step (i) completely saponified all glycerides and phosphoglycerides in the soapstock. Following water removal, the resulting free fatty acid sodium salts were rapidly and quantitatively converted to fatty acid methyl esters (FAME) by incubation with methanol and sulfuric acid at 35°C and ambient pressure. Minimum molar reactant ratios for full esterification were fatty acids/methanol/sulfuric acid of 1∶30∶5. The esterification reaction was substantially complete within 10 min and was not inhibited by residual water contents up to ca. 10% in the saponified soapstock. The product FAME contained >99% fatty acid esters, 0% triglycerides, <0.05% diglycerides, <0.1% monoglycerides, and <0.8% free fatty acids. Free fatty acid levels were further reduced by washing with dilute sodium hydroxide. Free and total glycerol were <0.01 and <0.015%, respectively. The water content was <0.04%. These values meet the current specifications for biodiesel, a renewable substitute for petroleum-derived diesel fuel. The identities and proportions of fatty acid esters in the FAME reflected the fatty acid content of soybean lipids. Solids formed during the reaction contained 69.1% ash and 0.8% protein. Their sodium content indicated that sodium sulfate was the prime inorganic component. Carbohydrate was the predominant organic constituent of the solid.     

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.     

Bio‐ and auto‐catalytic esterification of high acid mowrah fat and palm kernel oil

An alternative process for the deacidification of high acid palm kernel oil (PKO) and mowrah fat, MF, was investigated by autocatalytic and enzyme (Mucor miehei)‐catalyzed esterification of free fatty acids (FFA). In the process monoglyceride (MG) or glycerol was used as esterifying agent. The results of the autocatalytic esterification process were compared with that of bioesterification with respect to reduction of FFA. For the former process the optimum reaction temperature and oil to MG or glycerol proportion were established. The optimum reaction temperature for PKO free fatty acids with stoichiometric quantity of glycerol is 160—165 °C (at 10 mm Hg pressure (1.33 kPa)) and after 6 h the FFA content is reduced to 1.6%, w/w, (from 25.0%, w/w, original). However, if a stoichiometric amount of MG is used as an esterifying agent the optimum esterification temperature is found to be 195—200 °C (at 10 mm Hg pressure (1.33 kPa)), and after 8 h the FFA content is reduced to 3.4%, w/w. On the contrary, biorefining of PKO at 60 °C temperature and 10 mm Hg pressure (1.33 kPa) using optimum (40% excess) quantity of glycerol or 50% excess MG reduces the FFA level to 1.2% and 0.7%, w/w, respectively. Similar study on bio‐ and auto‐catalytic esterification of MF was also carried out and got comparable results. The final products were then characterized.     

Esterification kinetics of long-chain fatty acids and fatty alcohols with a surfactant-coated lipase in n-hexane

The esterification reaction kinetics of long-chain fatty acids and fatty alcohols catalyzed with a surfactant-coated lipase in a microaqueous n-hexane system were studied. The biocatalytic complex, surfactant-lipase adduct, showed 40 times the activity after a reaction time of 5 h compared to the unmodified lipase in the same reaction system. Various factors that may affect the activity of the modified lipase were studied, such as the influence of substrate fatty acid chainlength, water content, and temperature. By varying the concentration of each of the two substrates while keeping that of the other substrate constant, it was found that the esterification reaction follows Michaelis-Menten kinetics. The surfactant-enzyme complex kinetic parameters were determined with respect to both substrates. It was suggested that the kinetics of the lipase-catalyzed esterification reaction model follow a Ping-Pong Bi Bi mechanism with no substrate or product inhibition.     

Two-Step Production of Oil Enriched in Conjugated Linoleic Acids and Diacylglycerol

A two-step process was used to produce diacylglycerol-enriched structured lipid that contained mainly c9,t11 and t10,c12 isomers of conjugated linoleic acids (CLA). First, a structured triacylglycerol (TAG) was synthesized by lipase-catalyzed acidolysis of corn oil with CLA. This structured triacylglycerol contained 30.4 mol% CLA with 45.5% of the CLA mostly located at sn-1,3 positions of the glycerol backbone. Then, lipase-catalyzed glycerolysis was conducted between structured triacylglycerol and glycerol to produce diacylglycerol-enriched structured lipid. The final product contained 6.8% monoacylglycerol, 31.5% diacylglycerol and 61.1% TAG after 48 h reaction. The selected chemical (fatty acid composition, the content of mono-, di-, and triacylglycerol in the reaction product) and physical properties (melting profile) were determined by hihg-performance liquid chromatography (HPLC), gas chromatography (GC), and differential scanning calorimetry (DSC).     

Highly Efficient Enzymatic Synthesis of 2-Monoacylglycerides and Structured Lipids and their Production on a Technical Scale

We report here a two-step process for the high-yield enzymatic synthesis of 2-monoacylglycerides (2-MAG) of saturated as well as unsaturated fatty acids with different chain lengths. The process consists of two steps: first the unselective esterification of fatty acids and glycerol leading to a triacylglyceride followed by an sn1,3-selective alcoholysis reaction yielding 2-monoacylglycerides. Remarkably, both steps can be catalyzed by lipase B from Candida antarctica (CalB). The whole process including esterification and alcoholysis was scaled up in a miniplant to a total volume of 10 l. With this volume, a two-step process catalyzed by CalB for the synthesis of 1,3-oleoyl-2-palmitoylglycerol (OPO) using tripalmitate as starting material was established. On a laboratory scale, we obtained gram quantities of the synthesized 2-monoacylglycerides of polyunsaturated fatty acids such as arachidonic-, docosahexaenoic- and eicosapentaenoic acids and up to 96.4% of the theoretically possible yield with 95% purity. On a technical scale (>100 g of product, >5 l of reaction volume), 97% yield was reached in the esterification and 73% in the alcoholysis and a new promising process for the enzymatic synthesis of OPO was established.     

The Acrylation of Glycerol: A Precursor to Functionalized Lipids

With the objective of expanding the number of functionalized lipids available, the reactive vinyl group of acrylic acid was introduced to triacylglycerol by esterification of glycerol. Didecanoylacryloylglycerol was synthesized from decanoic and acrylic acids and glycerol using K₂O as catalyst. 2 g (21.7 mmol) glycerol, 11.3 g (65.4 mmol) decanoic acid, 6.2 g (86.1 mmol) acrylic acid, 60 ml hexane, and 400 mg K₂O were added to 300-ml closed stainless steel reactor and maintained at 200 °C for 5 h. The resulting product, designated DDA, was isolated at about 40% yield based on the acylglycerol products. The other products included tridecanoylglycerol, diacryloyldecanoylglycerol, and the diacylglycerols of these acids. DDA was then converted to functionalized lipids by the Michael addition and Heck reaction. The Michael addition of thiophenol and 4-bromothiophenol yielded the corresponding linear thioethers whereas and Heck reaction products from bromobenzene and bromoanisole yielded triacylglycerols containing trans-cinnamic acid and trans-(4-methoxy)cinnamic acid, respectively.     

The preparation and purification of monoglycerides. II. Direct esterification of fatty acids with glycerol

Studies have been made on the direct esterification reaction with various saturated and unsaturated fatty acids using more than theoretical concentration of glycerol (for maximum monoglyceride production) at 180C reaction temperature and in presence and absence of alkaline catalyst. The results show that the maximum monoglyceride formed is in the range of 55–60% of the fatty product at equilibrium stage of the reaction. The alkaline catalyst substantially increases the initial rate of reaction without appreciably lowering the time required for reaching the equilibrium concentration of monoglyceride in the resulting reaction mixture. Catalyst helps in depressing diglyceride formation.     

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.     

Esterification of polyunsaturated fatty acids by various forms of immobilized lipase from Rhizomucor miehei

Ethyl esterification specificity of a lipase from Rhizomucor miehei for polyunsaturated fatty acids (PUFA) was compared at 1 and 100 mM to study molecular recognition of PUFA. The chemical shift of methylene adjacent to carboxyl groups in the nuclear magnetic resonance spectrum of docosahexaenoic acid (DHA) in ethanol moved to a lower magnetic field as the concentration of DHA increased, suggesting that the degree of dissociation of DHA decreased. Specificity constants or apparent second-order rate constants (V _max/K _m or catalytic power) for 1 mM esterification by immobilized lipases were higher than the native lipase. Immobilized hydrophobic carrier of low mass transfer resistance for the esterification substrate may improve maximal velocity and affinity for the substrate. Higher specificity constants for 1 mM substrates were observed using immobilized lipases fixed on an anion exchange resin with glutaraldehyde and on a cation exchange carrier with carbodiimide. Activity yields measured with 1 mM PUFA substrate were high. For the substrates at a concentration of 100 mM, higher specific constants with these bifunctional reagents were not observed but higher activity yields were found.     

Modification of Vegetable oils. IV. Reesterification of fatty acids with glycerol

Summary 1. An investigation has been made of the esterification of glycerol and peanut oil fatty acids under reduced pressure, with and without the assistance of various metal chlorides and oxides as catalysts. 2. The uncatalyzed reaction is bimolecular in character but proceeds in two successive stages, of which the latter has the lower velocity constant. Velocity constants have been determined for the initial and final stages of the reaction, at intervals between 166° and 241° C. The calculated heats of activation for the initial and final stages of the reaction are respectively 12,300 and 10,800 calories per mole. The free fatty acid concentration corresponding to the termination of the first stage decreases progressively as the temperature of the reaction is increased. 3. Of a wide variety of metal oxides and chlorides tested, zinc and tin chlorides were outstanding in catalytic activity. The reaction, when catalyzed with these materials, is complex and no longer simply bimolecular. It is believed that tin and zinc chlorides react initially with free fatty acids and free glycerol to form metal soaps and chlorohydrins, and that esterification proceeds through interaction of these two initial reaction products. Other metal chlorides, including the chlorides of aluminum, antimony, mercury, nickel, magnesium, manganese, lead, iron, and cadmium, do not appear to be capable of reacting in this manner, and are relatively poor catalysts. The oxides of tin and zinc are also deficient in catalytic activity, as is hydrochloric acid. 4. The reaction proceeds at a reasonable speed, i.e., the FFA content of the product is reduced to about 3% in 6 hours, if 0.0008 mole of tin chloride per 100 g. of fatty acids is used as a catalyst at 175° C. or if a similar amount of zinc chloride is used as a catalyst at 200° C. Equally rapid esterification is obtained without a catalyst only above 250° C. Esterification is assisted by maintaining a vacuum upon the reaction vessel to remove water vapor from the reacting material as rapidly as it is formed. A vacuum of about 20 mm. pressure of mercury is satisfactory. 5. If zinc or tin chloride catalysts are employed, the metals may be completely removed from the esterified oils by ordinary alkali refining. These catalysts do not cause the oil to polymerize during the course of esterification, do not cause conjugation in the oils, and are not detrimental to the color of the product. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Identification of diacylglycerols and triacylglycerols in a structured lipid sample by atmospheric pressure chemical ionization liquid chromatography/mass spectrometry

Atmospheric pressure chemical ionization liquid chromatography/mass spectrometry was used in the identification of diacylglycerol and triacylglycerol (TAG) molecular species in a sample of a structured lipid. In the study of acylglycerol standards, the most distinctive differences between the diacylglycerol and TAG molecules were found to be the molecular ion and the relative intensity of monoacylglycerol fragment ions. All saturated TAG ranging from tricaproin to tristearin, and unsaturated TAG including triolein, trilinolein, and trilinolenin, had ammonium adduct molecular ions [M+NH₄]⁺. Protonated molecular ions were also produced for TAG containing unsaturated fatty acids and the intensity increased with increasing unsaturation. Diacylglycerol fragment ions were also formed for TAG. The ammonium adduct molecular ion was the base peak for TAG containing polyunsaturated fatty acids, whereas the diacylglycerol fragment ion was the base peak for TAG containing saturated and monounsaturated medium- and long-chain fatty acids; the relative intensities of the ammonium adduct molecular ions were between 14 and 58%. The most abundant ion for diacylglycerols, however, was the molecular ion [M−17]⁺, and the relative intensity of the monoacylglycerol fragment ion was also higher than that for TAG. These distinctive differences between the diacylglycerol and TAG spectra were utilized for rapid identification of the acylglycerols in the sample of a structured lipid.     

假丝酵母99-125脂肪酶促酯化合成生物柴油的研究

1 INTRODUCTION Biodiesel, that is long-chain fatty acid short-chain alcohol esters (methyl, ethyl, propyl and butyl ester), is produced by esterification of fatty acids or inter- esterification of oils and fats. These fatty acid alcohol esters are not only used as important industrial addi- tives and surfactants, but also used for biofuel. The biodiesel is a biodegradable, environmental friendly, renewable substitute of diesel fuel[1]. The traditional production of biodiesel is by chem- i…     

The decomposition of secondary esters of castor oil with fatty acids

In this study, thermal splitting of secondary fatty acid esters of castor oil was investigated to determine the reaction kinetics under various conditions. Zinc oxide,p toluenesulfonic acid and sulfuric acid were used as catalysts. Reactions were carried out at 260, 270, and 280°C. Experimental data fitted the first-order rate equation for the catalyzed and noncatalyzed reactions. In addition to the kinetic investigation, the splitting (pyrolysis) mixture was evaluated in the preparation of a synthetic drying oil. For this purpose, the mixed fatty acids of linseed, sunflower andEcballium elaterium seed oils were used in the esterification stage of the process. Pyrolysis mixtures were converted to drying oils by combining the liberated acids with equivalent amounts of glycerol. The oils thus obtained show good drying oil properties.