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 glycerol III. Lipase-catalyzed synthesis of regioisomerically pure 1,3-sn-diacylglycerols and 1 (3)-rac-monoacylglycerols derived from unsaturated fatty acids

Lipases that display high regioselectivities and broad substrate tolerance were used as catalysts for the efficient esterification of glycerol under the conditions of irreversible acyl transfer. A variety of unsaturated fatty acids, such as oleic, linoleic, erucic, ricinolic, hydroxystearic and coriolic acid, were used for this purpose in the form of their vinyl esters. Suitable biocatalysts were chosen on the basis of systematic screening experiments regarding their regioselectivities (RE) and substrate tolerances. Distinct differences were found and expressed in numerical RE values as a measure for differences of these biocatalysts as being specific, selective, and nonspecific. Based on these experiments, a variety of molecules were synthesized on a preparative scale (>150 mmol) in good yield (ca.85%) and with high regioisomerical purities (>95% RE).     

Enzymatic synthesis of symmetrical 1,3-diacylglycerols by direct esterification of glycerol in solvent-free system

1,3-Diacylglycerols were synthesized by direct esterification of glycerol with free fatty acids in a solvent-free system. Free fatty acids with relatively low melting points (<45°C) such as unsaturated and medium-chain saturated fatty acids were used. With stoichiometric ratios of the reactants and water removal by evaporation at 3 mm Hg vacuum applied at 1 h and thereafter, the maximal 1,3-diacylglycerol content in the reaction mixture was: 84.6% for 1,3-dicaprylin, 84.4% for 1,3-dicaprin, 74.3% for 1,3-dilinolein, 71.7% for 1,3-dieicosapentaenoin, 67.4% for 1,3-dilaurin, and 61.1% for 1,3-diolein. Some of the system’s parameters (temperature, water removal, and molar ratio of the reactants) were optimized for the production of 1,3-dicaprylin, and the maximal yield reached 98%. The product was used for the chemical synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol. The yield after purification was 42%, and the purity of the triacylglycerol was 98% (both 1,3-dicapryloyl-2-eicosapentaenoylglycerol and 1,2-dicapryloyl-3-eicosapentaenoylglycerol included) by gas chromatographic analysis, of which 90% was the desired structured triacylglycerol (1,3-dicapryloyl-2-eicosapentaenoylglycerol) as determined by silver ion high-performance liquid chromatographic analysis.     

Enzymatic synthesis and spectroscopic characterization of 1,3-divernoloyl glycerol fromVernonia galamensis seed oil

cis-12,13-Epoxy-cis-octadecenoic (vernolic) acid occurs in triglycerides of the seed oil ofVernonia galamensis. The seeds also contain a lipase capable of hydrolyzing the triglycerides. Previous investigators incubated the seed ofVernonia anthelmintica and isolated 5.6% yield of 1,3-divernoloyl glycerol. We used crude lipase extract fromV. galamensis seed to synthesize 1,3-divernoloyl glycerol from vernonia oil in pentane at 40% yield. A 94% conversion of the 1,3-divernoloyl glycerol to pure vernolic acid (5.34% oxirane = 98.9% purity) was achieved by a low-energy saponification process. The carbon-13 nuclear magnetic resonance (NMR) spectrum of the 1,3-divernoloyl glyceride indicates a potential for using carbon-13 NMR spectroscopy in the identification of isomeric diglycerides. Thus the paper describes the synthesis, spectroscopic and chemical characterization of 1,3-divernoloyl glycerol, in addition to providing quantitative carbon-13 NMR studies ofV. galamensis oil.     

Lipase-catalyzed synthesis of structured triacylglycerides from 1,3-diacylglycerides

A new method for the lipase-catalyzed synthesis of structured TAG (ST) is described. First, sn1,3-dilaurin or-dicaprylin were enzymatically synthesized using different published methods. Next, these were esterified at the sn2-position with oleic acid or its vinyl ester using different lipases. Key to successful enzymatic synthesis of ST was the choice of a lipase with appropriate FA specificity, i.e., one that does not act on the FA already present in the sn1,3-DAG, but that at the same time exhibits high selectivity and activity toward the FA to be introduced. Reactions were performed in the presence of organic solvents or in solvent-free systems under reduced pressure. With this strategy, mixed ST containing the desired compounds 1,3-dicaprylol-2-oleyl-glycerol or 1,3-dilauroyl-2-oleyl-glycerol (CyOCy or LaOLa) were obtained at 87 and 78 mol% yield, respectively, using immobilized lipases from Burkholderia cepacia (Amano PS-D) in n-hexane at 60°C. However, regiospecific analysis with porcine pancreatic lipase indicated that in CyOCy, 25.7% caprylic acid and in LaOLa 11.1% lauric acid were located at the sn2-position. Oleic acid vinyl ester was a better acyl donor than oleic acid. Esterification of sn1,3-DAG and free oleic acid gave very low yield (<20%) of ST in a solvent system and moderate yield (>50%) in a solvent-free system under reduced pressure.     

Enzymatic preparation of enantiomerically pure sn-2,3-diacylglycerols: A stereoselective ethanolysis approach

Stereoselective ethanolysis of monoacid TAG by immobilized Rhizomucor miehei lipase (RML) was studied for preparation of optically pure sn-2,3-DAG. Trioctanoylglycerol (TO) was used as a model substrate. The enantiomeric purity of the product, sn-2,3-dioctanoylglycerol (sn-2,3-DO), was very high (percent enantiomeric excess >99%) when an excess of ethanol was used. The result indicated that RML was highly stereoselective toward the sn-1 position of TO under conditions of excess ethanol. The stereoselectivity of RML depended on the amount of ethanol. The larger the amount of ethanol was, the higher the stereoselectivity became. After optimizing the parameters such as reactant molar ratio, water content, and temperature, (ethanol/TO molar ratio =31∶1 and water content =7.5 wt% of the reactants at 25°C), optically pure sn-2,3-DO was obtained at 61.1 mol% in the glyceride fraction in 20 min. The above conditions were further applied for ethanolysis of monoacid TAG with different acyl groups such as tridecanoylglycerol (C10∶0), tridodecanoylglycerol (C12∶0), tritetradecanoylglycerol (C14∶0) and trioctadecenoylglycerol [triolein, (C18∶1)]. The yields and enantiomeric purities of 1,2(2,3)-DAG were dramatically reduced when TAG with FA longer than decanoic acid were used.     

Lipase-catalyzed esterification of lactic acid with straight-chain alcohols

Enzymatic synthesis of esters of lactic acid and straight-chain alcohols with different chain lengths (C₆–C₁₈) were investigated in batch reactions with hexadecanol (C₁₆) as the model alcohol. Cyclohexane was the best solvent for higher ester yields, and the best biocatalyst was the immobilized Candida antarctica lipase B (Novozym 435) as well as the textile-immobilized Candida sp. lipase. A method was established to obtain ester yields in the range of 71 to 82% for the different alcohols, and the most favorable conditions for the esterification reaction using Novozym 435 were an equimolar ratio of lactic acid to alcohol, each at a concentration of 120 mM each; a 50°C reaction temperature; 190 rpm shaking speed; and the addition of 100 mg molecular sieves (4 Å) for drying. The ester yield increased with increasing lipase load, and a yield of 79.2% could be obtained after 24 h of reaction at 20 wt% of Novozym 435. The immobilized Candida sp. lipase prepared in the laboratory also could be used to produce esters of lactic acid and straight-chain alcohols, but it had a much lower activity than Novozym 435 with a temperature optimum of 40°C.     

Utilization of reaction medium-dependent regiospecificity of Candida antarctica lipase (Novozym 435) for the synthesis of 1,3-dicapryloyl-2-docosahexaenoyl (or eicosapentaenoyl) glycerol

A highly efficient enzymatic method for the synthesis of regioisomerically pure 1,3-dicapryloyl-2-docosahexaenoyl glycerol (CDC) in two steps was established. 2-Monoglyceride (2-MG) formation by ethanolysis of tridocosahexaenoylglycerol (DDD) with immobilized Candida antarctica lipase (Novozym 435) as catalyst was the key step of the synthesis. CDC was finally obtained by reesterification of 2-MG with ethylcaprylate (EtC) catalyzed by Rhizomucor miehei lipase (Lipozyme IM). The regiospecificity of Novozym 435 depended on the type of reaction and the initial composition of the reaction medium. It displayed strict 1,3-regiospecificity for ethanolysis at a high excess of ethanol in the reaction mixture although it displayed no regiospecificity in transesterification and esterification reactions. The highest yield of CDC (85.4%) was obtained by ethanolysis at a 4∶1 weight ratio of ethanol/DDD for 6 h followed by reesterification at a 20∶1 molar ratio of EtC/initial DDD for 1.5 h. The regioisomeric purity of CDC was 100%. Good results were obtained also for the synthesis of 1,3-dicapryloyl-eicosapentaenoylglycerol (CEC) by the same method: 84.2% yield and 99.8% regioisomeric purity at the same reactant ratios as above. The yield of the reesterification step and the regioisomeric purity of the product were influenced by the molar ratio of the reactants for both CDC and CEC syntheses: higher excess of EtC favored higher yields and regioisomeric purity of the products.     

Comparison of acyl donors for lipase-catalyzed production of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

Synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol (CEC) catalyzed by Lipozyme IM (immobilized Rhizomucor miehei lipase) was performed by interesterification of trieicosapentaenoylglycerol (EEE) with caprylic acid (CA) (acidolysis) and EEE with ethyl caprylate (EtC) (interesterification). Both methods involved two steps: (i) transesterification at an optimized water content and temperature for the high yield conversion of the substrate to CEC, 1-capryloyl-2-eicosapentaenoylglycerol (CEOH) and 2-eicosapentaenoylglycerol (OHEOH), and (ii) reesterification of CEOH and OHEOH to CEC by water removal under reduced pressure. Interesterification had clear advantages over acidolysis. The reaction rates for interesterification were higher and the reaction times shorter. The final yield of CEC by interesterification was higher, and the extent of acyl migration, indicated by the tricapryloylglycerol content, was lower. The disadvantage of the higher price of EtC used for interesterification (approximately 10 times higher than the price of CA) was overcome by synthesizing it directly in the same reaction vessel prior to the interesterification step. EtC was rapidly synthesized by esterification of CA with ethanol in high yield (92% obtained in 2.5 h). The amount of water added to the reaction mixture and the reaction temperature influenced the yields of CEC, CEOH, and OHEOH in the transesterification step for both interesterification and acidolysis methods. The regioisomeric purity of CEC was 100% for both methods at temperatures of 40°C or less. The highest yield of CEC (81%) was obtained for the interesterification of EEE with EtC, formed directly in the same reaction vessel, at a CA/EEE molar ratio of 20∶1 and 30°C.     

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.     

Enzymatic synthesis of 1,3-dicapryloyl-2-eicosapentaenoylglycerol

1,3-Dicapryloyl-2-eicosapentaenoylglycerol (CEC) was synthesized by interesterification of trieicosapentaenoylglycerol (EEE) with ethyl caprylate (EtC) catalyzed by Lipozyme^TM. After some of the reaction conditions were optimized, the maximal molar content of CEC in the glycerides of the reaction mixture was 91%. Among the parameters studied in the optimization, the critical ones were: (i) the water content, which influenced the conversion of EEE to CEC and 1-capryloyl-2-eicosapentaenoylglycerol (CEOH), and (ii) the timing of water removal under reduced pressure for the reesterification of CEOH to form CEC. The complete synthesis of CEC from ethyl eicosapentaenoate (EtE) was performed in three steps: (i) hydrolysis of EtE to free eicosapentaenoic acid (EPA), (ii) esterification of glycerol with EPA to form EEE, and (iii) interesterification of EEE with EtC under the optimized conditions. The first two steps were catalyzed by Novozym^TM and the third by Lipozyme^TM. The total yield over all the steps was 88%, and no purification of the intermediates was necessary. The regioisomeric purity of the product was 100% by silver-ion high-pressure liquid chromatography.     

Enzymatic synthesis of geranyl acetate inn-hexane withCandida antarctica lipases

Geranyl acetate is an important flavor and fragrance compound. Two immobilizedCandida antarctica lipases, SP382 and SP435, were investigated for their use in the synthesis of geranyl acetate by direct esterification. Yields between 95 and 99% molar conversion were obtained with 2 and 15% (w/w reactants) of SP435 and SP382 lipases, respectively. Optimum yields were obtained at 0.1M acetic acid and 0.12M geraniol after 16-h incubation. No inhibitory effect was observed at increasing concentrations of geraniol. Addition of 60% (w/w reactants) water led to 50 and 60% reduction in the esterification activity of SP382 and SP435 lipases, respectively. The best yields were obtained at added water contents between 0–5% (w/w reactants). Solvents with a logP value of 0.85 or more gave reaction yields of more than 80% molar conversion. Higher logP values did not necessarily lead to higher conversion yields. The immobilized lipase SP382 was still active after reusing ten times in the direct esterification reaction.     

Lipase-catalyzed synthesis of isoamyl butyrate: Optimization by response surface methodology

Immobilized lipase from Mucor miehei (Lipozyme IM-20) was employed in the esterification of butyric acid and isoamyl alcohol to synthesize isoamyl butyrate in n-hexane. Response surface methodology based on five-level, five-variable central composite rotatable design was used to evaluate the effects of important variables—enzyme/substrate (E/S) ratio (5–25 g/mol), acid concentration (0.2–1.0 M), alcohol concentration (0.25–1.25 M), incubation period (12–60 h), and temperature (30–50°C)—on esterification yield of isoamyl butyrate. In the range of parameters studied, the extent of esterification decreased with temperature, lower E/S ratios, and incubation periods. Excess acid and alcohol concentrations (i.e., acid/alcohol >1.4 or alcohol/acid >1.4) were found to decrease yield probably owing to inhibition of the enzyme by acid or alcohol, the former being more severe. The optimal conditions achieved are as follows: E/S ratio, 17 g/mol; acid concentration, 1.0 M; incubation period, 60 h; alcohol concentration, 1.25 M; and temperature, 30°C. With these conditions, the predicted value was 1.0 M ester, and the actual experimental value was 0.98 M.     

Enzymatic esterification and transesterification of N-protected Glu or Asp and synthesis of sweetener aspartam analogues by a new glutamic acid specific endopeptidase

Esterification and transesterification reactions are carried out in water miscible organic solvents by using a new glutamic acid specific endopeptidase, isolated from Bacillus lichenifonnis. Sweetener aspartam analogues syntheses are also described.     

Lipase-catalyzed esterification of citronellol with lauric acid in supercritical carbon dioxide/co-solvent media

Direct esterification of citronellol and lauric acid catalyzed by immobilized lipase B from Candida antarctica was performed in supercritical carbon dioxide with different organic solvents and ionic liquids serving as co-solvents. The highest concentration of citronellol laurate after 1 h of reaction performance (3.95 mmol/g substrates) was obtained in SC CO₂ with ethyl methylketone serving as a co-solvent. The optimal temperature and pressure for citronellol laurate synthesis in SC CO₂/EMK medium was determined to be 60 °C and 10 MPa.     

Enzymatic synthesis of oleyl oleate in dense fluids

Esterification between oleic acid and oleyl alcohol, catalyzed by theMucor miehei immobilized lipase in a batch-stirred tank reactor with supercritical carbon dioxide as solvent produced higher reaction rates at supercritical conditions than in the solvent-free system. A continuous fixed-bed reactor was designed based on the results obtained from batch experiments. At 150 bar, 40°C, and with water activity 0.46% w/w, the activity of the enzyme preparation is practically unchanged when CO₂ was used as solvent. The addition of small amounts of water increases the conversion rate. The higher conversion also was observed at longer residence time. Whenn-butane was used as reaction medium, a decrease in conversion was observed.     

Enzymatic synthesis of l-menthyl esters in organic solvent-free system

l-Menthol has been widely used as a food additive and an ingredient of cosmetics, and it is esterified to moderate the strong flavor. We attempted esterification of l-menthol with long-chain unsaturated fatty acid in an organic solvent-free enzymatic system. Commercially available lipases were screened, and Candida rugosa lipase was selected as a catalyst. Several factors affecting the esterification were investigated, and the reaction conditions were determined as follows: A reaction mixture of l-menthol/fatty acid (1:3, mol/mol), 30% water, and 700 units of the lipase per gram of reaction mixture was incubated at 30°C with stirring. After 24 h under these conditions, the esterification extents of l-menthol with oleic, linoleic, and α-linolenic acids reached 96, 88, and 95%, respectively. The structure of the esterified product was confirmed by mass, infrared, and nuclear magnetic resonance spectroscopies. Bacause Candida lipase acted strongly on l-menthol and very weakly on d-menthol, dl-menthol was esterified with oleic acid under the same conditions. The reaction showed high enantioselectivity; the enantiomeric ratio (E) was 31, and enantiomeric excess (ee) of l-menthyl oleate reached 88% after 32 h.     

Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol

The objective of this study was to investigate the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their FA selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40°C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the FA, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. As an example, when FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained after 48 h. The residual FFA contained 78% DHA and only 3% EPA, in 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration.     

Candida sp.99-125脂肪酶及其在化学品合成中的应用

传统的酯化或转酯化产品的合成通常需要高温、强酸、强碱等相对苛刻的条件,脂肪酶由于其生物催化过程具有高效、高选择性、条件温和和环境友好等特点,在化学品的合成中越来越受到人们的关注。本课题组开发了一种可以用于酯类合成的新脂肪酶,并且实现了该酶的工业化生产。来源于Candida sp. 99-125的脂肪酶在非水相中对酯化和转酯化反应具有高效的催化活性和稳定性。本文介绍了该脂肪酶的发酵生产及其在中长链脂肪酸酯、二元酸酯、维生素A棕榈酸酯、手性化合物以及生物柴油等多种化学品的合成中的应用。     

Enzymatic synthesis of high-purity structured lipids with caprylic acid at 1,3-positions and polyunsaturated fatty acid at 2-position

We attempted to synthesize high-purity structured triacylglycerols (TAG) with caprylic acid (CA) at the 1,3-positions and a polyunsaturated fatty acid (PUFA) at the 2-position by a two-step enzymatic method. The first step was synthesis of TAG of PUFA (TriP), and the second step was acidolysis of TriP with CA. Candida antarctica lipase was effective for the first reaction. When a reaction medium of PUFA/glycerol (3∶1, mol/mol) and 5% immobilized Candida lipase was mixed for 24 h at 40°C and 15 mm Hg, syntheses of TAG of γ-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids reached 89, 89, 88, and 83%, respectively. In these reactions, the lipase could be used for at least 10 cycles without significant loss of activity. In the second step, the resulting trieicosapentaenoin was acidolyzed at 30°C for 48h with 15 mol parts CA using 7% of immobilized Rhizopus delemar lipase. The CA content in the acylglycerol fraction reached 40 mol%. To increase the content further, the acylglycerols were extracted from the reaction mixture with n-hexane and were allowed to react again with CA under conditions similar to those of the first acidolysis. After three successive acidolysis reactions, the CA content reached 66 mol%. The content of dicapryloyl-eicosapentaenoyl-glycerol reached 86 wt% of acylglycerols, and the ratio of 1,3-dicapryloyl-2-eicosapentaenoyl-glycerol to 1(3),2-dicapryloyl-3(1)-eicosapentaenoyl-glycerol was 98∶2 (w/w). In this reaction, the lipase could be used for at least 20 cycles without significant loss of activity. Repeated acidolysis of the other TriP with CA under similar conditions synthesized 1,3-dicapryloyl-2-γ-linolenoyl-glycerol, 1,3-dicapryloyl-2-arachidonoyl-glycerol, and 1,3-dicapryloyl-2-docosahexaenoyl-glycerol in yields of 58, 87, and 19 wt%, respectively.