Enzymatic Synthesis of Structured Triacylglycerols Containing CLA Isomers Starting from <Emphasis Type="Italic">sn</Emphasis>-1,3-Diacylglycerols

The synthesis of structured triacylglycerols (TAG) by the enzymatic reaction between sn-1,3-diacylglycerols (sn-1,3-DAG) and conjugated linoleic acid (CLA) isomers was studied. Both the substrates of the reaction were produced from vegetable oils, the sn-1,3-DAG from extra virgin olive oil and the CLA isomers from sunflower oil. The enzymatic reactions between these substrates were catalyzed for 96 h by an immobilized lipase from Rhizomucor miehei (Lipozyme IM) and the reactions carried out in solvent were monitored every 24 h by using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD). The enzymatic reactions were carried out in different reaction media (hexane, isooctane and solvent free) and with different CLA/sn-1,3-DAG ratios. Total % acidic composition and structural analysis data were evaluated to verify the presence of CLA isomers in sn-2- position of synthesized TAG. The results showed good levels of CLA incorporation in sn-1,3-DAG, from 19.2% of TAG synthesized in solvent free conditions with a 0.5:1 substrate ratio, to 47.5% of TAG synthesized in isooctane with a 2:1 substrate ratio. It was observed that for all the reaction media, the best sn-2- acylic specificity was obtained with a 0.5:1 substrate ratio.     

Elucidation of acyl migration during lipase-catalyzed production of structured phospholipids

Elucidation of acyl migration was carried out in the Lipozyme RM IM (Rhizomucor miehei)-catalyzed transesterification between soybean phosphatidylcholine (PC) and caprylic acid in solvent-free media. A five-factor response surface design was used to evaluate the influence of five major factors and their relationships. The five factors—enzyme dosage, reaction temperature, water addition, reaction time, and substrate ratio—were varied on three levels together with two star points. Enzyme dosage, reaction temperature, and reaction time showed increased effect on the acyl migration into the sn-2 position of PC, whereas increased water addition and substrate ratio had no significant effect in the ranges tested. The best-fitting quadratic response surface model was determined by regression and backward elimination. The coefficient of determination (R ²) was 0.84, which indicates that the fitted quadratic model has acceptable qualities in expressing acyl migration for the enzymatic transesterification. Correlation was observed between acyl donor in the sn-2 position of PC and incorporation of acyl donor into the intermediate lysophosphatidylcholine. Furthermore, acyl migration into the sn-2 position of PC was confirmed by TLC-FID, as PC with caprylic acid was observed on both positions. Under certain conditions, up to 18% incorporation could be observed in the sn-2 position during the lipase-catalyzed transesterification.     

An enzymatic method for the synthesis of mixed-acid phosphatidylcholine

The enzymatic synthesis of PC with decanoic acid in the sn-1 and hexanoic acid in the sn-2 position is described. The procedure comprises the following enzymatic steps: (i) treatment of egg yolk with phospholipase A₂ (PLA₂) to hydrolyze egg yolk PC to 1-acyl lysophosphatidylcholine (LPC); (ii) esterification of 1-acyl LPC with hexanoic acid catalyzed by PLA₂ to yield PC with hexanoic acid in the sn-2 position; (iii) removal of the FA in the sn-1 position by lipase-catalyzed ethanolysis to yield 2-hexanoyl LPC; and finally (iv) introduction of decanoic acid in this position by lipase-catalyzed esterification of 2-hexanoyl LPC to yield 1-decanoyl-2-hexanoyl-PC. Two egg yolks with a weight of 16 g were required to obtain 160 mg of the desired product. The chemical purity of the PC product and the positional purity of the FA were around 99%. The method is applicable for the synthesis of other mixed-acid PC species as well.     

Interesterification of butterfat by commercial microbial lipases in a cosurfactant-free microemulsion system

Lipase-catalyzed interesterification of butterfat was carried out in a cosurfactant-free microemulsion system containing mixtures of Span 60 and Tween 60 (ICI Specialty Chemicals Altemix Inc., Brantford, Ontario, Canada) as surfactants. Four commercial lipases were used—Lipozyme 10,000L (Novo Nordisk, Copenhagen, Denmark) and N, D and MPA (Amano Pharmaceutical Co. Ltd., Nagoya, Japan). Stereospecific analyses of fractionated selected high-molecular weight triacylglycerols were performed by enzymatic deacylation with commercial pancreatic lipase, random generation ofrac-1,2-diacylglycerols by Grignard degradation, synthesis ofrac-phosphatidylcholines and a stereospecific release ofsn-1,2 diacylglycerols by phospholipase A₂. The results showed that the hydrolytic affinity of commercial lipases demonstrated an acyl-group specificity toward lower-molecular weight fatty acids C4–C14∶0. Stereospecific analyses of fatty acids of interesterified selected triacylglycerols of butterfat catalyzed by lipase N demonstrated a 46% increase in the proportion of C18∶1cis Δ⁹ at thesn-2 position, whereas those catalyzed by lipases MAP, D and Lipozyme 10,000L were enriched with C16∶0 at the same position by 21, 35 and 41%, respectively.     

Lipolytic synthesis of optically active 1,2-dibutyryl-sn-glycerol. Identification of diglyceride by solvent-dependent specific rotation

The objective was to determine whether the initial pregastric lipase catalyzed hydrolysis of a triacylglycerol to 1,2(2,3)-diacylglycerol was a consequence of sn-specific hydrolysis. The identity of the reaction products for the enzyme-assisted hydrolysis and uncatalyzed acyl-transfer reaction sequence of tributyrylglycerol was assigned by ¹³C nuclear magnetic resonance. The optical activity of the product 1,2-dibutyryl-sn-glycerol (yield >50%, pH 6.5, 35°C, 13 min) was solvent dependent, being −2.92° (c ∼1.3, CHCl₃) and +3.32° (c ∼1.2, pyridine), and confirmation of sn-3 specificity by pregastric lipase was obtained.     

Enzymatic Analysis of Positional Distribution of Fatty Acids in Solid Fat by 1,3-Selective Transesterification with Candida antarctica Lipase B

A protocol for the analysis of the positional distribution of fatty acids (FA) in solid triacylglycerols (TAG) was developed using sn-1(3) selective alcoholysis catalyzed by immobilized Candida antarctica lipase B (CALB). One part by weight of solid fat and ten parts by weight of ethanol (99.5 %) were warmed to liquefy the fat. After adding 0.44 parts by weight of CALB, the mixture was shaken at 50 °C for 10 min then at 30 °C for 2.8 h. The recovery of 2-MAG after the 3-h transesterification reaction was ca. 85 % of the maximum theoretical yield (33 mol%), with the loss of 15 % attributable to the acyl migration from sn-2 to sn-1(3). The recovery was similar to that of the solvent-free alcoholysis of structured lipids, 1,3-dipalmitoyl, 2-oleoyl glycerol and 1,3-dioleoyl, 2-palmitoyl glycerol, conducted at 30 °C for 3 h. In contrast, the acyl migration from sn-1(3) to sn-2 was hardly observed. Because the detected acyl migration was only in the direction of sn-2 to sn-1(3), and not vice versa, it is proposed to determine the FA composition of the sn-2 position of TAG by the gas chromatographic analysis of 2-MAG fraction recovered from the enzymatic reaction mixture, and the FA composition of sn-1(3) position by a mass balance using the FA composition of TAG and of the sn-2 position as inputs. The procedure was successfully applied to palm oil and shea butter, and docosahexaenoic acid (DHA)-rich single cell oil from Aurantiochytrium sp. KH105 for the first time.     

Transesterification of soy lecithin by lipase and phospholipase

Soy lecithin was modified by enzymatic transesterification in a solvent-free system. 1,3-SpecificRhizomucor miehei lipase was found to be efficient in the transesterification with lauric acid and oleic acid, where oleic acid was more incorporated into soy lecithin. Phospholipase A₂ incorporated lauric acid hardly at all, but it hydrolyzed lecithin efficiently. The mixture of lipase and phospholipase A₂ (1:1, w/w) incorporated lauric acid to the same extent as did 1,3-specific lipase alone at the same total enzyme concentration. The main fatty acids replaced were palmitic and linoleic acids by 1,3-specific lipase and its mixture with phospholipase A₂, and linoleic and linolenic acids by phospholipase A₂ alone, suggesting an improved oxidative stability of the resulting product. Hydrolysis could not be prevented, but it could be regulated by incubation time and by enzyme dosage. The minimal water content for significant incorporation of lauric acid into lecithin was below 0.5% of the weight of the reaction mixture.     

Lipase-catalyzed synthesis of chiral triglycerides

Under certain reaction conditions, the acidolysis of tripalmitin with oleic acid using immobilized lipase from Rhizomucor miehei resulted in a higher level of monosubstituted oleoyldipalmitoyl (OPP) triglycerides than had been predicted according to kinetic modeling. The reaction products were subjected to chiral analysis by high-performance liquid chromatography (HPLC), which indicated that the enzyme was more active at the sn-1 position of the triglyceride than at the sn-3 position, resulting in synthesis of the chiral triglyceride 1-oleoyl-2,3-dipalmitoyl-sn-glycerol. A kinetic model was developed and was correlated with the HPLC method to provide a simple means to predict the stereoselectivity of lipase-catalyzed reactions. By using the model, the stereoselectivity of immobilized Rhizomucor miehei lipase was found to depend strongly on the initial water activity (a _w) of the reaction mixture, with greater selectivity occurring at lower a _w. The sn-1 selectivity was essentially maintained using various solvents, or without solvent, when a _w was kept constantly low. Variation in the fatty acid composition of the triglyceride indicated that shorter-chain fatty acids result in greater stereoselectivity, while variation of the chainlength of the free fatty acid indicated an enhancement by the longest chainlength. The stereoselectivity of this lipase was confirmed using a new ¹³C nuclear magnetic resonance method. By using immobilized R. miehei lipase at low a _w approximately 80% of the chiral triglyceride found in the reaction mixture was the sn-1 enantiomer, at high reaction conversion.     

Lipase-catalyzed transesterification of phosphatidylcholine at controlled water activity

The incorporation of a free fatty acid into thesn-1 position of phosphatidylcholine by lipase-catalyzed transesterification was investigated. The thermodynamic water activity of both the enzyme preparation and the substrate solution was adjusted to the same value prior to the reaction. The reaction rate increased with increasing water activity but the yield of modified phosphatidylcholine decreased due to hydrolysis. By using a large excess of the free fatty acid (heptadecanoic acid), the hydrolysis reaction was slowed down, so a higher yield was obtained at a given degree of incorporation. The best results were obtained withRhizopus arrhizus lipase immobilized by adsorption on a polypropylene support. With this preparation, a yield of 60% and nearly 50% incorporation of heptadecanoic acid (100% incorporation in thesn-1 position) was obtained at a water activity of 0.064. The enzyme preparation had good operational stability and position specificity. Little incorporation (<1%) was observed in thesn-2 position, when almost all the fatty acid in thesn-1 position was exchanged.     

Determining the regioselectivity of immobilized lipases in triacylglycerol acidolysis reactions

Lipase regioselectivity is the ability to distinguish between primary (i.e., sn-1,3) and secondary (sn-2) ester functionalities in a triacylglycerol molecule, which is of importance in the manufacture of structured lipids. Unlike existing methods of assessment, which utilize hydrolysis reactions, an alternative technique to assess the regioselectivity of lipases in triacylglycerol transesterification reactions has been developed. An acidolysis reaction is performed between triolein and decanoic, lauric, or stearic acids under conditions that minimize acyl migration, and products are analyzed by silver-ion complexation liquid chromatography, enabling detection of specific triacylglycerol positional isomers. From the rate of formation of these isomers the relative selectivity of the lipase for sn-2 and sn-1,3 ester bonds is determined. With lipases known to lack regioselectivity, the rate of reaction at sn-2 was similar to that at sn-1,3 from the start of the reaction. With sn-1,3 selective lipases, the formation of triacylglycerol isomers with decanoic acid in the secondary position was not detected at any point in the reaction. Regioselectivity as a function of reaction progress was monitored. Two lipases from the genus Pseudomonas exhibited activity toward all positions, but the rate at sn-2 was much reduced, and no incorporation of decanoic acid into this position was detectable until a high degree of conversion had been achieved.     

Enzymatic and chemical synthesis of phosphatidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid

Regioselective incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into phosphatidylcholine (PC) was carried out using enzymatic and chemical synthesis. Incorporation at the sn‐1 position was successfully achieved by lipase‐catalysed esterification of 2‐palmitoyl‐lysophosphatidylcholine (LPC), although in most cases, the enzymes incorporated EPA and DHA at lower rates than other fatty acids. For the incorporation of DHA, Candida antarctica lipase B was the only useful enzyme, while incorporation of EPA was efficiently carried out using either this enzyme or Rhizopus arrhizus lipase. The highest yields in the lipase‐catalysed reactions were obtained at the lowest water activity (close to 0). However, by carrying out the reactions at a higher water activity of 0.22, more EPA and DHA were incorporated. Esterification of 2‐palmitoyl‐LPC with pure EPA at this water activity converted 66 mol‐% of LPC to PC using Rhizopus arrhizus lipase as catalyst. When the fatty acid was DHA and the catalyst Candida antarctica lipase B, 45 mol‐% of PC was obtained. For incorporation of EPA and DHA at the sn‐2 position, phospholipase A₂ was used, but the reaction was very slow. Chemical coupling of 1‐palmitoyl‐LPC and EPA or DHA was more efficient, resulting in complete conversion of LPC.     

Thermoxidation of substrate models and their behavior during hydrolysis by porcine pancreatic lipase

The behavior of thermoxidized triacylglycerols during hydrolysis catalyzed by porcine pancreatic lipase was evaluated using nonpolar triacylglycerols isolated from palm olein (NPTPO), triolein, and sn-1,3 diolein substrates. Substrates were thermoxidized at 180°C for 1 to 4 h. Owing to formation of polymers and dimers of triacylglycerols, the molecular weight of the thermoxidized substrates increased. After 1 h heating, the concentration of polymers and dimers was similar for the sn-1-3 diolein and triolein samples but higher in NPTPO samples. Conjugated double bonds were formed in all samples, and α,β-unsaturated carbonyl compounds developed through allylic oxidations. These caused increased ultraviolet absorbance at 232 nm. The hydrolysis of heated and unheated samples by the lipase can be described by a Michaelian equation. The enzyme showed a higher apparent V _max and K _M with heated sn-1,3 diolein and triolein than with their unheated counterparts. This was due to the generation of polar compounds which acted as emulsifiers and which favored the formation of an oil/water microemulsion. This behavior was not observed in NPTPO, where heating decreased the apparent V _max and K _M over the first 2 h. Later, a tendency to increase these values was observed. The results could be explained by a balance between concentration of surfactants and of natural emulsifiers in the thermoxidized samples.     

Enzymatic synthesis of low-calorie structured lipids in a solvent-free system

For the synthesis of low-calorie structured lipids (LCSL), transesterification between triacetin and stearic acid using immobilized lipase in a solvent-free system was investigated. Stearic acid, a long-chain saturated fatty acid, was incorporated mainly into the sn-1 and/or sn-3 positions of triacetin by lipase-catalyzed reaction. Three types of reactor systems (open, closed, and vacuum) were studied for the production of LCSL. The effects of various reaction variables such as water activity of substrates and lipase, molar ratio of substrates, stirring speed and reaction temperature were investigated. In the vacuum reactor system, a certain amount of water was added periodically to maintain the optimal water content of the reaction system. When a suitable amount of water (0.65 wt% of substrates) was added at every 1 h into the vacuum reactor system, more than 88% LCSL was obtained within 4 h using Chirazyme^® L-2.     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Optimization of the reaction conditions in the lipase-catalyzed synthesis of structured triglycerides

Structured triglycerides of the ABA-type, containing one type of fatty acid (A) in the sn-1 and sn-3 positions and a second type of fatty acid (B) in the sn-2 position of the glycerol, were synthesized using lipases. The highest yields and purities were achieved in a two-step process, where a triglyceride of the B-type was subjected to an alcoholysis reaction in an organic solvent catalyzed by sn-1,3-regiospecific lipases yielding the corresponding 2-monoglyceride (2-MG). Using this strategy, e.g., 2-monopalmitin (2-MP) was obtained in up to 88% yield at >95% purity by crystallization. Esterification of 2-MP with oleic acid resulted in the formation of 1,3-oleyl-2-palmitoyl-glycerol in up to 72% yield containing 94% palmitic acid in the sn-2 position. The best lipases were from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus. Water activity, solvent, and carrier for lipase immobilization strongly influenced the yield and purity of the products in both steps. Furthermore, 2-MG from fish oil were produced by alcoholysis in up to 84% yield at >95% purity.     

Identification of triacylglycerols in the enzymatic transesterification of rapeseed and butter oil

A blend of rapeseed and butter oil was transesterified using immobilized Thermomyces lanuginosus lipase (Lipozyme® TL IM) as catalyst. The reaction was followed by reversed-phase HPLC and the triacylglycerol peaks were tentatively identified from their elution times by calculating equivalent carbon numbers. Further identification was made using HPLC-electrospray tandem mass spectrometry. A few of the triacylglycerols detected were typical combinations of fatty acids originating from rapeseed oil, such as α-linolenic acid, and short-chain fatty acids from butter oil. Due to the regioselectivity of the lipase, the transesterification reaction involved mainly fatty acids in the sn-1 and sn-3 positions. However, significant changes in the fatty acid composition in the sn-2 position were detected after 6 h.     

Measuring stereoselectivity in lipase-catalyzed acidolysis reactions by ultra-high resolution 13C nuclear magnetic resonance

Elucidating the stereoselectivity of lipases in synthetic reactions of triacylglycerols has hitherto been carried out using traditional analytical techniques to determine the composition of the reaction products. These methods are laborious and are not always appropriate for analysis of certain triacylglycerol types. A direct method, utilizing a stereospecific deuterium-labeled triacylglycerol substrate, has been developed where the stereoisomeric composition of the reaction product is determined by ultra-high resolution ¹³C nuclear magnetic resonance (NMR) spectroscopy. Through lipase-catalyzed transesterification of deuterium-labeled trilauroylglycerol with oleic acid, chemical shifts were induced in the ¹³C NMR spectrum by the deuterium atom and olefinic double bonds, enabling unambiguous stereospecific assignment of triacylglycerol species. By this method of analysis, we found an effect of the degree of reaction conversion on the extent of stereoisomerism in the triacylglycerol product. Stereoselectivity was greatest (for sn-1) with lipase from Rhizomucor miehei. Lipases from Rhizopus niveus, Candida rugosa, Carica papaya, and the cutinase from Fusarium sp. were also found to exhibit stereoselectivity, with preference for either sn-1 or sn-3 acyl exchange.     

Production of margarine fats by enzymatic interesterification with silica-granulated Thermomyces lanuginosa lipase in a large-scale study

Interesterification of a blend of palm stearin and coconut oil (75∶25, w/w), catalyzed by an immobilized Thermomyces lanuginosa lipase by silica granulation, Lipozyme TL IM, was studied for production of margarine fats in a 1- or 300-kg pilot-scale batch-stirred tank reactor. Parameters and reusability were investigated. The comparison was carried out between enzymatic and chemical interesterified products. Experimentally, Lipozyme TL IM had similar activity to Lipozyme IM for the interesterification of the blend. Within the range of 55–80°C, temperature had little influence on the degree of interesterification for 6-h reaction, but it had slight impact on the content of free fatty acids (FFA). Drying of Lipozyme TL IM from water content 6 to 3% did not affect its activity, whereas it greatly reduced FFA and diacylglycerol contents in the products. Lipozyme TL IM was stable in the 1-kg scale reactor at least for 11 batches and the 300-kg pilot-scale reactor at least for nine batches. Due to regiospecificity of the lipase (sn-1,3 specific), enzymatically interesterified products had different fatty acid distribution at sn-2 position from the chemically randomized products, implying the potential nutritional benefits of the new technology. Presented at the 91st American Oil Chemists' Society Annual Meeting in San Diego, April 28, 2000.     

Synthesis and Structural Analysis of Structured Triacylglycerols with CLA Isomers in the <Emphasis Type="Italic">sn</Emphasis>-2- Position

The present research deals with the chemical esterification of the sn-2- position of sn-1,3-diacylglycerol (sn-1,3-DAG) with 9cis,11trans (c9,t11) and 10trans,12cis (t10,c12) conjugated linoleic acid (CLA) isomers to obtain structured triacylglycerols (TAG); the sn-1,3-DAG substrates were produced from extra virgin olive oil by means of enzymatic reactions while CLA isomers were obtained using a three-step procedure based on alkaline hydrolysis of sunflower oil, urea purification of linoleic acid (LA) and alkaline isomerization of LA. The results showed good levels of CLA incorporation in structured TAG at the tested temperatures: 37.5% at 4 °C and 39.1% at 14 °C. To evaluate the incorporation of CLA isomers in sn-2- position of sn-1,3-DAG structural analysis of the newly synthesized TAG was carried out using an enzymatic and a chemical method. The results of the structural analysis also showed up the occurrence of acyl migration. The pancreatic lipase method allowed the direct determination of the fatty acid composition of TAG sn-2- position but this enzymatic method showed different results (p < 0.05) in respect to the chemical one; this occurrence could be due to an acylic specificity of the lipase. High incorporation of CLA isomers in sn-2- position of TAG was observed, 77.0% at 4 °C and 81.5% at 14 °C, considering the results of the chemical procedure.     

Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides

Structured triacylglycerides with medium-chain fatty acids (caprylic acid) in sn1- and sn3-positions and a long-chain unsaturated fatty acid (oleic or linoleic acid) in the sn2-position of glycerol (MLM) were synthesized by lipase catalysis in a two-step process. First, pure 2-monoacylglycerides (2-MG) were synthesized by alcoholysis of triacylglycerides (triolein, trilinolein, or peanut oil) in organic solvents with 1,3-regiospecific lipases (from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus). The 2-MG were purified by crystallization and obtained in up to 71.8% yield. These 2-MG were esterified in a second reaction with caprylic acid in n-hexane to form almost pure MLM. For 2-MG obtained from peanut oil, the final product contained more than 90% caprylic acid in the sn1- and sn3-positions, whereas the sn2-position was composed of 98.5% unsaturated long-chain fatty acids. Reaction conditions for both steps were optimized with respect to source and immobilization of lipase, water activity, and solvent.