Glyceride Synthesis Research Articles

Response surface optimisation of lipase-catalysed esterification of glycerol and n-3 polyunsaturated fatty acids from salmon oil

Enzymatic synthesis of acylglycerols from glycerol and n-3 fatty acid concentrate, prepared from salmon oil, was optimised by response surface methodology (RSM). Salmon oil was hydrolysed using a stereospecific sn-1, sn-3 lipase from Aspergillus oryzae and filtered on hydrophobic membrane. Acylglycerols synthesis was performed using Lipozyme IM (Novo Nordisk) under different reaction conditions according to a Doehlert experimental design. Effects of temperature, water content, glycerol/fatty acid molar ratio, and enzyme/substrate ratio were studied. Pathway maximisation indicated that an esterification degree of 97% was obtained under the following conditions: glycerol/oil 1.2, temperature 46 °C, molecular sieves 3.5 g per 100 ml and enzyme/substrate 8.1.

Synthetic Applications of Enzymes Entrapped in Reverse Micelles & Organo-GelsΨ

The reverse micellar medium consists of tiny water droplets stabilized by surfactants in a bulk water-immiscible, organic solvent. The solutions are optically transparent and almost any enzyme can be solubilized in reverse micelles without loss of its specific activity. Reverse micelles provide a unique microenvironment for enzymes to react with water insoluble or poorly soluble compounds present in the organic phase. It is possible to use even multi-enzyme systems to carry out a sequence of transformations. The presence of surfactant in the medium helps to overcome diffusion resistance and enzymes entrapped in hydrophilic gels such as polyacrylamide or calcium alginate can also be used for biotransformation of hydrophobic substrates and recycled several times. The solubilization of gelatin in reverse micelles to form organogels and solubilization of microbial cells in water-in-oil microemulsions without loss of viability have opened up new possibilities. Entrapped enzymes in such organogels or ȁ 9;gelozymes‘ can be used in organic solvents without the use of surfactant, simplifying the workup procedure. Enzymatic reactions requiring co-factors such as reduction with alcohol dehydrogenase and epoxidation with monooxygenase can be carried out with whole cells. This review discusses various applications of enzymes entrapped in reverse micelles and organogels for resolution of amino acids, peptide synthesis, reduction of prochiral ketones, synthesis of glycerides and chiral intermediates useful in production of agrochemicals and pharmaceuticals. The possibilities of substrate isolation, surfactant reuse and enzyme recycling with the help of bioreactors are also discussed. Keywords: enzymes, reverse micelles, organo-gels, peptide synthesis, acylase, cephalexin, chymotrypsinbetamethasone

Chloroplastic Glyceride Isoform of Dihydroxyacetone Phosphate Reductase from Dunaliella tertiolecta: Purification and Characterization

The chloroplastic glyceride isoform of dihydroxyacetone phosphate reductase (Gly-DHAPR) in the photosynthetic unicellular green algae, Dunaliella, plays key role in the synthesis of glycerol-P and glycerides. A four-step procedure has been developed to purify the Gly-DHAPR from the chloroplasts of Dunaliella tertiolecta. The enzyme was purified 462-fold to apparent electrophoretic homogeneity by precipitation of Rubisco by polyethylene glycol-4000, and successive chromatography on DEAE cellulose, Sephacryl S-200, and Red Agarose. The overall yield of the purified enzyme was 5.1% with a specific activity of 425 μmol. min−1. mg−1 protein, and a subunit molecular mass of 37 kD. The Gly-DHAPR had little preference for NADH or NADPH, but was highly specific for DHAP. The purified enzyme was slightly stimulated by 50 mM NaCl, KCl or by 25 mM MgCl2. Detergents, lipids, fatty acids, or long-chain acyl-CoA derivatives inhibited the Gly-DHAPR. The Gly-DHAPR differs in properties from the other chloroplastic osmoregulatory isoform of DHAP reductase from Dunaliella, but has significant similarities with the glyceride isoforms from higher plants for glycerol-P and triglyceride synthesis.

Comparative Fatty Acid Selectivity of Lipases in Esterification Reactions with Glycerol and Diol Analogues in Organic Media

Reaction selectivity of Pseudomonas cepacia, Rhizomucor miehei, and Candida antarctica B lipases was assessed in multicompetitive esterification reaction mixtures containing an homologous series of n-chain even carbon number fatty acid (FA; C4-C18) substrates and a single alcohol cosubstrate (glycerol, 1,2-propanediol (1,2-PD), or 1, 3-propanediol (1,3-PD)) in tert-butyl methyl ether at water activity of 0.69 or 0.90 and a reaction temperature of 35 degrees C. For P. cepacia lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C16 > other FA; with 1,2-PD and 1, 3-PD, C16 > C8 > C14 > other FA. For R. miehei lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C12 > C10, C14 > other FA; with 1,2-PD and 1,3-PD, C8 > C12 > other FA. For C. antarctica B lipase, the ordinal patterns of FA selectivities observed were, with glycerol, C8 > C10, C6, C12 > other FA; with 1, 2-PD, C8 > C10, C6 > other FA; and with 1,3-PD, C8 > C10 > C6 > other FA. The differences in selectivity among FA ranged up to 16-fold, depending upon the lipase and alcohol cosubstrate used. These findings represent intrinsic and substrate-modulated features of FA selectivities that are of particular relevance to the use of lipases for acylglycerol synthesis reactions.

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil.

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.

Sterol carrier protein-2 suppresses microsomal acyl-CoA hydrolysis.

Although sterol carrier protein 2 (SCP-2) has long been regarded primarily as a sterol transfer protein, its actual physiological function is not known. The recent discovery that SCP-2 binds long chain fatty acyl-CoAs (LCFA-CoAs) with high affinity suggests additional roles for SCP-2 in cellular utilization of LCFA-CoAs for synthesis of glycerides and cholesterol esters. Concomitant to these anabolic pathways, LCFA-CoAs are also degraded by cellular hydrolases. The purpose of the work presented herein was to determine if SCP-2 altered the aqueous pool of LCFA-CoA by (i) extracting LCFA-CoA from microsomal membranes, and (ii) protecting LCFA-CoA from microsomal hydrolase activity. The data demonstrated for the first time that SCP-2 increases the aqueous pool of oleoyl-CoA by increasing the aqueous/membrane distribution oleoyl-CoA by 2.4-fold. In addition, SCP-2 inhibited the hydrolysis of oleoyl-CoA by microsomal acyl-CoA hydrolase 1.6-2.4 fold, depending on the concentration of oleoyl-CoA. By simultaneously extracting LCFA-CoA from membranes and inhibiting LCFA-CoA degradation SCP-2 may potentiate LCFA-CoA transacylation and modulate the role of LCFA-CoAs as intracellular signaling molecules.

Bacterial Biocatalysts: Molecular Biology, Three-Dimensional Structures, and Biotechnological Applications of Lipases

Bacteria produce and secrete lipases, which can catalyze both the hydrolysis and the synthesis of long-chain acylglycerols. These reactions usually proceed with high regioselectivity and enantioselectivity, and, therefore, lipases have become very important stereoselective biocatalysts used in organic chemistry. High-level production of these biocatalysts requires the understanding of the mechanisms underlying gene expression, folding, and secretion. Transcription of lipase genes may be regulated by quorum sensing and two-component systems; secretion can proceed either via the Sec-dependent general secretory pathway or via ABC transporters. In addition, some lipases need folding catalysts such as the lipase-specific foldases and disulfide-bond-forming proteins to achieve a secretion-competent conformation. Three-dimensional structures of bacterial lipases were solved to understand the catalytic mechanism of lipase reactions. Structural characteristics include an alpha/beta hydrolase fold, a catalytic triad consisting of a nucleophilic serine located in a highly conserved Gly-X-Ser-X-Gly pentapeptide, and an aspartate or glutamate residue that is hydrogen bonded to a histidine. Four substrate binding pockets were identified for triglycerides: an oxyanion hole and three pockets accommodating the fatty acids bound at position sn-1, sn-2, and sn-3. The differences in size and the hydrophilicity/hydrophobicity of these pockets determine the enantiopreference of a lipase. The understanding of structure-function relationships will enable researchers to tailor new lipases for biotechnological applications. At the same time, directed evolution in combination with appropriate screening systems will be used extensively as a novel approach to develop lipases with high stability and enantioselectivity.

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 Km 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

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 10h. 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.

AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target

AMP-activated kinase (AMPK) is activated in response to metabolic stresses that deplete cellular ATP, and in both liver and skeletal muscle, activated AMPK stimulates fatty acid oxidation. To determine whether AMPK might reciprocally regulate glycerolipid synthesis, we studied liver and skeletal-muscle lipid metabolism in the presence of 5-amino-4-imidazolecarboxamide (AICA) riboside, a cell-permeable compound whose phosphorylated metabolite activates AMPK. Adding AICA riboside to cultured rat hepatocytes for 3 h decreased [14C]oleate and [3H]glycerol incorporation into triacylglycerol (TAG) by 50% and 38% respectively, and decreased oleate labelling of diacylglycerol by 60%. In isolated mouse soleus, a highly oxidative muscle, incubation with AICA riboside for 90 min decreased [14C]oleate incorporation into TAG by 37% and increased 14CO2 production by 48%. When insulin was present, [14C]oleate oxidation was 49% lower and [14C]oleate incorporation into TAG was 62% higher than under basal conditions. AICA riboside blocked insulin's antioxidative and lipogenic effects, increasing fatty acid oxidation by 78% and decreasing labelled TAG 43%. Similar results on fatty acid oxidation and acylglycerol synthesis were observed in C2C12 myoblasts, and in differentiated C2C12 myotubes, AICA riboside also inhibited the hydrolysis of intracellular TAG. These data suggest that AICA riboside might inhibit sn-glycerol-3-phosphate acyltransferase (GPAT), which catalyses the committed step in the pathway of glycerolipid biosynthesis. Incubating rat hepatocytes with AICA riboside for both 15 and 30 min decreased mitochondrial GPAT activity 22-34% without affecting microsomal GPAT, diacylglycerol acyltransferase or acyl-CoA synthetase activities. Finally, purified recombinant AMPKalpha1 and AMPKalpha2 inhibited hepatic mitochondrial GPAT in a time-and ATP-dependent manner. These data show that AMPK reciprocally regulates acyl-CoA channelling towards beta-oxidation and away from glycerolipid biosynthesis, and provide strong evidence that AMPK phosphorylates and inhibits mitochondrial GPAT.

Lipase-catalyzed esterification of glycerol with n-3 polyunsaturated fatty acids from winterized fish oil Esterificación catalítica de glicerol con ácidos grasos 3-n poliinsaturados de aceite de pescado

Menhaden oil was hydrolyzed using a lipase from Pseudomonas sp. The hydrolysate was cold frac tionated at-72°C. Glyceride synthesis was performed using the same lipase under different reaction environments. The best conditions for the esterification reaction were 39 °C for 18 h in a reaction mixture containing anhydrous glycerol, n-3 polyunsaturated fatty acids (PUFA) enriched solution (2% lipids in hexane), hexane, and phosphate buffer-lipase solution (1% w/v). Product composition was 81.33% triacylglycerides and 18.67% of free fatty acids (w/w). Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) accounted for 36.18% of the esterified fatty acids, of which 58% was EPA and 42% was DHA. This method offers an alternative to produce glycerides rich in n-3 PUFA.

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO 3 powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO 3 powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.

Enzymatic glyceride synthesis in a foam reactor

AbstractWe report the results of our study on Rhizomucor miehei lipase‐catalyzed lauric acid‐glycerol esterification in a foam reactor. A satisfactory yield of glyceride synthesis can be achieved with an unusually high initial water content (50% w/w). We found that product formation could be regulated by controlling foaming. Foaming was a function of the air flow rate, reaction temperature, pH value, ionic strength, and substrate molar ratio. Monolaurin and dilaurin, which constituted nearly 80% of the total yield, were the two dominant products in this reaction; trilaurin was also formed at the initial stages of the reaction. A study of pH and ionic strength effects on an independent basis revealed that they affect the interfacial mechanism in different manners. On varying the ratio of lauric acid and glycerol, only a slight change in the degree of conversion was detected and the consumption rate of fatty acid was approximately the same.

Alcoholysis and Glyceride Synthesis with Immobilized Lipase on Controlled-Pore Glass of Varying Hydrophobicity in Supercritical Carbon Dioxide

Lipases from Humicola lanuginosa and Candida antarctica lipase B were adsorbed onto methylated controlled-glass beads with varying degrees of hydrophobicity. The effect of support hydrophobicity on the amount of lipase adsorbed was studied. The immobilized lipases were examined for their activity in an alcoholysis reaction of cod liver oil with ethanol in supercritical carbon dioxide. Furthermore, the activity of the immobilized Humicola lanuginosa lipase in glyceride synthesis in supercritical carbon dioxide was studied. For both lipases investigated, the amount adsorbed decreased with decreasing hydrophobicity of the support; moreover, for Humicola lanuginosa lipase, the lipolytic activity was found to depend largely on the substrate and the hydrophobicity of the immobilization support. In the alcoholysis reaction, a highly hydrophobic carrier enhanced lipase activity whereas in glyceride synthesis, the reverse effect was observed. For Candida antarctica lipase, the support hydrophobicity did not affect the activity of the lipase. In addition, the results showed that the lipid class composition of the reaction mixture reflected the lipolytic activity of the lipases immobilized on the different supports tested.

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

AbstractEnzymatic 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.

Glyceride synthesis in a solvent‐free system

AbstractSynthesis of partial glycerides in a solvent‐free system has been investigated with various acyl donors and glycerol as substrates and a 1,3‐specific immobilized lipase to catalyze the reaction. Capric acid was the most efficient acyl donor, compared with ethyl caprate and tricaprin. However, to obtain a high yield of dicaprin and a low amount of tricaprin, ethyl caprate was the acyl donor of choice. The composition of the product mixture was determined by the ratio of ethyl caprate to glycerol; a molar ratio of 3∶1 was optimum for dicaprin synthesis. The water content in glycerol did not influence the final yield of dicaprin, but initial production of capric acid increased with increasing water content. The reaction was found to be controlled entirely by external mass transfer. The yield of diglyceride could be increased from 70 to 90% by lowering the reaction temperature, so that the diglyceride precipitated during the reaction.

Glyceride synthesis catalyzed by cutinase using the monomolecular film technique.

The monomolecular film technique previously used to study the kinetics of lipase hydrolysis was adapted to synthesizing oleoyl glycerides (monoolein, diolein, and triolein). The water subphase was replaced by glycerol, and a film of oleic acid was initially spread on the glycerol surface. In this system a recombinant cutinase from Fusarium solani was able to catalyze oleoyl glyceride synthesis. More than 50% of the oleic acid film was acylated after 7 min of reaction. The surface pressure applied to the monomolecular film acts as a physical selectivity factor since glyceride synthesis can be steered so as to produce either diolein or triolein.

Effects of insulin treatment of diabetic rats on hepatic partitioning of fatty acids between oxidation and esterification, phospholipid and acylglycerol synthesis, and on the fractional rate of secretion of triacylglycerol in vivo

1. The hypothesis that insulin treatment of streptozotocin-diabetic rats does not alter acutely the ability of acylcarnitine synthesis to compete successfully for cytosolic long-chain acyl-CoA [Grantham and Zammit (1988) Biochem. J. 249, 409-414], was tested in vivo by using the technique of selective labelling of hepatic fatty acids in awake unrestrained rats. In the same animals, the partitioning of hepatic fatty acids between acylglycerol and phospholipid synthesis, and of newly labelled triacylglycerol between secretion into the plasma and retention in the liver, was also studied. 2. In untreated diabetic animals, the ratio of fatty acid oxidation to esterification was double that found in normal fed animals, whereas there were no differences in the values of the above-mentioned parameters of glycerolipid metabolism. Thus the insulin status of the rats only has chronic effects on specific aspects of fatty acid metabolism in the liver. 3. Treatment of diabetic rats with insulin resulted in no change in the oxidation/esterification ratio for the first 5 h after the start of insulin administration. Thereafter, there were reciprocal changes in the 14CO2 expired (an index of oxidation) and 14C label recovered in hepatic and plasma glycerolipids. However, the pattern of partitioning observed in normal fed rats was still not re-established after 8 h of insulin treatment. 4. There was a small and transient decrease in the fractional rate of triacylglycerol secretion by the liver at the start of insulin treatment and an increase in the proportion of labelled fatty acid that was utilized for phospholipid synthesis such that phospholipid labelling as a proportion of that of total glycerolipids was doubled after 8 h of insulin treatment. 5. The data are discussed in relation to the roles of insulin in mediating acute changes in hepatic fatty acid metabolism and very-low-density-lipoprotein triacylglycerol secretion by the liver.

Fatty acid synthesis: a potential selective target for antineoplastic therapy.

OA-519 is a prognostic molecule found in tumor cells from breast cancer patients with markedly worsened prognosis. We purified OA-519 from human breast carcinoma cells, obtained its peptide sequence, and unambiguously identified it as fatty acid synthase through sequence homology and enzymology. Tumor fatty acid synthase is an approximately 270-kDa polypeptide which specifically abolished immunostaining of human breast cancers by anti-OA-519 antibodies. Tumor fatty acid synthase oxidized NADPH in a malonyl-CoA-dependent fashion and synthesized fatty acids composed of 80% palmitate, 10% myristate, and 10% stearate from acetyl-CoA, malonyl-CoA, and NADPH with a specific activity of 624 nmol of NADPH oxidized per min per mg. Tumor cell lines with elevated fatty acid synthase showed commensurate increases in incorporation of [U-14C]acetate into acylglycerols demonstrating that fatty acid synthase increases occur in the context of overall increases in endogenous fatty acid synthesis. Cerulenin inhibited acylglycerol synthesis in tumor cells and fibroblast controls in a dose-dependent fashion and also caused a growth inhibition which generally paralleled the level of endogenous fatty acid synthesis. Supraphysiologic levels of palmitate, 14 microM in dimethyl sulfoxide, significantly reversed the growth inhibition caused by cerulenin at concentrations of up to 5 micrograms/ml, indicating that cerulenin-mediated growth inhibition was due to fatty acid synthase inhibition.

A comparison of different strategies for lipase-catalyzed synthesis of partial glycerides

Four different approaches for the synthesis of monolaurylglycerol (MLG) by non specificPseudomonas cepacia lipase in a crude and purified form have been studied: a. The direct esterification of glycerol by lauric acid in bis-(2-ethylhexyl)sulfosuccinate sodium salt (AOT)/isooctane microemulsion systems; b. the transesterification of glycerol by vinyl laurate in the presence or not of any solvent; c. solid-phase glycerolysis of trilaurin; and, d. transesterification of protected glycerol, 1,2-O-isopropylidene glycerol, by vinyl laurate, in the presence or not of any solvent. It was found that in the two latter cases (d and c) the formation of pure MLG was occurred, while in the first two cases (a and b) apart of MLG the formation of DLG was also observed.