Rapid enzymatic production of acylglycerols from conjugated linoleic acid and glycerol in a solvent-free system

Rates and product distributions have been determined for the consecutive esterification reactions of conjugated linoleic acid (CLA) with glycerol in the presence of an immobilized Candida antarctica lipase from Novo Nordisk (Lipozyme IM). In a solvent-free environment, both rates and product distributions are affected by the ratio of reactants, temperature, and hydration level. For mole ratios of CLA to glycerol ranging from 1/1 to 3/1, incorporation of at least 95% of the original CLA into the product acylglycerols was achieved in less than 7 h of reaction at 50°C. © Rapid Science Ltd. 1998     

The biocatalytic activity of bromelain in ester synthesis reaction: difference between the intrinsic lipase activity and thermal catalysis

Biocatalytic activities of bromelain preparations were carried out in proteolytic (4500 units g^–1), lipolytic (67 units g^–1) and, more particularly, in fatty acid ester synthetic reactions. The ester synthesis reactions were studied and several thermodynamic parameters and non-biological reference reactions were also investigated. Only temperature had a strong influence on the maximum reaction yield (30% after 10 days) and revealed that thermal catalysis, which exists in esterification, raises doubts concerning the real biocatalytic activity of the plant extract. When this thermal catalysis is taken into account, the intrinsic lipase activity of the bromelain preparations in esterification reactions is nil.     

Growth Inhibition by Phenylalanine in Euglena gracilis

The hydrolysis and esterification by a thermostable lipase from Humicola lanuginosa No. 3 were investigated. Both reactions occurred readily at temperatures between 45~50°C. Esterification by the enzyme with glycerol was observed to be specific towards fatty acids with carbon numbers of C12~C18. Laurie acid esters with different alcohols such as primary alcohols, terpene alcohols, eie., were also synthesized readily. Esterification by the enzyme was adversely affected by the water content (optimum, ca. 7%), however, the hydrolysis rate increased rapidly with increasing water content (optimum, az. 60%). The enzyme showed increased activity in organic solvent-aqueous reaction systems. Nevertheless, hydrolysis in complete organic phase reactions was found not to be feasible. Hydrolysis at a higher temperature (50 or 55°C) in a solvent free phase was almost the same as that in organic solvent-aqueous phase reactions. The components of glycerides varied considerably during hydrolysis, whereby esterification resulted in a higher quantity of mono- and diglycerides (about 40%), compared to in the case of hydrolysis, for which the value was about 10~20%.     

Emulsifiers from solid and liquid polyols: different strategies for obtaining optimum conversions and selectivities

The present paper provides a general overview of the factors involved in both the kinetics and the selectivity of partial acylation reactions of polyols (sugars). Different kinetic strategies for maximum production of intermediate esters of various polyols and monosaccharides are reported and discussed. Physicochemical requirements for obtaining maximum selectivities and complementary strategies for reducing reaction times are discussed. The reactions studied include glycerol, glucose, fructose, mannose, sorbitol and an alkyl glucoside as precursors. The high selectivity towards the monoglyceride in the presence and absence of a solvent has been attributed to a combination of the precipitation of the desired ester, use of glycerol in excess and the relatively low solubility of the fatty acid in the system. Unlike the reaction in the presence of a solvent, the reaction in a solvent-free medium produces the diester first. The monoester only accumulates in the medium as a consequence of disproportionation and glycerolysis reactions of the diester formed earlier. Selective esterification of solid sugars (polyols) which have an intermediate solubility in acetone is favored at low temperatures at which a sufficient amount of polyol dissolves and concurrent precipitation of the desired product can be achieved. By contrast, use of elevated temperatures is more appropriate for selective partial esterification of polyols, which are the most soluble in the solvent employed. Polyols (sugars) which are the less soluble into the liquid reaction phase cannot be easily esterified. Diffusional limitations on the rate of dissolution of the solid precursor can be minimized by increasing the surface area of the solid polyol.     

The control of lipase-catalysed transesterification and esterification reaction rates. Effects of substrate polarity, water activity and water molecules on enzyme activity

The reaction rate of two lipase-catalysed reactions, esterification and transesterification, were studied in a liquid/solid two-phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents. Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non-specific C. cylindracea lipase-catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of water-saturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity. Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.     

Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida sp. 99–125 in organic solvent system

In this study, hydrolysis and methanolysis of glycerol trioleate (TG) by lipase Candida sp. 99–125 were investigated under different water conditions. Both the reaction rates were relatively low without water, while increasing water content to 5 wt.% (or more, from 10–20%) based on the TG amount caused remarkable higher TG conversion for both reactions. Moreover, comparing the time course curves of the hydrolysis and methanolysis, it could be concluded that the methanolysis reaction catalyzed by this Candida sp. 99–125 appeared to accord with the successive reaction mechanism. TG was first hydrolyzed to partial glycerides and oleic acid (OA), then oleic acid methyl ester (OAME) was produced by esterification of the OA with methanol. This water effect was also confirmed by the experiments that water substitutions such as t-butanol and some surfactants added into the system did not get such high yields as that of the water included system. So these results showed that water took part in the methanolysis reaction, and successive hydrolysis–esterification process might be the catalytic mechanism of this lipase.     

Rice bran lipase catalyzed esterification of palm oil fatty acid distillate and glycerol in organic solvent

Rice bran lipase (RBL) was delipidated to enhance its stability in organic solvent and its esterification activity at elevated temperature. The esterification activity of delipidated RBL increased as temperature was increased from 45 to 65°C. The esterification activity of delipidated RBL at 65°C was about 14 times greater than that of the non-delipidated RBL. As temperature was further increased to 75°C, the non-delipidated RBL lost all esterification activity, whereas the delipidated RBL retained approximately 48% of its esterilication activity. The delipidated RBL maintained a relative esterification activity greater than 80% after 16 h of incubation in hexane, whereas the non-delipidated RBL maintained a relative esterification activity of only 50%. A method for production of acylglycerol using delipidated RBL to esterify palm oil fatty acid distillate (PFAD) with glycerol in hexane was successfully developed. The effects of reaction temperatures and type of water removal agents (silica gel and molecular sieve) on the degree of esterification were also examined. A 4 h reaction at 65°C, catalyzed by delipidated RBL and using silica gel as the water removal agent resulted in 53.8% esterification. Thin layer chromatography analysis suggested that the esterified product was primarily comprised of mono-and di-acylglycerols.     

非水相酶促合成癸酸偏甘油酯的研究

对无溶剂非水相中癸酸与甘油的酶促酯化反应进行了研究,发现Pseudomonas fluoresces脂肪酶（PFL）、Mucor miehei脂肪酶（MML）和Candida antarictica脂肪酶（CAL）均有较好的催化活性。CAL酶促转化癸酸的最适反应条件为：60℃,加酶量为20～100u/g,初始加水量为甘油质量的12%。CAL的1,3位置专一性在最终产物中未表达。CAL酶催化剂的失活主要与机械磨损有关,反应5批次后酶活残留量为96.4%。敞开物系、真空脱水或分子筛脱水均为有效脱水方式。敞开物系中反应物量比不影响平衡转化率而会影响单甘酯平衡产率。用碳酸氢钠水溶液萃取可有效脱除产品中的残余癸酸,终产品酸价为0.68mg KOH/g。提高甘油比例并使用非脱水原料,无外加水结合部分流加癸酸的工艺,可以减少减压脱水或敞开反应的时间,5h后癸酸最高转化率可达96.9%。      

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Synthesis of fatty acid esters and diacylglycerols at elevated temperatures by alkalithermophilic lipases from Thermosyntropha lipolytica

LipA and LipB of Thermosyntropha lipolytica DSM 11003 as previously published are the most alkalithermophilic (pH _opt ^25°C  = 9.4–9.6, T _opt = 96°C) and thermostable (T _1/2 ^24 h  = 74–76°C) lipases currently known. The purified enzymes were analyzed in organic solvents for their ability to catalyze synthesis of diacylglycerols and various alcohol fatty acids. To obtain 100% recovery and avoid a 40% and 50% loss of catalytic activity during lyophilization of purified LipA and LipB, respectively, addition of 1 mg/ml bovine serum albumin (BSA) and 25% polyethylene glycol (PEG400) was required. LipA and LipB catalyzed esterification of fatty acids and alcohols with the highest yields for octyl oleate (LipA) and lauryl oleate (LipB) and also catalyzed synthesis of 1,3-dioleoyl glycerol, 1-oleoyl-3-lauroyl glycerol, and 1-oleoyl-3-octoyl glycerol. Isooctane was the most efficient solvent for esterification reactions at 85°C. Similar to the positional specificity for the hydrolytic reaction in aqueous solutions, LipA and LipB catalyzed in organic solvents the synthesis of diacylglycerol with esterification of position 1 and 3 with a yield of 62% for di-oleoyl glycerol. The reported conversion rates do not represent the full potential of these enzymes, since only 1/100th–1/1,000th of the protein concentrations usually used in commercial processes were available. However, use of slightly increased protein concentrations confirmed the trend to higher yields with higher protein concentrations. The obtained specificity and variety of the reactions catalyzed by LipA and LipB, and their high thermostability allowing synthesis to occur at 90°C, demonstrate their great potentials for industrial applications, particularly in structured lipid biosynthesis for substrates that are less soluble at mesobiotic temperatures.     

Thermodynamics of the lipase-catalyzed esterification of glycerol and n-octanoic acid in organic solvents and in the neat reaction mixture

The thermodynamics of the lipase-catalyzed esterification of glycerol with n-octanoic acid have been investigated with acetonitrile, benzene, and toluene as solvents and in the neat reaction mixture (no organic solvent added). This esterification reaction leads to five products: 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol. This, in turn leads to a total of 12 reactions. Values of the equilibrium constants for these reactions have been measured (HPLC, GC, and LC/MS) at 37°C in the above mentioned media. The equilibrium constants range from 0.9 to 20.7, 0.20 to 8.0, 0.23 to 10.0, and 0.57 to 2.2 in acetonitrile, benzene, toluene, and neat media, respectively. Relative standard molar Gibbs free energies of formation Δ_fG_m⁰ of 1-monooctanoyl glycerol, 2-monooctanoyl glycerol, 1,2-dioctanoyl glycerol, 1,3-dioctanoyl glycerol and 1,2,3-trioctanoyl glycerol in the organic solvents and in the neat reaction mixture have been calculated and used to compactly summarize the thermodynamics of these reactions. The results show an approximate correlation with the permittivities of the solvents.     

Adsorptive control of water in esterification with immobilized enzymes: I. Batch reactor behavior

Reducing the influence of an undesired product in an enzymatic reaction could have a significant impact on the productivity of such systems. Here, we focus on the removal of water formed during an enzymatic esterification in a batch reactor. A commercial immobilized lipase preparation, known as Lipozyme, is used as the biocatalyst and propionic acid and isoamyl alcohol dissolved in hexane are the substrates. In this system, the water formed will partition between the catalyst and the medium. As the more polar reactants are converted into the less polar ester product, the water is partitioned more towards the biocatalyst and the accumulation of water eventually causes lower reaction rates. Addition of a strong-acid cation exchange resin in sodium form is found to control the water accumulation on the biocatalyst without stripping the essential water needed for the enzyme to function and substantial improvements in conversion are achieved. A mathematical model is developed to describe the batch reaction behavior with and without added absorbent, which successfully predicts the behavior of water and its effects.     

Reversal of enzymatic hydrolysis: Rate and extent of ester synthesis as catalyzed by chymotrypsin and subtilisin Carlsberg at low water concentrations

Immobilized chymotrypsin catalyzes esterification of N-acetyltyrosine in a medium containing high concentrations of alcohols. The hydrophilic support and inclusion of glycerol protect the enzyme activity and allow catalysis to proceed in the presence of only 10% (v/v) water. The same equilibrium concentration of ester is obtained whether reaction proceeds from ester or from free acid. Hates of ester synthesis and hydrolysis are similar when measured under the same conditions, but are at least one order of magnitude slower than optimal rates of hydrolysis. Subtilisin Carlsberg in the free, unmodified form catalyzes ester synthesis at even lower water concentrations; optimal rates are obtained at 5–15% H₂O. Hydrolytic enzymes can thus be utilized as catalysts of synthesis reactions in nonaqueous solvents where synthesis is thermodynamically favored over hydrolysis; in some cases this may provide economic and/or energetic advantages over conventional techniques.     

Optimisation of n-octyl oleate enzymatic synthesis over Rhizomucor miehei lipase

Octyl oleate is a useful organic compound with several applications in cosmetic, lubricant and pharmaceutical industry. At first, the enzymatic synthesis of n-octyl oleate by direct lipase-catalysed esterification of oleic acid and 1-octanol was investigated in a stirred batch reactor in solvent-free system. A systematic screening and optimisation of the reaction parameters were performed to gain insight into the kinetics mechanism. Particularly, enzyme concentration, reaction temperature, stirrer speed, water content, substrates concentration and molar ratio were optimised with respect to the final product concentration and reaction rate. The kinetics mechanism of the reaction was investigated. Finally, a comparison of the experimental results obtained in a solvent free-system with those using two different solvents, supercritical carbon dioxide (SC-CO₂) and n-hexane, was proposed. It resulted that in SC-CO₂ higher concentration of the desired product was attained, requiring lower enzyme concentrations to achieve comparable conversion of free fatty acid into fatty acid ester.     

Candida Antarctica Lipase B-Catalysed Synthesis Of Dihydroxyacetone Fatty Acid Esters

The enzymatic syntheses of 1-lauroyl-dihydroxyacetone and 1, 3-dilauroyl-dihydroxyacetone were investigated. Lipase B from Candida Antarctica (SP435) was used to catalyse the acylation of dihydroxyacetone (DHA) with lauric acid in organic solvent media at controlled water activity. High conversions of dihydroxyacetone (< 90%) are achieved when the water activity is 0.11 or below in solvents of various hydrophobicities, such as diethyl ether, methyl-terr-butyl ether (MTBE) and diphenyl ether. The main product in the esterification of DHA with lauric acid is 1-lauroyl-DHA, while the amount of 1, 3-dilauroyl-DHA that is produced can be increased by changing the reaction conditions. Thus, hasing the water activity from 0.75 to 0.06 resulted in an increase in the total yield of 1, 3-dilauroyl-DHA from 3% to 20%. Solvents which have high logP values favoured the acylation of 1-lauroyl-DHA and thereby the formation of 1, 3-dilauroyl-DHA. Thus, when diphenyl ether was used in this reaction, the yield of 1, 3-dilauroyl-DHA was 45%. Complete acylation to 1, 3-dilauroyl-DHA was achieved when a fatty acid vinyl ester was used as acyl donor in a closed reactor.     

A new irreversible enzyme-aided esterification method in organic solvents

A new irreversible esterification method for carboxylic acids catalyzed by a lipase from Candida antarctica (Novozyme 435) in organic solvents has been developed. The water produced during the process is chemically destroyed by a corresponding ester of acetoacetate, which acts as a sacrificial substrate in this reaction. The flavour esters isobutyl acetate, methyl butyrate, ethyl butyrate and benzyl butyrate were synthesized either in small scale (0.05 mol) or large scale (1 mol). The yields range from 82 to 92% within 24 h at 52°C. Optimal molar ratios of reactants were 1:1:1 (carboxylic acid:alcohol:acetoacetate).     

Abiotic Condensation Synthesis of Glyceride Lipids and Wax Esters Under Simulated Hydrothermal Conditions

Precursor compounds for abiotic proto cellular membranes are necessary for the origin of life. Amphipathic compounds such as fatty acids and acyl glycerols are important candidates for micelle/bilayer/vesicle formation. Two sets of experiments were conducted to study dehydration reactions of model lipid precursors in aqueous media to form acyl polyols and wax esters, and to evaluate the stability and reactions of the products at elevated temperatures. In the first set, mixtures of n-nonadecanoic acid and ethylene glycol in water, with and without oxalic acid, were heated at discrete temperatures from 150 ^∘C to 300 ^∘C for 72 h. The products were typically alkyl alkanoates, ethylene glycolyl alkanoates, ethylene glycolyl bis-alkanoates and alkanols. The condensation products had maximum yields between 150 ^∘C and 250 ^∘C, and were detectable and thus stable under hydrothermal conditions to temperatures < 300 ^∘C. In the second set of experiments, mixtures of n-heptanoic acid and glycerol were heated using the same experimental conditions, with and without oxalic acid, between 100 ^∘C and 250 ^∘C. The main condensation products were two isomers each of monoacylglycerols and diacylglycerols at all temperatures, as well as minor amounts of the fatty acid anhydride and methyl ester. The yield of glyceryl monoheptanoates generally increased with increasing temperature and glyceryl diheptanoates decreased noticeably with increasing temperature. The results indicate that condensation reactions and abiotic synthesis of organic lipid compounds under hydrothermal conditions occur easily, provided precursor concentrations are sufficiently high.     

Synthesis of phosphatidylcholine with defined fatty acid in the sn-1 position by lipase-catalyzed esterification and transesterification reaction

The incorporation of caproic acid in the sn-1 position of phosphatidylcholine (PC) catalyzed by lipase from Rhizopus oryzae was investigated in a water activity-controlled organic medium. The reaction was carried out either as esterification or transesterification. A comparison between these two reaction modes was made with regard to product yield, product purity, reaction time, and byproduct formation as a consequence of acyl migration. The yield in the esterification and transesterification reaction was the same under identical conditions. The highest yield (78%) was obtained at a water activity (a(w)) of 0.11 and a caproic acid concentration of 0.8 M. The reaction time was shorter in the esterification reaction than in the transesterification reaction. The difference in reaction time was especially pronounced at low water activities and high fatty acid concentrations. The loss in yield due to acyl migration and consequent enzymatic side reactions was around 16% under a wide range of conditions. The incorporation of a fatty acid in the sn-1 position of PC proved to be thermodynamically much more favorable than the incorporation of a fatty acid in the sn-2 position.     

Green synthesis of isoamyl acetate via silica immobilized novel thermophilic lipase from <Emphasis Type="Italic">Bacillus aerius</Emphasis>

Isoamyl acetate, a pear or banana flavor, is widely used in food, beverage, cosmetic, and pharmaceutical industries. In the present work, lipase from Bacillus aerius was immobilized on silica gel matrix in the presence of a cross-linking agent, glutaraldehyde, and its efficiency in synthesizing isoamyl acetate using esterification reaction was studied. The esterification of acetic acid and isoamyl alcohol by silica-bound lipase was studied as a function of time and temperatures. The incubation time of 10 h, temperature of 55°C, substrate molar ratio 1: 1, and the amount of lipase as 1% were found to be optimal for the esterification reaction. The bound lipase catalyzed the esterification of acetic acid by isoamyl alcohol with the yield of about 68% under the optimized reaction conditions. The product was identified as isoamyl acetate using gas-liquid chromatography, nuclear magnetic resonance, and Fourier transform IR spectroscopy analysis by the presence of an ester group at the wavenumber of 1720.5 cm^–1.