Studies of lipase-catalyzed esterification reactions of some acetylenic fatty acids

Esterification of five positional isomers of acetylenic fatty acids [viz. 9:1(2a), 11:1(10a), 18:1(6a), 18:1(9a) and 22:1(13a)] of different chain lengths with n-butanol in n-hexane in the presence of eight different lipases [Lipozyme IM 20 (Rhizomucor miehei), Lipolase 100T (R. miehei), Novozyme 435 (Candida antarctica), PPL (porcine pancreatic lipase), CCL (C. cylindracea), PS-D (Pseudomonas cepacia), Lipase A-12 (Aspergillus niger) and Lipase AY-30 (C. rugosa)] was studied. 2-Nonynoic acid was not esterified except when catalyzed by the lipase from C. antarctica (Novozyme 435) to give 42% butyl ester after 48 h. The lipases from A. niger (Lipase A-12) and C. rugosa (Lipase AY-30) showed poor biocatalytic behavior in the esterification of the acetylenic fatty acids studied. 10-Undecynoic acid gave the highest conversion rate of esterification with each kind of lipase used. 6-Octadecynoic acid showed a marked degree of resistance to esterification carried out in the presence of C. cylindracea (CCL), P. cepacia (PS-D), or porcine pancreatic (PPL) lipase but not significantly in the presence of the lipases of R. miehei (Lipozyme IM 20), R. miehei (Lipolase 100T), or Novozyme 435. 9-Octadecynoic acid and 13-docosynoic acid were not discriminated and were readily esterified by the remaining six lipases, but when compared to oleic acid the acetylenic fatty acids were comparatively much slower in conversion to the esters.     

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

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

Acylation of glucose catalysed by lipases in supercritical carbon dioxide

The acylation of glucose with lauric acid in a reaction catalysed by two Candida lipases and a Mucor miehei lipase in supercritical carbon dioxide (SCCO₂) was investigated. A linear dependence of the reaction rate on enzyme concentration was observed. Studies on the effect of temperature on enzyme activity showed that Candida antarctica lipase remains stable at temperatures as high as 70°C. Non-immobilised Candida rugosa lipase was found to have a temperature optimum at 60°C. The acylation reaction rate depended on the initial water activity of both substrates and enzyme; the optimum was 0·75 for Candida antarctica lipase, 0·53 for Candida rugosa lipase, and between 0·3 and 0·5 for Mucor miehei lipase. Candida rugosa lipase was most active at a molar ratio of sugar: acyl donor of 1: 3, while the optimum ratio was found to increase to 1: 6 when the reaction was catalysed by Candida antarctica and Mucor miehei lipases. © 1998 SCI     

Esterifications of 1- andrac-2-octanols with selected acids and acid derivatives using lipases

Several aliphatic acids and their ethyl, isopropenyl and 2,2,2-trichloroethyl esters were allowed to react with 1- and 2-octanols catalyzed by commercial lipase preparations of porcine pancreas and the fungiCandida rugosa, Aspergillus niger andMucor miehei. Comparisons of reactivity of the acids and esters were made in common organic solvents using the primary alcohol. Reactions of octanoic acid and its esters with 2-octanol in hexane allowed an evaluation of stereoselectivity of the lipases with different substrates that carried the same (octanoyl) residue. A partial resolution ofrac-2-hexadecanol withA. niger lipase is described, and the utility of lipase selectivities (stereo, positional, fatty acid and ester) is discussed with reference to the data presented.     

Lipase-catalyzed synthesis and properties of estolides and their esters

Eight lipases were screened for their ability to synthesize estolides from a mixture that contained lesquerolic (14-hydroxy-11-eicosenoic) acid and octadecenoic acid. With the exception ofAspergillus niger lipase, all 1,3-specific enzymes (fromRhizopus arrhizus andRhizomucor miehei lipases) were unable to synthesize estolides.Candida rugosa andGeotrichum lipases catalyzed estolide formation at >40% yield, with >80% of the estolide formed being monoestolide from one lesquerolic and one octadecenoic acyl group:Pseudomonas sp. lipase synthesized estolides at 62% yield, but the product mixture contained significant amounts of monoestolide with two lesquerolic acyl groups as well as diestolide. ImmobilizedR. miehei lipase was chosen to catalyze the esterification of mono-and polyestolide, derived synthetically from oleic acid, with fatty alcohols or α,ω-diols. Yields were >95% for fatty alcohol reactions and >60% for diol reactions. In addition, the estolide linkage remained intact through the course of the esterification process. Esterification of estolides improved the estolide’s properties—for example, lower viscosity and higher viscosity index—but slightly raised the melting point. Estolides and, particularly, estolide esters may be suitable as lubricants or lubricant additives.     

Solvent-free lipase-catalyzed thioesterification and transthioesterification of fatty acids and fatty acid esters with alkanethiols in Vacuo

Palmitic acid hexadecylthioester and other long-chain acyl thioesters have been prepared in high yield (80–85%, purity >98%) by solvent-free lipase-catalyzed thioesterification of fatty acids with alkanethiols in vacuo. A lipase B preparation from Candida antarctica was more effective than a lipase preparation from Rhizomucor miehei and, particularly, those from papaya latex and porcine pancreas. Lipase-catalyzed transthioesterification of fatty acid methyl esters with alkanethiols was less effective than thioesterification for the preparation of acyl thioesters. However, in transthioesterification, a lipase preparation from R. miehei was more effective than a lipase B preparation from C. antarctica. Lipases from papaya latex and porcine pancreas led to moderate conversions to acyl thioesters in both thioesterification and transthioesterification reactions, whereas only small proportions of thioesters were formed using lipase from Rhizopus arrhizus. Lipases from Chromobacterium viscosum and Candida rugosa were not effective at all.     

Enzymatic Incorporation of Selected Long-Chain Fatty Acids into Triolein

Acidolysis of triolein (tri C18:1) with selected long-chain fatty acids (LCFA) was carried out using Candida antarctica (Novozym 435), Rhizomucor miehei (Lipozyme RM IM), Pseudomonas sp. (PS-30), Aspergillus niger (AP-12), and Candida rugosa (AY-30). A better incorporation of stearic acid (SA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (AA), and docosapentaenoic acid (DPA) was achieved using lipase from Rhizomucor miehei. Lipase from Pseudomonas sp. catalyzed a better incorporation of linoleic acid (LA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into triolein. Thus, Rhizomucor miehei and to a lesser extent Pseudomonas sp. might be considered as providing the most effective enzymes for acidolysis of triolein with selected LCFA. In general, incorporation of LCFA into triolein (tri C18:1) may be affected by chain length, number of double bonds, and the location and geometry of the double bonds as well as reaction conditions and reactivity and specificity of lipases used. As the ratio of the number of moles of a mixture of equimole quantities of C18 FA to triolein changed from 1 to 3, incorporation of C18 FA into triolein increased accordingly with Rhizomucor miehei lipase. Similarly, incorporation of n-3 FA into triolein increased when ALA, DPA, DHA, and EPA were used. The same trend was noticed for a mixture of n-6 FA (LA + GLA + AA) and triolein.     

Enzymatic synthesis of fatty esters in 5‐caffeoyl quinic acid

The fatty esters of 5‐caffeoyl quinic acid were synthesized by direct esterification biocatalysed by an immobilised lipase obtained from Candida antarctica. Esterification yields (from 40 to 75%) depended on the carbon chain length of the fatty alcohols and whether or not solvent (2‐methyl 2‐butanol) was present in the reaction medium.     

Lipase-catalyzed modification of borage oil: Incorporation of capric and eicosapentaenoic acids to form structured lipids

Two immobilized lipases, nonspecific SP435 from Candida antarctica and sn-1,3 specific IM60 from Rhizomucor miehei, were used as biocatalysts for the restructuring of borage oil (Borago officinalis L.) to incorporate capric acid (10:0, medium-chain fatty acid) and eicosapentaenoic acid (20:5n-3) with the free fatty acids as acyl donors. Transesterification (acidolysis) reactions were carried out in hexane, and the products were analyzed by gas-liquid chromatography. The fatty acid profiles of the modified borage oil were different from that of unmodified borage oil. Higher incorporation of 20:5n-3 (10.2%) and 10:0 (26.3%) was obtained with IM60 lipase, compared to 8.8 and 15.5%, respectively, with SP435 lipase. However, SP435 lipase was able to incorporate both 10:0 and 20:5n-3 fatty acids at the sn-2 position, but the IM60 lipase did not. Solvents with log P values between 3.5 and 4.5 supported the acidolysis reaction better than those with log P values between −0.33 and 3.0.     

Selectivity of lipases: isolation of fatty acids from castor,coriander, and meadowfoam oils

The lipase‐catalyzed hydrolysis of castor, coriander, and meadowfoam oils was studied in a two‐phase water/oil system. The lipases from Candida rugosa and Pseudomonas cepacia released all fatty acids from the triglycerides randomly, with the exception of castor oil. In the latter case, the P. cepacia lipase discriminated against ricinoleic acid. The lipase from Geotrichum candidum discriminated against unsaturated acids having the double bond located at the Δ‐6 (petroselinic acid in coriander oil) and Δ‐5 (meadowfoam oil) position or with a hydroxy substituent (ricinoleic acid). The expression of the selectivities of the G. candidum lipase was most pronounced in lipase‐catalyzed esterification reactions, which was exploited as part of a two‐step process to prepare highly concentrated fractions of the acids. In the first step the oils were hydrolyzed to their respective free fatty acids, in the second step a selective lipase was used to catalyze esterification of the acids with 1‐butanol. This resulted in an enrichment of the targeted acids to approximately 95—98% in the unesterified acid fractions compared to the 70—90% content in the starting acid fractions.     

A Bi-Enzymatic Cascade Pathway towards Optically Pure FAHFAs**

Recently discovered endogenous mammalian lipids, fatty acid esters of hydroxy fatty acids (FAHFAs), have been proved to have anti-inflammatory and anti-diabetic effects. Due to their extremely low abundancies in vivo, forging a feasible scenario for FAHFA synthesis is critical for their use in uncovering biological mechanisms or in clinical trials. Here, we showcase a fully enzymatic approach, a novel in vitro bi-enzymatic cascade system, enabling an effective conversion of nature-abundant fatty acids into FAHFAs. Two hydratases from Lactobacillus acidophilus were used for converting unsaturated fatty acids to various enantiomeric hydroxy fatty acids, followed by esterification with another fatty acid catalyzed by Candida antarctica lipase A (CALA). Various FAHFAs were synthesized in a semi-preparative scale using this bi-enzymatic approach in a one-pot two-step operation mode. In all, we demonstrate that the hydratase-CALA system offers a promising route for the synthesis of optically pure structure-diverse FAHFAs.     

Wax ester-synthesizing activity of lipases

The synthesis/hydrolysis of wax esters was studied in an aqueous solution using purified rat pancreatic lipase, porcine pancreatic carboxylester lipase, and Pseudomonas fluorescens lipase. The equilibrium between wax ester synthesis and hydrolysis favored ester formation at neutral pH. The synthesizing activities were measured using free fatty acid or triacylglycerol as the acyl donor and an equimolar amount of long-chain alcohol as the acyl acceptor. When oleic acid and hexadecanol emulsified with gum arabic were incubated with these lipases, was ester was synthesized, in a dose- and time-dependent manner, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was about 0.9/0.1. These lipases catalyzed the hydrolysis of palmityl oleate emulsified with gum arabic, and the apparent equilibrium ratio of palmityl oleate/free oleic acid was also about 0.9/0.1. The apparent equilibrium ratio of wax ester/free fatty acid catalyzed by lipase depended on incubation pH and fatty alcohol chain length. When equimolar amounts of trioleoylglycerol and fatty acyl alcohol were incubated with pancreatic lipase, carboxylester lipase, or P. fluorescens lipase, wax esters were synthesized dose-dependently. These results suggest that lipases can catalyze the synthesis of wax esters from free fatty acids or through degradation of triacylglycerol in an aqueous medium.     

Changes in hydrolysis specificities of lipase from Rhizomucor miehei to polyunsaturated fatty acyl ethyl esters in different aggregation states

Hydrolysis specificities of lipase from Rhizomucor miehei were compared for various fatty acyl ethyl esters. Activity yields of immobilized lipases, measured with 1 mM substrate, were more than 100%. Differences in hydrolysis rate and affinity for the substrates between lipase preparations were also typically higher during hydrolysis of substrates at 100 mM than at 1 mM, indicating better mass transfer effects for 1-mM substrates. The native lipase showed higher affinity for polyunsaturated fatty acid substrates at 1 mM than at 100 mM. Hydrolysis rates for 1-mM substrates were observed with immobilized lipases, fixed on anion exchange resin with glutaraldehyde and on cation exchange carrier with carbodiimide, and suggested some modification of the basic amino acid related to the lid of R. miehei lipase. Activation with these bifunctional reagents was not observed for 100-mM substrates, indicating that interfacial activation always occurred because of aggregation of 100-mM substrates. These results show that lipase from R. miehei recognizes molecular aggregation of lipids, and that various changes occur in the hydrolysis specificities for fatty acids.     

Regioselective acylation of flavonoids catalyzed by lipase in low toxicity media

Flavonoid fatty esters were prepared by acylation of flavonoids (rutin and naringin) by fatty acids (C8, C10, C12), catalyzed by immobilized lipase from Candida antarctica in various solvent systems. The reaction parameters affecting the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the acyl donor concentration have been investigated. At optimum reaction conditions, the conversion of flavonoids was 50—60% in tert‐butanol at a_w less than 0.11. In all cases studied, only flavonoid monoester was identified, which indicates that this lipase‐catalyzed esterification is regioselective.     

Enzymatic production and physicochemical characterization of uncommon wax esters and monoglycerides

Wax esters from fatty alcohols and uncommon fatty acids were synthesized in yields up to 90% when commercially available microbial lipases fromRhizomucor miehei (Lipozyme^™) andCandida antarctica (SP 435) were used with limited water content in nonpolar solvents under mild conditions. The corresponding fatty acids were prepared by chemical conversion of naturally occurring resources (agricultural surpluses). Also, when phenylboronic acid was added as solubilizing agent in a nonpolar solvent, the direct enzymatic monoacylation of glycerol with uncommon fatty acids was successful. The measurement of π/A-isotherms by means of a Langmuir film balance indicated medium film pressures, medium or large molecular areas, and interesting phase behavior. The monolayer of a wax ester at the air/water interface could be directly visualized by Brewster angle microscopy.     

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

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

Utilization of acid oils in making valuable fatty products by microbial lipase technology

Microbial lipase-catalyzed hydrolysis, esterification, and alcoholysis reactions were carried out on acid oils of commerce such as coconut, soybean, mustard, sunflower, and rice bran for the purpose of making fatty acids and various monohydric alcohol esters of fatty acids of the acid oils. Neutral glycerides of the acid oils were hydrolyzed byCanadida cylindracea lipase almost completely within 48 h. Acid oils were converted into fatty acid esters of short- and long-chain alcohols like C₄, C₈, C₁₀, C₁₂, C₁₆, and C₁₈ in high yields by simultaneous esterification and alcoholysis reactions withMucor miehei lipase as catalyst. Acid oils of commerce can be utilized as raw materials in making fatty acids and fatty acid esters using lipase-catalyzed methodologies.     

Enzymatic fractionation of fatty acids: Enrichment of γ-linolenic acid and docosahexaenoic acid by selective esterification catalyzed by lipases

Immobilized lipase preparations from seedlings of rape (Brassica napus L.) andMucor miehei (lipozyme) used as biocatalysts in esterification and hydrolysis reactions discriminate strongly against γ-linolenic and docosahexaenoic acids/acyl moieties. Utilizing this property, γ-linolenic acid contained in fatty acids of evening primrose oil has been enriched seven to nine-fold, from 9.5 to almost 85% by selective esterification of the other fatty acids with butanol. Similarly, docosahexaenoic acid of cod liver oil has been enriched four to five-fold, from 9.4 to 45% by selective esterification of fatty acids (other than docosahexaenoic acid) with butanol. As long as the reaction is stopped before reaching equilibrium, very little of either γ-linolenic acid or docosahexaenoic acid are converted to butyl esters, which results in high yields of these acids in the unesterified fatty acid fraction.     

Methyl-branched octanoic acids as substrates for lipase-catalyzed reactions

Hydrolyses of racemic methyl-branched octanoic acid thiolesters are described using six commercial lipases as catalysts. Branching at positions 2, 4 and 5 greatly reduced activity; branching at the 3-position virtually eliminated activity. The reactivities of the racemic branched thiolesters relative to the unbranched ester were very similar for each lipase preparation examined. In reactions involving configurationally pure 2-methyloctanoic acids, the S-enantiomer reacted faster both in esterification of aliphatic alcohols and in hydrolyses of aliphatic alcohol esters with all of the lipases examined. Stereobiases in hydrolyses of the octanoic acid esters branched at other positions were low and variable. In sharp contrast to the hydrolyses of the thiolesters of 2-methyloctanoic acid, two aryl esters of 2-methyloctanoic acid catalyzed byR. miehei lipase hydrolyzed with a bias for the R-configuration. A view of the ester-enzyme complex is offered, to explain the relative rates of reaction of the racemic esters. Reference to a brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Immobilization of lipases on porous polypropylene: Reduction in esterification efficiency at low loading

Rhizomucor miehei, Humicola sp., Rhizopus niveus, and Candida antarctica B lipases were immobilized by physical adsorption onto a macroporous polypropylene support. In an esterification reaction, the enzyme efficiency, and therefore cost-effectiveness, is greatly affected by enzyme loading, with an apparent suppression of efficiency at low lipase loadings for both R. miehei and Humicola sp. lipases. This results in the appearance of a pronounced maximum in the efficiency-loading relationship at approximately 100,000 lipase units (LU)/g for R. miehei lipase (10% of its saturation loading) and at approximately 200,000 LU/g for Humicola sp. lipase (50% of its saturation loading). The other lipases studied do not show similar trends. At low loadings, only a small portion of the surface area is occupied and gives the lipase the opportunity to spread; it is hypothesized that the reduction in efficiency at low loadings is due to a distortion of the active molecular conformation caused by the lipase maximizing its contact with the support as a result of its high affinityfor the support surface. The relationship between efficiency and loading was different for each of the lipases studied, which may reflect both differences in the strength of the affinity of the lipase for the support and in the ease at which the molecular conformation of the lipase can be distorted.