The influence of water on the synthesis of n-butyl oleate by immobilizedMucor miehei lipase

The influence of initial water concentration on the synthesis of n-butyl oleate was investigated. The synthesis was done with immobilizedMucor miehei lipase—Lypozyme^™—at various reaction conditions. The activity of the enzyme is lower at higher amounts of water. Initial reaction rates, as well as equilibrium conversion, increase at low initial water concentration. Optimal water concentration for the activity of immobilized lipase is temperature dependent at the pressure of 1 bar. Low initial water concentration barely effects equilibrium esterification at 0.032 bar. At high initial water concentrations equilibrium conversion, as well as initial reaction rates, decrease at both pressures.     

Enzymatic synthesis of fatty alcohol esters by alcoholysis

Lipase-catalyzed conversions of some minor oils and fats like mowrah (Madhuca latifolia), mango (Mangifera indica) kernel, and sal (Shorea robusta) fats into low, medium, and high molecular weight alcohol esters have been investigated. In solvent-free medium, alcoholysis of the above-mentioned fats with 10% (w/w) Mucor miehei lipase produced alcohol esters in good yield. The percentage molar conversions of C₄, C₈, C₁₀, C₁₂, C₁₄, C₁₆, C₁₈, and C_18:1 alcohols into corresponding alcohol esters ranged from 86.8 to 99.2, while the percentage molar conversions on the basis of oil were in the range of 108.0 to 123.5.     

Enzymatic synthesis of steryl esters of polyunsaturated fatty acids

Steryl esters of long-chain fatty acids have water-holding properties, and polyunsaturated fatty acids (PUFA) have various physiological functions. Because steryl ester of PUFA can be expected to have both features, we attempted to synthesize steryl esters of PUFA by enzymatic methods. Among lipases used, Pseudomonas lipase was the most effective for the synthesis of cholesteryl docosahexaenoate. When a mixture of cholesterol/docosahexaenoic acid (3:1, mol/mol), 30% water, and 3000 units/g of lipase was stirred at 40°C for 24 h, the esterification extent attained 89.5%. Under the same reaction conditions, cholesterol, cholestanol, and sitosterol were also esterified efficiently with docosahexaenoic, eicosapentaenoic, arachidonic, and γ-linolenic acids.     

Enzymatic glyceride synthesis in a foam reactor

We 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.     

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

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

Incorporation of eicosapentaenoic and docosahexaenoic acids into groundnut oil by lipase-catalyzed ester interchange

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were incorporated into groundnut oil by interesterification with a 1,3-specific lipase fromMucor miehei. The resultant EPA and DHA concentrations of the groundnut oil were 9.5 and 8.0%, respectively.     

Enzymatic synthesis of position-specific low-calorie structured lipids

An immobilized sn-1,3-specific lipase from Rhizomucor miehei (IM 60) was used to catalyze the interesterification of tristearin (C_18:0) and tricaprin (C_10:0) to produce low-calorie structured lipids (SL). Acceptable product yields were obtained from a 1:1 mole ratio of both triacylglycerols with 10% (w/w of reactants) of IM 60 in 3 mL hexane. The SL molecular species, based on total carbon number, were 44.2% C₄₁ and 40.5% C₄₉, with 3.8 and 11.5% unreacted tristearin C₅₇ and tricaprin C₂₇, respectively, remaining in the product mixture. The best yield of C₄₁ species (44.3%) was obtained with zero added water. Tricaprylin (C_8:0) was also successfully interesterified with tristearin in good yields at 1:1 mole ratio. Products were analyzed by reverse-phase high-performance liquid chromatography with an evaporative light-scattering detector. Reaction parameters, such as substrate mole ratio, enzyme load, time course, added water, reaction media, and enzyme reuse, were also investigated. Hydrolysis by pancreatic lipase revealed the specific fatty acids present at the sn-1,3 positions of SL. Biocatalysis Symposium Paper, presented at the AOCS Annual Meeting & Expo, Seattle, Washington. May 11–14, 1997.     

Soy lecithin-monoester interchange reaction by microbial lipase

Modification of the fatty acid composition of soy lecithin, principally at its 1-position, was investigated by interchange reaction with the methyl ester of individual fatty acids and a lipase as the catalyst. The consequent effect on the surface activity of soy lecithin was also examined. The interchange reaction was carried out by heating a mixture of soy lecithin and methyl ester of a fatty acid at 60°C for 48 h with 10% (by weight of the reactants) Mucor miehei lipase. The lipase was filtered from the reaction mixture, and the product was isolated by combination of acetone extraction, which removed the methyl ester fraction, and by preparative thin-layer chromatography separation. The soy lecithin showed distinct change in its fatty acid composition in the sn-1 position. Capric acid was incorporated by 8.4%, while lauric acid and myristic acid were introduced at 14.1 and 15.7%, respectively. The linolenic acid percentage was increased by about 10 units. The interfacial tension of soy lecithin changed significantly after incorporation of various saturated fatty acids.     

Isolation of tocopherol and sterol concentrate from sunflower oil deodorizer distillate

The isolation of tocopherols and sterols together as a concentrate from sunflower oil deodorizer distillate was investigated. The sunflower oil deodorizer distillate was composed of 24.9% unsaponifiable matter with 4.8% tocopherols and 9.7% sterols, 28.8% free fatty acid (FFA) and 46.3% neutral glycerides. The isolation technology included process steps such as biohydrolysis, bioesterification and fractional distillation. The neutral glycerides of the deodorizer distillates were hydrolyzed byCandida cylindracea lipase. The total fatty acids (initial FFA plus FFA from neutral glycerides) were converted into butyl esters withMucor miehei lipase. The esterified product was then fractionally distilled in a Claisen-vigreux flask. The first fraction, which was collected at 180–230°C at 1.00 mm of Hg for 45 min, contained mainly butyl esters, hydrocarbons, oxidized products and some amount of free fatty acids. The fraction collected at 230–260°C at 1.00 mm Hg for 15 min was rich in tocopherols (about 30%) and sterols (about 36%). The overall recovery of tocopherols and sterols after hydrolysis, esterification and distillation were around 70% and 42%, respectively, of the original content in sunflower oil deodorizer distillate.     

Enzymatic alcoholysis reaction of soy phospholipids

Lipase-catalyzed alcoholysis of soy phospholipids was investigated to simultaneously make lysophospholipids and fatty acid esters of individual alcohols. Alcoholysis was carried out by stirring a mixture of soy phospholipids and individual alcohols in equimolar proportions with 10% (by weight of reactants) Mucor miehei lipase at 55°C for 24 h. The products were isolated by column chromatography after removal of the lipase. Lysophospholipids (in 69–78% molar yield) were obtained from soy phospholipids, and the yield of esters of various alcohols also conformed nearly with theoretical yields.     

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

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

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.     

Medium-chain fatty acid-rich glycerides by chemical and lipase-catalyzed polyester-monoester interchange reaction

Medium-chain triglycerides (MCT) that contain caprylic acid (C_8:0) and capric acid (C_10:0) have immense medicinal and nutritional importance. Coconut oil can be used as a starting raw material for the production of MCT. The process, based on the interchange reaction between triglycerides and methyl esters of medium-chain fatty acids by chemical catalyst (sodium methoxide) or lipase (Mucor miehei) catalyst, appears to be technically feasible. Coconut oils with 25–28.3% (w/w) and 22.1–25% (w/w) medium-chain fatty acids have been obtained by chemical and lipase-catalyzed interchange reactions. Coconut olein has also been modified with C_8:0 and C_10:0 fatty acids, individually as well as with their mixtures, by chemical and lipase-catalyzed interchange reactions. Coconut olein is a better raw material than coconut oil for production of mediumchain fatty acid-rich triglyceride products by both chemical and lipase-catalyzed processes.     

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

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

Enzymatic enrichment of conjugated linoleic acid isomers and incorporation into triglycerides

A method was developed for the enrichment of either the cis9,trans11 or the trans10,cis12 isomer of conjugated linoleic acid (CLA) from a synthetic CLA mixture consisting predominantly of these isomers in equal amounts. Lipases were screened for their ability to selectively esterify one isomer at a significantly greater rate than the other isomer. An immobilized lipase from Rhizomucor miehei was nonselective, but a lipase from Geotrichum candidum esterified the cis9,trans11 isomer more rapidly than the trans10,cis12 isomer. This selectivity was exploited at the kilogram scale to prepare an ester fraction with a content of 91% cis9,trans11 CLA and an unreacted free fatty acid fraction consisting of 82% trans10,cis12 CLA, based on total CLA content. The components of the reaction mixture were separated by molecular distillation. Each enriched fraction was then incorporated into palm oil triglycerides by interesterification with the non-selective lipase from R. miehei. Two triglyceride fats resulted, which were enriched in either cis9,trans11 CLA (26.5% cis9,trans11 and 1.7% trans10,cis12) or trans10,cis12 CLA (3.5% cis9,trans11 and 22.9% trans10,cis12).     

Utilization of acid oils in making surface-active compounds by lipase-catalyzed hydrolysis and esterification reactions

Nonionic surface-active molecules were made from acid oils such as mustard, sunflower, rice bran, soybean, coconut, and polyethylene glycols (PEG) of varying molecular weights (200, 300, 400, 600), using processes based on lipasecatalyzed hydrolysis with Candida cylindracea lipase and esterification with Mucor miehei lipase. Both the PEG molecular weight and the acid oil influence the yield of ester. Molar concentration of reatants also influences the rate and yield of ester. Surface tension values of ester products reveal that maximal lowering of surface tension of water occurs in the case of PEG 600 and coconut acid oil ester. This work demonstrates an important technological advance in the synthesis of nonionic surfactants of the alkyl ethoxylate type from a variety of acid seed oils in high yields by using lipase technology involving first hydrolysis and then esterification with another lipase.     

Lipase-catalyzed incorporation of n−3 polyunsaturated fatty acids into vegetable oils

The ability of immobilized lipases IM60 fromMucor miehei and SP435 fromCandida antarctica to modify the fatty acid composition of selected vegetable oils by incorporation of n−3 polyunsaturated fatty acids into the vegetable oils was studied. The transesterification was carried out in organic solvent with free acid and ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) as acyl donors. With free EPA as acyl donor, IM60 gave higher incorporation of EPA than SP435. However, when ethyl esters of EPA and DHA were the acyl donors, SP435 gave higher incorporation of EPA and DHA than IM60. When IM60 and free acid were used, the addition of 5 μL water increased EPA incorporation into soybean oil by 4.9%. With ethyl ester of EPA as acyl donor, addition of 2 μL water increased EPA incorporation by 3.9%. For SP435, addition of water up to 2μL resulted in increased EPA incorporation, but the incorporation declined when the added water exceeded this amount. The addition of water increased the EPA incorporation into Trisun 90 after 24 h reaction but not the reaction rate at early stages of the reaction.     

A comparative study between biorefining combined with other processes and physical refining of high-acid mohua oil

The biorefining process under optimum conditions de-acidified the high-acid mohua oil by nearly 85% with considerable improvement of color. The process, in combination with alkali-refining, bleaching and deodorization, yielded excellent oil with respect to color, unsaponifiable matter content and triglyceride content. The combination of biorefining and physical refining significantly reduced the loss of oil, and the color, unsaponifiable matter and diglyceride content increased while triglyceride content decreased. The physical refining process alone, on the other hand, produced oil with considerably darker color, increased unsaponifiable matter and diglycerides, and decreased triglyceride. Biorefining followed by alkali-refining, bleaching and deodorizing steps or by physical refining can be regarded as a much better alternative refining process than the physical refining process alone for oils of high acidity.     

Utilization of high-melting palm stearin in lipase-catalyzed interesterification with liquid oils

Palm stearin with a melting point (m.p.) of 49.8°C was fractionated from acetone to produce a low-melting palm stearin (m.p.=35°C) and a higher-melting palm stearin (HMPS, m.p.=58°C) fraction. HMPS was modified by interesterification with 60% (by weight) of individual liquid oils from sunflower, soybean, and rice bran by means of Mucor miehei lipase. The interesterified products were evaluated for m.p., solid fat content, and carbon number glyceride composition. When HMPS was interesterified individually with sunflower, soybean or rice bran at the 60% level, the m.p. of the interesterified products were 37.5, 38.9, and 39.6°C, respectively. The solid fat content of the interesterified products were 30–35 at 10°C, 17–19 at 20°C, and 6–10 at 30°C, respectively. The carbon number glyceride compositions also changed significantly. C₄₈ and C₅₄ glycerides decreased remarkably with a corresponding increase of the C₅₀ and C₅₂ glycerides. All these interesterified products were suitable for use as trans acid-free and polyunsaturated fatty acid-rich shortening and margarine fat bases.