Triglyceride specificity ofCandida cylindracea lipase: Effect of docosahexaenoic acid on resistance of triglyceride to lipase

Tuna oil was hydrolyzed withCandida cylindracea lipase. After 70% hydrolysis of the oil, the docosahexaenoic acid (DHA) content in the glyceride mixture [a mixture of TG (triglyceride), DG (diglyceride) and MG (monoglyceride)] was twice that of the original oil. DHA-rich TG and DG were observed, but DHA-rich MG was absent.C. cylin-dracea lipase seemed to have a “triglyceride specificity,” and it favors TG without DHA over TG containing DHA. In accordance with this hypothesis, TG containing a mixture of oleic acid (OA) and DHA was synthesized and then hydrolyzed withC. cylindracea lipase. TGs in the hydrolysis product were fractionated and analyzed quantitatively by high-performance liquid chromatography. Four kinds of TGs were obtained. TG with three molecules of OA was hydrolyzed most easily. Increasing the DHA content of TG resulted in less hydrolysis of TG. The results suggested thatC. cylindracea lipase had a TG specificity for the whole structure of TG in preference to the individual ester bonds; OA coexisting with DHA in TG was resistant toC. cylindracea lipase due to the TG structure.     

Separation of eicosapentaenoic acid and docosahexaenoic acid in fish oil by kinetic resolution using lipase

The objective of this study was to investigate the use of lipases as catalysts for separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in fish oil by kinetic resolution. Transesterification of various fish oil triglycerides with a stoichiometric amount of ethanol by immobilized Rhizomucor miehei lipase under anhydrous solvent-free conditions resulted in a good separation. When free fatty acids from the various fish oils were directly esterified with ethanol under similar conditions, greatly improved results were obtained. By this modification, complications related to regioselectivity of the lipase and nonhomogeneous distribution of EPA and DHA into the various positions of the triglycerides were avoided. As an example, when tuna oil comprising 6% EPA and 23% DHA was transesterified with ethanol, 65% conversion into ethyl esters was obtained after 24 h. The residual glyceride mixture contained 49% DHA and 6% EPA (8:1), with 90% DHA recovery into the glyceride mixture and 60% EPA recovery into the ethyl ester product. When the corresponding tuna oil free fatty acids were directly esterified with ethanol, 68% conversion was obtained after only 8h. The residual free fatty acids comprised 74% DHA and only 3% EPA (25:1). The recovery of both DHA into the residual free fatty acid fraction and EPA into the ethyl ester product remained very high, 83 and 87%, respectively.     

Enrichment of arachidonic acid: Selective hydrolysis of a single-cell oil fromMortierella withCandida cylindracea lipase

Three lipases, isolated previously in our laboratory, and a known lipase fromCandida cylindracea were screened for the enrichment of arachidonic acid (AA). The enzyme fromC. cylindracea was the most effective for the production of oil with high concentration of AA. When a single-cell oil fromMortierella alpina, containing 25% AA, was hydrolyzed with this lipase for 16 h at 35°C, the resulting glycerides contained 50% AA at 52% hydrolysis. After this, no further hydrolysis occurred, even with additional lipase. However, when the glycerides were extracted from the hydrolyzate and were hydrolyzed again with new lipase, the resulting oil contained 60% AA, with a recovery of 75% of its initial AA content. Triglycerides were the main components of the resulting oil. The release of each fatty acid from the oil depended on the hydrolysis rate of its ester. The fatty acid, whose ester is the poorest substrate for the enzyme, is concentrated in the glycerides.     

Synthesis of docosahexaenoic acid-rich triglyceride with immobilizedChromobacterium viscosum lipase

Docosahexaenoic acid (DHA) in the free fatty acid (FFA) derived from enzymically hydrolyzed tuna oil was concentrated by partial titration and precipitation of other FFA as sodium salts with acetone. A triglyceride containing up to 46.2% DHA was synthesized from the DHA-rich glyceride mixture and FFA by use of an immobilizedChromobacterium viscosum lipase.     

The preparation of concentrates of eicosapentaenoic acid and docosahexaenoic acid by lipase-catalyzed transesterification of fish oil with ethanol

The objective of this study was to investigate the use of lipases as catalysts for producing concentrates of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil as an alternative to conventional chemical procedures. Transesterification of fish oil with ethanol was conducted under anhydrous solvent-free conditions with a stoichiometric amount of ethanol. Among the 17 lipases tested, the results showed that Pseudomonas lipases had the highest activity toward the saturated and monounsaturated fatty acids in the fish oil, much lower activity toward EPA and DHA and, at the same time, good tolerance toward the anhydrous alcoholic conditions. With 10 wt% of lipase, based on weight of the fish oil triacylglycerol substrate (15% EPA and 9% DHA initial content), a 50% conversion into ethyl esters was obtained in 24 h at 20°C, in which time the bulk of the saturated and monounsaturated fatty acids reacted, leaving the long-chain n-3 polyunsaturated fatty acids unreacted in the residual mixture as mono-, di-, and triacylglycerols. This mixture comprised approximately 50% EPA+DHA. Total recovery of DHA and EPA was high, over 80% for DHA and more than 90% for EPA. The observed fatty acid selectivity, favoring DHA as a substrate, was most unusual because most lipases favor EPA.     

Enrichment of polyunsaturated fatty acids withGeotrichum candidum lipase

Three lipases, isolated previously in our laboratory, each with different fatty acid and positional specificities, and a known lipase fromCandida cylindracea were screened for concentrating docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids in glycerides.Geotrichum candidum lipase was found to be suitable for their concentration in glycerides. Tuna oil was treated at 30°C with this lipase for 16 h, and 33.5% hydrolysis resulted in the production of glycerides containing 48.7% of DHA and EPA. The hydrolysis was not increased despite adding further lipase, so the glycerides were extracted, and the reaction was repeated. The second hydrolysis produced glycerides containing 57.5% of DHA and EPA in a 54.5% yield, with recovery of 81.5% of initial DHA and EPA. Of the total glycerides, 85.5% were triglycerides. These results showed thatG. candidum lipase was effective in producing glycerides that contained a high concentration of polyunsaturated fatty acids in good yield.     

Preparation of highly purified concentrates of eicosapentaenoic acid and docosahexaenoic acid

Because of the complexity of marine lipids, polyunsaturated fatty acid (PUFA) derivatives in highly purified form are not easily prepared by any single fractionation technique. The products are usually prepared as the ethyl esters by esterification of the body oil of fat fish species and subsequent physicochemical purification processes, including short-path distillation, urea fractionation, and preparative chromatography. Lipase-catalyzed transesterification has been shown to be an excellent alternative to traditional esterification and short-path distillation for concentrating the combined PUFA-content in fish oils. At room temperature in the presence of Pseudomonas sp. lipase and a stoichiometric amount of ethanol without any solvent, efficient transesterification of fish oil was obtained. At 52% conversion, a concentrate of 46% eicosapentaenoic acid (EPA) plus docosahexaenoic acid (DHA) was obtained in excellent recovery as a mixture of mono-, di-, and triacylglycerols. The latter can be easily separated from the saturated and monounsaturated ethyl esters and converted into ethyl esters either by conventional chemical means or enzymatically by immobilized Candida antarctica lipase. Urea-fractionation of such an intermediary product can give an EPA+DHA content of approximately 85%.     

Purification of docosahexaenoic acid by selective esterification of fatty acids from tuna oil with Rhizopus delemar lipase

To purify docosahexaenoic acid (DHA), we attempted the selective esterification of fatty acids originating from tuna oil with lipases. Tuna oil was hydrolyzed in NaOH-ethanol solution, and the resulting fatty acid mixture [DHA, 23.2%; named tuna-free fatty acid (FFA)] was used as a starting material. Rhizopus delemar which acted lightly on DHA, was a suitable catalyst for the selective esterification of tuna-FFA, and lauryl alcohol was the best substrate. The reaction proceeded most effectively when a mixture of 2.4 g lauryl alcohol/tuna-FFA (2:1, mol/mol), 0.6 g water, and 600 U Rhizopus lipase was incubated at 30°C for 20 h with stirring at 500 rpm. Under these conditions 72% of tuna-FFA was esterified, and 84% of DHA was recovered in the unesterified fatty acid fraction. The DHA content in the fatty acid fraction rose from 23 to 73% with this reaction. To further elevate the DHA content, the unesterified fatty acids were extracted, and then esterified again under the same conditions. By this repeated esterification, DHA was purified to 89% with a recovery of 71% of its initial content.     

Production of triglycerides enriched in long-chain n-3 polyunsaturated fatty acids from fish oil

Processes that combine enzymic and physical techniques have been studied for concentrating and separating eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil.Candida rugosa lipase was used in hydrolysis reactions to concentrate these acids in the glyceride fraction. By controlling the degree of hydrolysis, two products have been obtained, one enriched in total n-3(∼50%), the other enriched in DHA and depleted in EPA (DHA∼40%, EPA∼7%). The glyceride fraction from these reactions was recovered by evaporation and converted back to triglycerides by partial enzymic hydrolysis, followed by enzymic esterification. Both reactions were carried out withRhizomucor miehei lipase. DHA-depleted free fatty acids from aC. rugosa hydrolysis were fractionated to increase the EPA level (∼30%) and re-esterified to triglycerides by reaction with glycerol andR. miehei.     

Purification of docosahexaenoic acid from tuna oil by a two-step enzymatic method: Hydrolysis and selective esterification

Purification of docosahexaenoic acid (DHA) was attempted by a two-step enzymatic method that consisted of hydrolysis of tuna oil and selective esterification of the resulting free fatty acids (FFA). When more than 60% of tuna oil was hydrolyzed with Pseudomonas sp. lipase (Lipase-AK), the DHA content in the FFA fraction coincided with its content in the original tuna oil. This lipase showed stronger activity on the DHA ester than on the eicosapentaenoic acid ester and was suitable for preparation of FFA rich in DHA. When a mixture of 2.5 g tuna oil, 2.5 g water, and 500 units (U) of Lipase-AK per 1 g of the reaction mixture was stirred at 40°C for 48 h, 83% of DHA in tuna oil was recovered in the FFA fraction at 79% hydrolysis. These fatty acids were named tuna-FFA-Ps. Selective esterification was then conducted at 30°C for 20 h by stirring a mixture of 4.0 g of tuna-FFA-Ps/lauryl alcohol (1:2, mol/mol), 1.0 g water, and 1,000 U of Rhizopus delemar lipase. As a result, the DHA content in the unesterified FFA fraction could be raised from 24 to 72 wt% in an 83% yield. To elevate the DHA content further, the FFA were extracted from the reaction mixture with n-hexane and esterified again under the same conditions. The DHA content was raised to 91 wt% in 88% yield by the repeated esterification. Because selective esterification of fatty acids with lauryl alcohol proceeded most efficiently in a mixture that contained 20% water, simultaneous reactions during the esterification were analyzed qualitatively. The fatty acid lauryl esters (L-FA) generated by the esterification were not hydrolyzed. In addition, L-FA were acidolyzed with linoleic acid, but not with DHA. These results suggest that lauryl DHA was generated only by esterification.     

Isolation of erucic acid from rapeseed oil by lipase-catalyzed hydrolysis

Three lipases were compared for their ability to hydrolyze high erucic acid rapeseed oil, with the objective of concentrating the erucic acid in a single glyceride fraction. Lipase fromPseudomonas cepacia released all fatty acids rapidly and did not result in selective distribution of erucic acid.Geotrichum candidum lipase released C20 and C22 fatty acids extremely slowly, resulting in their accumulation in the di- and triglyceride fractions. Less than 2% of the total erucic acid was found in the free fatty acid (FFA) fraction. Lipase fromCandida rugosa released erucic acid more slowly than C20 and C18 fatty acids at 35°C but only resulted in a limited accumulation of the erucic acid in the di- and triglyceride fractions. However, when hydrolysis catalyzed byC. rugosa lipase was carried out below 20°C, the reaction mixture solidified and was composed solely of FFAs and diglycerides. The diglyceride fraction contained approximately 95% erucic acid while about 20% of the total erucic acid was found in the FFA fraction. It is concluded that hydrolysis at low temperature withC. rugosa lipase results in a higher purity of erucic acid in the glyceride fraction than can be obtained withG. candidum lipase, but with considerable loss of erucic acid to the FFA fraction.     

Selective hydrolysis of borage oil with Candida rugosa lipase: Two factors affecting the reaction

A 46% γ-linolenic acid (GLA)-containing oil was produced by selective hydrolysis of borage oil (GLA content, 22%) at 35°C for 15 h in a mixture containing 50% water and 20 units (U)/g reaction mixture of Candida rugosa lipase. The GLA content was not raised over 46%, even though the hydrolysis extent was increased by extending the reaction time and by using a larger amount of the lipase. However, 49% GLA-containing oil was produced by hydrolysis in a reaction mixture with 90% water. This result suggested that free fatty acids (FFA) that accumulated in the mixture affected the apparent fatty acid specificity of the lipase in the selective hydrolysis and interfered with the increase of the GLA content. To investigate the kinetics of the selective hydrolysis in a mixture without FFA, glycerides containing 22, 35, and 46% GLA were hydrolyzed with Candida lipase. The result showed that the hydrolysis rate decreased with increasing GLA content of glycerides, but that the release rate of GLA did not change. Thus, it was found that the apparent fatty acid specificity of the lipase in the selective hydrolysis was also affected by glyceride structure. When 46% GLA-containing oil was hydrolyzed at 35°C for 15 h in a mixture containing 50% water and 20 U/g of the lipase, GLA content in glycerides was raised to 54% at 20% hydrolysis. Furthermore, GLA content in glycerides was raised to 59% when the hydrolysis extent reached 60% using 200 U/g of the lipase. These results showed that repeated hydrolysis was effective to produce the higher concentration of GLA oil. Because film distillation was found to be extremely effective for separating FFA and glycerides, large-scale hydrolysis of borage oil was attempted. As a result, 1.5 kg of 56% GLA-containing oil was obtained from 7 kg borage oil by repeated reaction.     

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.     

脂肪酶法浓缩鱼油中EPA和DHA的研究现状和发展趋势

本文评述了脂肪酶浓缩鱼油中ＥＰＡ、ＤＨＡ的几种方法,并介绍了ＤＰＡ,ＤＨＡ的分离提纯手段。     

Immobilization of <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase by sol-gel entrapment and its application in the hydrolysis of soybean oil

Immobilization of lipase AY from Candida rugosa by entrapment within a chemically inert hydrophobic sol-gel support was studied. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and isobutyltrimethoxysilane. Certain modifications were incorporated into the conventional immobilization procedure, including the use of glucose as additive and the application of vacuum during the drying and aging stages. The activity and thermostability of immobilized enzyme were subsequently determined in hydrolyzing soybean oil. Hydrolysis results showed more than 95 mol% of the theoretical yield for the formation of FF after 1 h of reaction at 40°C. The level of FFA was 3.3 times greater than that seen when an immobilized enzyme was prepared by the conventional sol-gel process. The immobilized enzyme retained most of its hydrolytic activity compared to the free enzyme and kept more than 95% activity after 120 h of incubation at 40°C, whereas the free enzyme lost 67% of its activity after 24 h of incubation and almost all of its activity after 96 h of incubation at 40°C. The immobilized enzyme also proved to be very stable, as it retained more than 90% of the initial activity after 16 one-hour reactions. Surface characterization studies suggested that the enzyme-containing sol-gel particles have amorphous morphology and are void of micro/meso pores.     

Enrichment of eicosapentaenoic acid and docosahexaenoic acid in saponified menhaden oil

Eicosapentaenoic acid (EPA, 20∶5n−3) and docosahexaenoic acid (DHA, 22∶6n−3) in free fatty acids (FFA) derived from saponified menhaden oil were concentrated by the solubility differences of FFA-salts in organic solvent. FFA-salts were formed by adding NaOH to a solution containing FFA. A Buchner funnel was used to separate solid phases from liquids containing FFA-salts. FFA that are rich in EPA and DHA can be recovered from the liquid phase by the addition of 12 N HCl. The effects of reaction time, the amount of NaOH, and solvent used on the concentration of EPA and DHA were systematically investigated. With a total volume of 112 mL, made up of 1.85% 15 N NaOH, 88.1% acetone, and 10.0% FFA, a reaction temperature of 30°C, and a reaction time of 1 h, the resulting liquid phase contained 65.4 wt% EPA and DHA, with a corresponding yield of 41.5%. By replacing the acetone with a mixture of 45% acetone and 55% acetonitrile and then storing the liquid phase at −70°C overnight, the content and yield of EPA and DHA in the final liquid phase were 61.4 wt% and 66.2%, respectively.     

Separation of EPA and DHA in fish oil by lipase-catalyzed esterification with glycerol

The objective of this study was to investigate the use of lipases as catalysts for separating EPA and DHA in fish oil by kinetic resolution based on their FA selectivity. Esterification of FFA from various types of fish oils with glycerol by immobilized Rhizomucor miehei lipase under water-deficient, solvent-free conditions resulted in a highly efficient separation of EPA and DHA. Reactions were conducted at 40°C with a 10% dosage of the lipase preparation under vacuum to remove the coproduced water, thus rapidly shifting the reaction toward the products. The bulk of the FA, together with EPA, were converted into acylglycerols, whereas DHA remained in the residual FFA. As an example, when FFA from tuna oil comprising 5% EPA and 25% DHA were esterified with glycerol, 90% conversion into acylglycerols was obtained after 48 h. The residual FFA contained 78% DHA and only 3% EPA, in 79% DHA recovery. EPA recovery in the acylglycerol fraction was 91%. The type of fish oil and extent of conversion were highly important parameters in controlling the degree of concentration.     

Utilization of rice hull ash as a support material for immobilization of Candida cylindracea lipase

Rice hull ash was heated in a muffle furnace at 700°C for 2 h and metallic oxides were leached with 10% sulfuric acid. The acid-activated ash was then examined for immobilization of Candida cylindracea lipase. Immobilization was carried out by direct addition of the enzyme solution to the activated ash suspended in hexane. The immobilized lipase retained 30% of its hydrolytic activity, but thermal stability was greatly increased. Half-lives of the immobilized enzyme at 50, 60, and 70°C were 45, 17, and 4 min, respectively. Optimal pH of the immobilized enzyme was 7.2. The apparent K_m and V_max for olive oil were 41 mM and 99.5 μmol/h-mg solid, respectively.     

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.     

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.