Copolymeric polythioesters by lipase-catalyzed thioesterification and transthioesterification of alpha,omega-alkanedithiols.

Linear copolymeric polythioesters [PTE; poly(alpha,omega-alkanedioic acid-co-alpha,omega-alkanedithiols)] were formed in good yield (approximately 69%) by thioesterification of 1,12-dodecanedioic acid with 1,6-hexanedithiol and 1,8-octanedithiol, respectively, catalyzed by immobilized lipase from Rhizomucor miehei (Lipozyme RM IM) in vacuo without a solvent. Similarly, transthioesterification (thiolysis) of diethyl 1,12-dodecanedioate with 1,6-hexanedithiol led to the formation of approximately 66% PTE. Poly (1,12-dodecanedioic acid-co-1,6-hexanedithiol) and poly (1,12-dodecanedioic acid-co-1,8-octanedithiol) were extracted from the reaction mixture using methyl-t-butylether, precipitated at -20 degrees C and the precipitates extracted with boiling i-hexane to yield two fractions of PTE. The i-hexane-insoluble fraction of poly (1,12-dodecanedioic acid-co-1,6-hexanedithiol) shows an average molecular mass (Mw) of 1,212 Da, corresponding to a molecular weight range of up to 13,200 Da and a degree of polymerization of up to 38 monomer units. The i-hexane-insoluble fraction of poly (1,12-dodecanedioic acid-co-1,8-octanedithiol) shows a Mw of 2,360 Da, corresponding to a molecular weight range of up to 19,500 Da and a maximum degree of polymerization of up to 52 monomer units. The low-molecular weight (<800 Da) reaction products of thioesterification of 1,12-dodecanedioic acid with 1,6-hexanedithiol, elucidated by gas chromatography-mass spectroscopy, show the following intermediates: (1) 9,20-dioxo-1,8-dithiacycloeicosane; (2) 17,28-dioxo-1,8,9,16-tetrathiacyclooctacosane; (3) 1,12-dodecanedioic acid methyl(O)ester 6'-S-mercaptohexyl thio(S)ester; and (4) oligomeric linear thioester, formed by thioesterification of two molecules of 1,12-dodecanedioic acid with one molecule of 1,6-hexanedithiol.

Mono-thioesters and di-thioesters by lipase-catalyzed reactions of alpha,omega-alkanedithiols with palmitic acid or its methyl ester.

1- S-Mono-palmitoyl-hexanedithiol and 1- S-mono-palmitoyl-octanedithiol were prepared in high yield (80-90%) by solvent-free lipase-catalyzed thioesterification of palmitic acid with the corresponding alpha,omega-alkanedithiols in vacuo. Similarly, 1,6-di- S-palmitoyl-hexanedithiol and 1,8-di- S-palmitoyl-octanedithiol were prepared in moderate yield (50-60%) by solvent-free lipase-catalyzed thioesterification of palmitic acid with 1- S-Mono-palmitoyl-hexanedithiol and 1- S-mono-palmitoyl-octanedithiol, respectively. An immobilized lipase preparation from Rhizomucor miehei (Lipozyme RM IM) was more effective than a lipase B preparation from Candida antarctica (Novozym 435) or a lipase preparation from Thermomyces lanuginosus (Lipozyme TL IM). Lipase-catalyzed transthioesterifications of methyl palmitate with alpha,omega-alkanedithiols using the same enzymes were less effective than thioesterification for the preparation of the corresponding 1- S-mono-palmitoyl thioesters.

Steryl and stanyl esters of fatty acids by solvent-free esterification and transesterification in vacuo using lipases from Rhizomucor miehei, Candida antarctica, and Carica papaya.

Sitostanol has been converted in high to near-quantitative extent to the corresponding long-chain acyl esters via esterification with oleic acid or transesterification with methyl oleate or trioleoylglycerol using immobilized lipases from Rhizomucor miehei (Lipozyme IM) and Candida antarctica (lipase B, Novozym 435) as biocatalysts in vacuo (20-40 mbar) at 80 degrees C, whereas the conversion was markedly lower at 60 and 40 degrees C. Corresponding conversions observed with papaya (Carica papaya) latex lipase were generally lower. High conversion rates observed in transesterification of sitostanol with methyl oleate at 80 degrees C using Lipozyme IM were retained even after 10 repeated uses of the biocatalyst. Saturated sterols such as sitostanol and 5alpha-cholestan-3beta-ol were the preferred substrates as compared to Delta(5)-unsaturated cholesterol in transesterification reactions with methyl oleate using Lipozyme IM. Transesterification of cholesterol with dimethyl 1,8-octanedioate using Lipozyme IM in vacuo yielded methylcholesteryl 1,8-octanedioate (75%) and dicholesteryl 1,8-octanedioate (5%). However, transesterification of cholesterol with diethyl carbonate and that of oleyl alcohol with ethylcholesteryl carbonate, both catalyzed by Lipozyme IM, gave ethylcholesteryl carbonate and oleylcholesteryl carbonate, respectively, in low yield (20%). Moreover, cholesterol was transesterified with ethyl dihydrocinnamate using Lipozyme IM to give cholesteryl dihydrocinnamate in moderate yield (56%), whereas the corresponding reaction of lanosterol gave lanosteryl oleate in low yield (14%).

High-yield preparation of wax esters via lipase-catalyzed esterification using fatty acids and alcohols from crambe and camelina oils.

Fatty acids obtained from seed oils of crambe (Crambe abyssinica) and camelina (Camelina sativa) via alkaline saponification or steam splitting were esterified using lipases as biocatalysts with oleyl alcohol and the alcohols derived from crambe and camelina oils via hydrogenolysis of their methyl esters. Long-chain wax esters were thus obtained in high yields when Novozym 435 (immobilized lipase B from Candida antarctica) and papaya (Carica papaya) latex lipase were used as biocatalysts and vacuum was applied to remove the water formed. The highest conversions to wax esters were obtained with Novozym 435 (> or =95%) after 4-6 h of reaction, whereas with papaya latex lipase such a high degree of conversion was attained after 24 h. Products obtained from stoichiometric amounts of substrates were almost exclusively (>95%) composed of wax esters having compositions approaching that of jojoba (Simmondsia chinensis) oil, especially when crambe fatty acids in combination with camelina alcohols or camelina fatty acids in combination with crambe alcohols were used as substrates.

Fatty acid steryl, stanyl, and steroid esters by esterification and transesterification in vacuo using Candida rugosa lipase as catalyst.

Sterols (sitosterol, cholesterol, stigmasterol, ergosterol, and 7-dehydrocholesterol) and sitostanol have been converted in high to near-quantitative yields to the corresponding long-chain acyl esters via esterification with fatty acids or transesterification with methyl esters of fatty acids or triacylglycerols using lipase from Candida rugosa as biocatalyst in vacuo (20-40 mbar) at 40 degrees C. Neither organic solvent nor water is added in these reactions. Under similar conditions, cholesterol has been converted to cholesteryl butyrate and steroids (5alpha-pregnan-3beta-ol-20-one or 5-pregnen-3beta-ol-20-one) have been converted to their propionic acid esters, both in moderate to high yields, via transesterification with tributyrin and tripropionin, respectively. Reaction parameters studied in esterification include the temperature and the molar ratio of the substrates as well as the amount and reuse properties of the C. rugosa lipase. Lipases from porcine pancreas, Rhizopus arrhizus, and Chromobacterium viscosum are quite ineffective as biocatalysts for the esterification of cholesterol with oleic acid under the above conditions.

Antioxidants eliminate stereomutation and thioether formation during lipase-catalyzed thioesterification and transthioesterification for the preparation of uniform cis- and trans-unsaturated thioesters.

The lipase-catalyzed preparation of acyl thioesters from unsaturated fatty acids and alkanethiols is accompanied by the formation of geometrical isomers via stereomutation and of thioether derivatives via addition at the olefinic bond, both induced by thiyl radicals. Therefore, a method was developed in order to inhibit radical generation by the addition of antioxidants and thus prevent the formation of geometrical isomers and thioether derivatives during the lipase-catalyzed preparation of unsaturated acyl thioesters. In the presence of antioxidants such as 2,6-di-t-butyl-4-methylphenol (BHT) and octyl gallate thioesterification of oleic and elaidic acids with 1-tetradecanethiol as well as transthioesterification of methyl linoleate with 1-tetradecanethiol led to the corresponding geometrically uniform thioesters without radical-induced side reactions. In the absence of antioxidants rapid stereomutation of unsaturated acyl moieties as well as formation of high proportions of thiyl radical-induced addition products such as isomeric 9(10)-S-tetradecyl stearic acids and 9(10)-S-tetradecyl stearic acid tetradecyl thioesters were observed.

Specificity of papaya lipase in esterification with respect to the chemical structure of substrates.

Esterification, catalyzed by papaya (Carica papaya) lipase (CPL), was studied with various alcohols and carboxylic acids under competitive conditions. Acids studied were straight-chain saturates of different chain lengths, with octanoic acid as the reference. Alcohols chosen were aliphatic straight-chain, branched, secondary, tertiary, terpene, and aromatic alcohols of different chain lengths, using 1-hexanol as the reference. The initial reaction rate increased with increasing chain length of the acid from C4:0 to C18:0, followed by a slight decrease with C20:0. In the case of alcohols, an optimum chain length of 8 carbon atoms was obtained for the straight-chain aliphatic group (C2 to C16). Ethanol, 1-propanol, and secondary and tertiary alcohols showed rather low reactivity. Branching of the alcohols was found not to affect the reactivity in esterification; among the terpenes, beta-citronellol [(2E)-3, 7-dimethyl-6-octenol] and geraniol [(2E)-3,7-dimethylocta-2, 6-dien-1-ol] were found to be more reactive than nerol [(2Z)-3, 7-dimethylocta-2,6-dien-1-ol]. The highest reaction rate was found for the aromatic benzyl alcohol (phenylmethanol).

Papaya (Carica papaya) lipase with some distinct acyl and alkyl specificities as compared with microbial lipases.

Lipase from papaya (Carica papaya) latex (CPL), Candida antarctica lipase B (Novozym 435, NOV) and Rhizomucor miehei lipase (Lipozyme IM 20, LIP) were used as biocatalysts for the esterification of caprylic acid with straight-chain saturated C(4)-C(18) alcohols and unsaturated C(18) alcohols, such as cis-9-octadecenyl (oleyl, C(18:1), n-9), cis-6-octadecenyl (petroselinyl, C(18:1), n-12), cis-9,cis-12-octadecadienyl (linoleyl, C(18:2), n-6), all-cis-9,12,15-octadecatrienyl (alpha-linolenyl, C(18:3), n-3) and all-cis-6,9,12-octadecatrienyl (gamma-linolenyl, C(18:3), n-6) alcohols. With CPL, highest activity was found in the esterification of octanol and decanol, whereas both NOV and LIP showed a broad chain-length-specificity for the alcohols. CPL, as opposed to the microbial lipases, strongly discriminated against all the saturated long-chain ( > C(12)) and unsaturated C(18) alcohols.

Adipose tissue triacylglycerols of rats are modulated differently by dietary isomeric octadecenoic acids from coriander oil and high oleic sunflower oil.

Earlier feeding studies of rats revealed that petroselinic acid [18:1(n-12)] from triacylglycerols of coriander (Coriandrum sativum) oil is extensively incorporated into the lipids of heart and liver and metabolized via beta-oxidation and chain elongation. We report here the composition and stereospecific distribution of acyl moieties, particularly isomeric octadecenoyl moieties, in adipose tissue triacylglycerols of male weaned Wistar rats fed diets containing, in addition to 20 g corn oil/kg feed, 120 g coriander oil per kg feed at a level of 63 g 18:1(n-12) moieties/100 g acyl moieties of the oil for 10 wk. For comparison, a group of rats was fed a similar corn oil-containing isocaloric diet with large proportions of oleoyl moieties [18:1(n-9)] from high oleic sunflower oil [72 g 18:1(n-9)/100 g acyl moieties of the oil]. The composition of the triacylglycerols of epididymal, subcutaneous and perirenal adipose tissues was very similar for each feeding group, broadly reflecting the composition of the dietary oils. Feeding coriander oil, compared with high oleic sunflower oil, led to extensive incorporation of 18:1(n-12) into the triacylglycerols of the adipose tissues with a concomitant significantly and dramatically lower 18:1(n-9) concentration and, as a consequence, to the generation of triacylglycerol species containing 18:1(n-12) moieties. Petroselinoyl moieties from coriander oil were esterified predominantly at the sn-1,3 positions of the adipose tissue triacylglycerols; 18:1(n-9) moieties from high oleic sunflower oil were fairly evenly distributed between the sn-1,3 and sn-2 positions. We suggest that acyltransferases involved in the biosynthesis of adipose tissue triacylglycerols direct 18:1(n-12) moieties preferentially to sn-1,3-positions.

Fat infiltration in liver of rats induced by different dietary plant oils: high oleic-, medium oleic- and high petroselinic acid-oils.

Beneficial effects on health parameters by ingestion of plant oils having high levels of monounsaturated fatty acids such as oleic (cis-9-octadecenoic) acid have been described in several studies. Recently, we have shown that feeding of coriander oil containing high proportions of a positional isomer of oleic acid, i.e. petroselinic (cis-6-octadecenoic) acid, led to significantly decreasing proportions of arachidonic (all cis-5,8,11,14-eicosatetraenoic) acid in the cellular lipids. Here, we report histopathological findings in male Wistar rats fed isocaloric diets containing 2% corn oil and 12% (w/w), each, of different plant oils (high-oleic and conventional sunflower oil, olive oil, rapeseed oil as well as coriander oil) containing varying levels of monounsaturated fatty acids, i.e. oleic or petroselinic acids. One group of animals was fed a standard diet containing 4% (w/w) of fat as control. Liver, heart, aorta, stomach, and spleen were anatomically and histologically examined. Livers of animals from all the groups that had received high-fat diets-except the coriander oil group-showed moderate non-degenerative fat infiltrations of the hepatocytes beginning in the periportal fields. Marked to severe fat infiltration was observed in hepatocytes of animals fed coriander oil. In this experimental group the livers were found to contain fatty cysts in addition to hepatocytes with mixed-size lipid vesicles. Moreover, enlarged nuclei were observed in numerous hepatocytes without fat infiltration. Fat infiltration was not observed in livers of animals fed a rat standard diet. In the cells of all other organs and tissues studied, in particular heart and aorta, neither non-degenerative fat infiltrations nor other degenerative changes were observed for any group. Our results show that feeding of fat-rich diets containing varying proportions of C18 cis-monoenoic fatty acids induced different histopathological alterations in the livers of rats, depending on position of the double bond. Plant oils containing varying proportions of oleic acid lead exclusively to moderate fat infiltration in the hepatocytes, whereas in the animals fed high-petroselinic coriander oil degenerative alterations (fatty cysts) as well as -in several cases-enlarged nuclei were found that are likely due to a non-specific stimulation of the hepatocytes resulting from the cell degeneration observed.

Petroselinic acid from dietary triacylglycerols reduces the concentration of arachidonic acid in tissue lipids of rats.

Studies in vitro have revealed that triacylglycerols containing petroselinoyl [18:1(n-12)] moieties are hydrolyzed by pancreatic lipase at much lower rates than other triacylglycerols. To assess the lipolysis and absorption in vivo of such unusual triacylglycerols, diets containing 120 g seed oil triacylglycerols of coriander (Coriandrum sativum) per kg diet at a level of 72 g 18:1(n-12) moieties/100 g oil were fed to a group of weaned male Wistar rats without restriction for a period of 10 wk. For comparison, groups of rats were fed similar isocaloric diets containing plant oil triacylglycerols with various levels of oleoyl [18:1(n-9)] moieties, e.g., high oleic sunflower seed oil [75 g 18:1(n-9)/100 g oil], olive oil [(66 g 18:1(n-9)/100 g oil], medium oleic rapeseed oil [54 g 18:1(n-9)/100 g oil] and conventional high linoleic sunflower seed oil [25 g 18:1(n-9)/100 g oil]. All diets were supplemented with 20 g corn oil/kg diet. Consumption of coriander oil, compared with the other oils, led to significantly greater liver weights. No significant differences were observed among the groups fed various levels of oleic acid in body weight, the weights of heart, liver, kidneys, spleen or testes, lipid content of heart, or total cholesterol, HDL cholesterol and triacylglycerol concentrations of blood plasma. Ingestion of coriander oil led to incorporation of 18:1(n-12) into heart, liver and blood lipids and to a significant reduction in the concentration of arachidonic acid in the lipids of heart, liver and blood with a concomitant increase in the concentration of linoleic acid compared with results for the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Solubilization and partial purification of constituents of acyl-CoA elongase from Lunaria annua.

All the constituent enzymes of acyl-CoA elongase, i.e., beta-ketoacyl-CoA synthase, beta-ketoacyl-CoA reductase, beta-hydroxyacyl-CoA dehydrase and trans-2-enoyl-CoA reductase, have been solubilized from a 15,000 x g particulate fraction from developing seeds of honesty (Lunaria annua) using Triton X-100. All these activities were retained upon subsequent precipitation of the solubilized protein with polyethylene glycol and resuspension of the precipitate followed by ion exchange chromatography of the resulting protein on DEAE-cellulose. A 4.2-fold enrichment of the acyl-CoA elongase was thus obtained. Further chromatography of the DEAE fraction containing all the constituents of acyl-CoA elongase on Ultrogel yielded a major protein fraction exhibiting the activities of beta-ketoacyl-CoA synthase and beta-ketoacyl-CoA reductase only. Almost 30-fold purification of the beta-ketoacyl-CoA synthase was thus achieved. The beta-ketoacyl-CoA synthase was inhibited only at high concentrations of cerulenin, but at very low concentrations of iodoacetamide. Inhibition could be reduced by preincubation with thioesters, indicating that an enzyme thioester intermediate is involved in the condensation reaction of the acyl-CoA elongation.

Stereoselectivity of lipases: esterification reactions of octadecylglycerol.

Stereoselectivity of several triacylglycerol lipases (EC 3.1.1.3) has been investigated in the enzymatic esterification of rac-1-O-octadecylglycerol with oleic acid in the presence of organic solvents, such as hexane. X-1(3)-O-Octadecylmonooleoylglycerols were the only products formed with most lipases; considerable proportions of X-1(3)-O-octadecyldioleoylglycerols were also formed with the lipase from Candida cylindracea. The mixtures of unesterified enantiomeric substrates, i.e., X-1(3)-O-octadecylglycerols were converted to their 3,5-dinitrophenylurethane derivatives and subsequently resolved into sn-1 and sn-3 enantiomers by HPLC on a chiral stationary phase (Sumichiral OA 2100). The data on enantiomeric excess (ee) and enantiomeric ratio (E) in the unesterified substrate revealed for the lipases from porcine pancreas, Rhizopus sp., Pseudomonas sp., Candida cylindracea, Chromobacterium viscosum and Penicillium cyclopium a distinct preference for 1-O-octadecyl-sn-glycerol over its enantiomer indicating stereoselectivity for the sn-3 position. For the lipase from Rhizomucor miehei a slight stereoselectivity for the sn-1 position was observed. Solvents, such as diethyl ether and dichloromethane, strongly inhibited the esterification reaction, but the enzymatic activity could be restored upon removal of such solvents by washing with hexane indicating reversible inhibition.

Acyl-CoA elongase from a higher plant (Lunaria annua): metabolic intermediates of very-long-chain acyl-CoA products and substrate specificity.

A particulate fraction (15,000 x g pellet) from developing seeds of honesty (Lunaria annua) was found to synthesize very-long-chain acyl-CoA thioesters in a manner similar to mammalian systems, i.e., via condensation of an acyl-CoA with malonyl-CoA yielding beta-ketoacyl-CoA, which is reduced to beta-hydroxyacyl-CoA, the latter dehydrated to trans-2-enoyl-CoA that is finally reduced to very-long-chain acyl-CoA. Reduced pyridine nucleotides (NADH/NADPH) are required for the reduction steps. In the absence of reduced pyridine nucleotides only the condensation reaction occurs. The acyl-CoA elongase does not exhibit any pronounced specificity for any of the saturated (14:0 to 20:0) or (n - 9)cis-monounsaturated (14:1 to 22:1) acyl-CoA substrates, although both the saturated and monounsaturated acyl-CoA substrates having chain lengths of C18 and C20 are elongated somewhat faster.

Triacylglycerol lipase from rape (Brassica napus L.) suitable for biotechnological purposes.

Triacylglycerol lipase (EC 3.1.1.3) from rape (Brassica napus L. cv Ceres) is quite easily prepared from the 100,000 x g supernatant of cotyledon homogenates. The lipase is present in a high-molecular-mass fraction (greater than 1.5 x 10(6) dalton by gel filtration), but it can be rapidly extracted from the 100,000 x g supernatant by precipitation with polyethyleneglycol 8000 (4%, w/v) and MgCl2 (40 mM) giving about a 10-fold purification. After delipidation, the lipase has an Mr of about 300,000. It hydrolyzes triacylglycerols to fatty acids and glycerol, although the fatty acids from the sn-1 or -3 positions are hydrolyzed first to yield 1,2(2,3)-diacylglycerols. Lipase immobilized onto Celite by precipitation with acetone at -20 degrees C catalyzes the esterification of oleic acid with butanol dissolved in hexane.

Biosynthesis of triacylglycerols containing very long chain monounsaturated acyl moieties in developing seeds.

Particulate (15,000g) fractions from developing seeds of honesty (Lunaria annua L.) and mustard (Sinapis alba L.) synthesize radioactive very long chain monounsaturated fatty acids (gadoleic, erucic, and nervonic) from [1-(14)C]oleoyl-CoA and malonyl-CoA or from oleoyl-CoA and [2-(14)C]malonyl-CoA. The very long chain monounsaturated fatty acids are rapidly channeled to triacylglycerois and other acyl lipids without intermediate accumulation of their CoA thioesters. When [1-(14)C]oleoyl-CoA is used as the radioactive substrate, phosphatidylcholines and other phospholipids are most extensively radiolabeled by oleoyl moieties rather than by very long chain monounsaturated acyl moieties. When [2-(14)C]malonyl-CoA is used as the radioactive substrate, no radioactive oleic acid is formed and the newly synthesized very long chain monounsaturated fatty acids are extensively incorporated into phosphatidylcholines and other phospholipids as well as triacylglycerols. The pattern of labeling of the key intermediates of the Kennedy pathway, e.g. lysophosphatidic acids, phosphatidic acids, and diacylglycerols by the newly synthesized very long chain monounsaturated fatty acids is consistent with the operation of this pathway in the biosynthesis of triacylglycerols.

Lipase from Brassica napus L. discriminates against cis-4 and cis-6 unsaturated fatty acids and secondary and tertiary alcohols.

Lipase (EC 3.1.1.3) from oilseed rape (Brassica napus L., cv Ceres) hydrolyzes triacylglycerols containing a broad range of fatty acids at similar rates. In esterification reactions carried out in hexane, rape lipase also uses a wide range of fatty acids and alcohols as reaction partners. However, the rates of esterification of petroselinic, gamma-linolenic, stearidonic and docosahexaenoic acids are only between 2 and 7% that of oleic acid. The common feature of these fatty acids is that the first double bond is cis-4 or cis-6. Petroselaidic acid with a trans-6 double bond is esterified about 10-times faster than petroselinic acid. Arachidonic and eicosapentaenoic acids, both with the first double bond being cis-5, are esterified about 20-times faster than docosahexaenoic acid. By analogy, tripetroselinin and tri-gamma-linolenin are hydrolyzed at 14% and 1.5%, respectively, of the rate of triolein hydrolysis. The rape lipase esterifies primary alcohols but cannot esterify secondary and tertiary alcohols.

Assay for triacylglycerol lipase by a rapid thin-layer chromatographic technique.

A rapid and accurate assay for lipase-catalyzed hydrolysis of radioactively labeled triacylglycerols has been developed. Aliquots of reaction mixtures are applied directly, i.e., without extraction of the lipolysis products, to thin-layer chromatography plates coated with Silica Gel H containing 5% Na2CO3 (w/w), heated for 10 sec, and developed with diethyl ether-methanol 97:3 (v/v) to a height of 4-5 cm. About 98.5% of the fatty acids are immobilized as sodium salts at the origin of the chromatogram, whereas tri-, di-, and monoacylglycerols migrate close to the solvent front. Adsorbent at the origin and that at the remaining part of the chromatogram are then assayed for radioactivity without prior staining.