Long-chain acyl thioesters prepared by solvent-free thioesterification and transthioesterification catalysed by microbial lipases

Long-chain acyl thioesters (thio wax esters) have been prepared in high (80% to more than 90%) yields by solvent-free esterification of fatty acids (lauric, myristic, palmitic and stearic acids) with long-chain thiols, such as decane thiol, dodecane thiol, tetradecane thiol and hexadecane thiol, catalysed by lipases from Candida antarctica (Novozym) and Rhizomucor miehei (Lipozyme) in the presence of a 0.4-nm molecular sieve. In the thioesterification reaction Novozym was a more effective biocatalyst than Lipozyme. The extent of thioesterification increased with increasing molar ratio of fatty acid to alkane thiol (1:1 to 3:1) and with temperature (40 °C compared to 60 °C), as well as with the amount of the enzyme preparation and the amount of 0.4-nm molecular sieve. Decreasing the chain length of the alkane thiol from C₁₆ to C₁₀ also increased the extent of thioesterification. Lipase-catalysed solvent-free transthioesterification of fatty acid methyl esters with alkane thiols was less effective for the preparation of acyl thioesters than was thioesterification of fatty acids with alkane thiols. In transthioesterification, Lipozyme was slightly more effective as a biocatalyst than Novozym. Received: 3 September 1998 / Received revision: 18 November 1998 / Accepted: 21 November 1998     

Enrichment of γ-linolenic acid from fungal oil by lipase-catalysed reactions

Summary Various lipases have been evaluated as biocatalysts for the enrichment of -linolenic acid from a commercial fungal oil derived from Mucor sp. by selective esterification of the fungal oil fatty acids with n-butanol or by selective hydrolysis of the oil. Lipase from M. miehei (Lipozyme), as compared to lipases from Candida cylindracea, Penicillium cyclopium, and Rhizopus arrhizus, was found to be most effective in the enrichment of -linolenic acid in unesterified fatty acids upon esterification of the fungal oil fatty acids with n-butanol. Thus, the -linolenic acid content could be raised from 10.4% in the starting material to 68.8% in the unesterified fatty acids. Selective hydrolysis of the fungal oil triacyglycerols using Lipozyme resulted in about 1.5-fold enrichment of -linolenic acid in the unhydrolysed acylglycerols. Other lipases tested, such as those from P. cyclopium, C. cylindracea, R. arrhizus, Penicillium sp. (Lipase G), porcine pancreas and Chromobacterium viscosum, were also rather ineffective in the enrichment of -linolenic acid by selective hydrolysis of the fungal oil triacylglycerols. Offprint requests to: K. D. Mukherjee     

Preparation of o-palmitoyl alkyl lactates through lipase atalysis

Preparation of o-palmitoyl alkyl lactates with methyl, ethyl, propyl, isopropyl and butyl lactates were attempted in a complex esterification reaction using lipases as catalysts. Compared to lactic acid, alkyl lactates were found to be less inhibitory in nature as they resulted in slightly better yields at shake-flask level. Of the alkyl lactates tested, butyl lactate showed better esterification. Porcine pancreas lipase gave higher yields of esters than Rhizomucor miehei lipase (Lipozyme IM20).     

Changes in fatty acid composition of lipid classes in developing mustard seed

Maturation of mustard (Sinapis alba) seed proceeds with a sharp decrease in the amounts of palmitic and linoleic acids in the total lipids up to 6 weeks after flowering (WAF). Concomitantly, the concentration of oleic acid increases, reaching a plateau at 4 WAF, which is followed by chain elongation of oleic acid to gadoleic and erucic acids. Compositional changes in constituent fatty acids of individual lipid classes indicate that the very long-chain monounsaturated fatty acids (C₂₀ and C₂₂), as opposed to common long-chain fatty acids (C₁₆ and C₁₈), are metabolized to triacylglycerols mainly by esterification to preformed diacylglycerols and monoacylglycerols, rather than via esterification to glycerol-3-phosphate or lysophosphatidic acids.     

Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid. Received: 29 October 1998 / Received revision: 18 December 1998 / Accepted: 21 December 1998     

Water as a competitive inhibitor of lipase-catalysed esterification in organic media

Summary Kinetic parameters were determined for esterification of dodecanol and decanoic acid in hexane catalysed by lipases from Rhizomucor miehei and Candida rugosa, after pre-equilibration to different values of thermodynamic water activity (a_w). V_m increases with increasing a_w, but so do the K_m values for both substrates. The effect on K_m for the alcohol probably represents competition between the first product and the second substrate, as expected for Ping-Pong kinetics. The rise in K_m for the acid probably reflects the displacement of water molecules during substrate binding.     

Selective lipid hydrolysis byGeotrichum candidum NRRL Y-553 lipase

Summary A crude preparation of lipases from the fungusGeotrichum candidum NRRL Y-553, which shows specificity toward cis-9 unsaturated fatty acids, was used to hydrolyze natural fats and oils, with the goal of producing industrially useful fatty acid and/or mono- and diacylglycerols products. Tallow and soy oil were hydrolyzed, producing free fatty acids high in oleic or cis-9 unsaturates, respectively. Hydrolysis of rapesced oil produced acylglycerols enriched in erucic acid.     

Enzymatic fatty esters synthesis in aqueous medium with lipase from Candida parapsilosis (Ashford) Langeron and Talice

Summary The synthesis of fatty esters in aqueous medium by alcohoiysis catalysed by lipase from Candida parapsilosis (EC.3.1.1.3) is described. The transesterification of rapeseed oil with methanol leads to an equilibrium state after eight hours, with a yield (methyl esters formed/ total fatty acids initially present in the acylglycerols) of at least 53% (73, 45, 65% of the linolenic, linolek and oleic acids respectively). Yield was already about 42% after four hours of catalysis. This transesterification was permitted by methanol inhibition of hydrolysis of the esters.     

Lipase-catalyzed synthesis of partial glyceride

Summary Mucor miehei (IM 20) and Candida antarctica (SP 382) lipases were used for esterification of free fatty acids in the absence of organic solvent or transesterification of fatty acid methyl esters in hexane with isopropylidene glycerols. Acid catalyzed cleavage of the isopropylidene groups resulted in the formation of monoacyl glycerol (MAG) and diacyl glycerol (DAG). Both oleic (18:1 n-9) and eicosapentaenoic acid, EPA (20:5 n-3) were successfully incorporated into glycerides. Total acyl donor conversion ranged from 46.9 – 96.9% with MAG content of up to 88.5%.     

Acyl donors for sterol esterification by cell-free preparations from Sinapis alba roots

Homogenates or crude 300-16 000 g membrane fractions from Sinapis alba roots catalysed esterification of [4-¹⁴C]cholesterol with utilization of endogenous acyl sources. With acetone powder preparations cholesterol esterification was distinctly stimulated by a neutral lipid fraction isolated from S. alba roots. Among neutral lipids triacylglycerols were the most active in this process. Experiments with various acyl-labelled acylglycerols as acyl donors and non-labelled sterols as acceptors confirmed that triacylglycerols are directly utilized as the source of fatty acids for sterol esterification. Di- and mono-acylglycerols were much less effective.     

Synthesis of glycerides containing n-3 fatty acids and conjugated linoleic acid by solvent-free acidolysis of fish oil

Menhaden oil, a rich source of n-3 fatty acids, was interesterified with conjugated linoleic acid (CLA) in a reaction medium composed solely of substrates and either free or immobilized commercial lipase preparations. Of five lipases tested, an immobilized preparation from Mucor miehei provided the fastest rate of incorporation of CLA into fish oil acylglycerols; however, and as observed with most of the lipases utilized, a significant proportion of the n-3 fatty acid residues were liberated in the process. A soluble lipase from Candida rugosa converted free CLA to acylglycerol residues while leaving the n-3 fatty acid residues virtually untouched. Even though the reaction rate was slower for this enzyme than for the other four lipase preparations, the specificity of the free C. rugosa lipase gives it the greatest potential for commercial use in preparing fish oils enriched in CLA residues but still retaining their original n-3 fatty acid residues.     

The Metabolism of the Germinating Oil Palm (Elaeis guineensis) Seedling : In Vivo Studies

The metabolism of ¹⁴C-labeled fatty acids and triacylglycerols was followed in intact germinating oil palm seedlings as well as in tissue slices. In the germinating seedling, the shoot contained a normal pattern of membrane fatty acids (mainly C₁₆, C_18:1, C_18:2) but the kernel contained about 68% C₁₂ and C₁₄ fatty acids. Haustorium fatty acids were intermediate between the two. [¹⁴C]Acetate was actively metabolized by shoot and haustorium slices but not so actively by the kernel. Approximately 9% to 17% was converted to water-soluble substances, 4% to 6% to CO₂, and 0.5% to 5.9% to lipids. The fatty acids synthesized in the shoot and haustorium were mainly C₁₆, C₁₈, and C_18:1 fatty acids but in the kernel about 18% to 32% of the ¹⁴C-fatty acids were C₁₂ fatty acids.

[¹⁴C]Lauric acid was absorbed and metabolized by haustorium slices and by the haustorium in intact seedlings; it was partly esterified to triacylglycerols and also converted to water-soluble substances and insoluble tissue material. In contrast, tri-[¹⁴C]laurin was absorbed but not metabolized. The haustorium also absorbed other fatty acids but the longer chain (C₁₆ and C₁₈) fatty acids were not esterified or metabolized further. Preincubation of the haustorium with plant hormones or in the presence of kernel tissue did not alter its inactivity towards tri-[¹⁴C]laurin.

When tri-[¹⁴C]laurin or [¹⁴C]lauric acid were injected into the seed or the shoot, there was no movement or radioactivity to other parts of the seedling. When injected into the shoot, but not into the seed, tri-[¹⁴C] laurin was hydrolyzed and partly metabolized to water-soluble substances.

     

Lipases in organic solvents: The fatty acid chain length profile

Summary Competitive lipase-catalysed hydrolyses of triacylglycerols were used for the quantitative determination of the fatty acid profile of several lipases. In contrast to previously described methods a complete and reliable activity profile for triacylglycerols containing fatty acid residues with chain lengths from C₄ to C₁₈₁ can be obtained in one single experiment.     

Substrate selectivity in esterification of less common fatty acids catalysed by lipases from different sources

Lipases from microorganisms (Candida cylindracea, Rhizopus arrhizus and Rhizomucor miehei), animal tissue (porcine pancreas) and a higher plant (rape, Brassica napus) have been evaluated as biocatalysts in the esterifications with n-butanol with regard to their substrate specificity towards fatty acids having a cis-4 unsaturation, e.g. docosahexaenoic (n-3 22:6), cis-6 unsaturation, e.g. petroselinic (n-12-18:1), -linolenic (n-6 18:3) and stearidonic (n-3 18:4), as well as cis-8 unsaturation, e.g. dihomo--linolenic (n-6 20:3) acid, and fatty acids having unusual structures, e.g.cyclopentenyl (hydnocarpic and chaulmoogric), hydroxy (ricinoleic and 12-hydroxystearic) and epoxy (cis- and trans-9,10-epoxystearic) acids. A common feature of all the lipases tested is their ability to discriminate strongly against unsaturated fatty acids having a cis-4, cis-6 or a cis-8 unsaturation as substrates for esterification, whereas cyclopentenyl, hydrocy and epoxy acids are very well accepted as substrates. Esterification of the fatty acids of Hydnocarpus wightiana seed oil with n-butanol, catalysed by each of the above lipases, revealed that, as compared to the cyclopentenyl fatty acids having saturated alkyl chains, gorlic acid — a cyclopentenyl acid having a cis-6 unsaturation — is also strongly discriminated against as a substrate for esterification.This paper is Part of the doctoral thesis of Iván Jachmanián to be submitted to Facultad de Química, Universidad de la República, Montevideo, Uruguay     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Sources of C20-polyunsaturated fatty acids for biotechnological use

Summary Polyunsaturated fatty acids with 20 carbon atoms exhibit unique physiological activities in the human body, for example lowering of cholesterol and triacylglycerols in plasma, prevention of atherosclerosis and other cardiovascular diseases and reduction of colagen-induced thrombocyte aggregation. Moreover, these fatty acids are of great value in the nutrition of edible marine animals reared in mariculture, and as precursors of eicosanoid hormones. Potential sources of such fatty acids include fungi, mainly lower Phycomycetes, microalgae, viz. dinoflagellates, diatoms and unicellular red algae, marine macroalgae, particularly Phaeophyta and Rhodophyta, and mosses. The biomass may be enriched with C₂₀-polyunsaturated fatty acids by chilling, nitrogen starvation, controlled illumination and incubation with lipophilic compounds. Offprint requests to: S. S. Radwan     

Lipase-catalyzed regioselective synthesis of steryl (6′-<Emphasis Type="Italic">O</Emphasis>-acyl)glucosides

The regioselective acylation of cholesteryl β-d-glucoside, at the C-6 of the glucose moiety, was achieved using microbial lipases in organic solvents. With palmitic acid as an acyl donor 81 or 63% conversions of cholesteryl glucoside to its 6′-O-palmitoyl derivative were obtained using Candida antarctica or Rhizomucor miehei enzymes, respectively. High yields (64–92%) were also obtained with fatty acids 6:0–22:0 and 16:1 (n-7). The synthesis of cholesteryl (6′-O-palmitoyl)glucoside was also achieved via transesterification, using mono-, di- and tri-palmitoylglycerols or methyl and ethyl palmitate as acyl sources. With R. miehei lipase transesterification between methyl palmitate (80 mM) and cholesteryl glucoside (1 mM) proceeded after 24 h with a nearly quantitative yield (97%).     

Esterification of phenolic acids catalyzed by lipases immobilized in organogels

Lipases from Rhizomucor miehei and Candida antarctica B were immobilized in hydroxypropylmethyl cellulose organogels based on surfactant-free microemulsions consisting of n-hexane, 1-propanol and water. Both lipases kept their catalytic activity, catalyzing the esterification reactions of various phenolic acids including cinnamic acid derivatives. High reaction rates and yields (up to 94%) were obtained when lipase from C. antarctica was used. Kinetic studies have been performed and apparent kinetic constants were determined showing that ester synthesis catalyzed by immobilized lipases occurs via the Michaelis–Menten mechanism.     

Enhancement of activity and selectivity of Candida rugosa lipase and Candida antarctica lipase A by bioimprinting and/or immobilization for application in the selective ethanolysis of fish oil

Candida rugosa lipase (CRL) and Candida antarctica lipase A (CALA) with improved activity and selectivity were prepared for use in organic solvent media. CRL bioimprinted with fatty acids exhibited eightfold enhanced transesterification activity in hexane. Combination of bioimprinting and coating with lecithin or with immobilization did not improve the activity further. CALA was immobilized with and without bioimprinting, none of which improved the activity. All modified lipases were tested for selective ethanolysis of fish oil to concentrate omega-3 polyunsaturated fatty acids (PUFA). None of the preparations, except the immobilized ones catalysed ethanolysis. Immobilized CRL-catalyzed ethanolysis giving 27% (v/v) ethyl esters (EE) in 48 h, of which 43 mol% was oleic acid but no PUFA was detected in the EE fraction. Fatty acid selectivity of CALA was significantly improved by immobilization combined with bioimprinting, resulting in 5.5-fold lower omega-3 PUFA in EE.     

Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth

In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl₂, or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein.