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.     

Lipid and fatty acid composition of the fat body during the female reproductive cycle of Labidura riparia (Insecta Dermaptera)

During the reproductive cycle of the female Labidura riparia, cytological observations show cyclical modifications of lipid droplets in the periovarian adipocyte. Fat body lipids and their constitutive fatty acids are analyzed. The lipids are predominantly triacylglycerols, which increase after adult ecdysis during vitellogenic and non-vitellogenic periods. Small amounts of diacylglycerols and phospholipids are found. Diacylglycerols increase during vitellogenesis and decrease during the non-vitellogenic period. Cytological modifications of lipid droplets are probably related to diacylglycerol fluctuations. Gas-liquid chromatography of fatty acid methyl esters shows oleic acid to be the predominant fatty acid in total lipids and triacylglycerols; unsaturated acids are approximately twice as abundant as saturated acids all along the reproductive cycle. Fatty acid composition of diacylglycerols and phospholipids differs from triacylglycerols and total lipids composition. Palmitic, stearic, oleic and linoleic acids represent the major fatty acids; their relative amounts vary during the different periods of the reproductive cycle. The correlations between fat body lipid changes and ovarian development were discussed and compared with observations made on other insect species. Accepted: 23 April 1997     

Lipid storage compounds in marine bacteria

Forty psychrophile or psychrotrophic crude-oil-utilizing marine bacteria were investigated for their ability to accumulate lipid storage compounds in the cytoplasm during cultivation under nitrogen-limiting conditions. Most of them (73%) were able to accumulate specialized lipids like polyhydroxyalkanoic acids (PHA) while other lipids such as wax esters occurred in two isolates. Accumulation of PHA occurred predominantly at low temperatures (4–20 °C) as demonstrated for three isolates. Electron microscopy revealed polyphosphate inclusions occurring in two isolates in addition to PHA. Cells of the isolate Acinetobacter sp. 211 were able to synthesize and accumulate lipid inclusions during growth on acetate, ethanol, olive oil, hexadecanol and heptadecane. The composition of the lipid inclusions depended on the compounds provided as carbon source. Wax esters and acylglycerols occurred mainly during the cultivation on olive oil; in contrast, wax esters and free alcohols occurred during cultivation on hexadecanol. Total fatty acids in cells of the Acinetobacter sp. 211 amounted to 25% of the cellular dry weight in olive-oil-grown cells. Palmitic acid was the main fatty acid in the lipids when the cells were cultivated on acetate or ethanol (44% and 32% of total fatty acids respectively). In contrast, fatty acids occurring in the lipids during cultivation on hexadecanol, heptadecane or olive oil were related to the carbon source. The fatty acids present in the accumulated lipids consisted predominantly of saturated and unsaturated straight-chain fatty acids with a chain length ranging from 12 to 18 carbon atoms. Analysis of the lipid-granule-associated proteins in cells of Acinetobacter sp. 211 revealed a protein of 39 kDa as the predominant protein species. Received: 2 July 1996 / Received revision: 3 September 1996 / Accepted: 28 September 1996     

Conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel by immobilized Candida antarctica lipase

Acid oil, which is a by-product in vegetable oil refining, mainly contains free fatty acids (FFAs) and acylglycerols, and is a candidate of materials for production of biodiesel fuel. A mixture (acid oil model) of refined FFAs and vegetable oil was recently reported to be converted to fatty acid methyl esters (FAMEs) at >98% conversion by a two-step reaction system comprising methyl esterification of FFAs and methanolysis of acylglycerols using immobilized Candida antarctica lipase. The two-step system was thus applied to conversion of acid oil by-produced in vegetable oil refining to biodiesel fuel. Under similar conditions that were determined by using acid oil model, however, the lipase was unstable and was not durable for repeated use. The inactivation of the lipase was successfully avoided by addition of excess amounts of methanol (MeOH) in the first-step reaction, and by addition of vegetable oil and glycerol in the second-step reaction. Hence, the first-step reaction was conducted by shaking a mixture of 66 wt% acid oil (77.9 wt% FFAs, 10.8 wt% acylglycerols) and 34 wt% MeOH with 1 wt% immobilized lipase, to convert FFAs to their methyl esters. The second-step reaction was performed by shaking a mixture of 52.3 wt% dehydrated first-step product (79.7 wt% FAMEs, 9.7 wt% acylglycerols), 42.2 wt% rapeseed oil, and 5.5 wt% MeOH using 6 wt% immobilized lipase in the presence of additional 10 wt% glycerol, to convert acylglycerols to FAMEs. The resulting product was composed of 91.1 wt% FAMEs, 0.6 wt% FFAs, 0.8 wt% triacylglycerols, 2.3 wt% diacylglycerols, and 5.2 wt% other compounds. Even though each step of reaction was repeated every 24 h by transferring the immobilized lipase to the fresh substrate mixture, the composition was maintained for >100 cycles.     

Inhibitory effect of medium-chain-length fatty acids on synthesis of polyhydroxyalkanoates from volatile fatty acids by Ralstonia eutropha

Medium-chain-length fatty acids, such as nonanoic (9:0) and octanoic (8:0) acids, are more toxic to Ralstonia eutropha than volatile fatty acids such as acetic, propionic and butyric acids. Nonanoic acid was degraded to acetic and propionic acids via -oxidation by Ralstonia eutropha for cell growth and synthesis of polyhydroxyalkanoates (PHAs). In a mixture of the fatty acids, utilization of nonanoic acid was depressed by acetic and propionic acids, and vice versa. The PHA accumulation from the volatile fatty acids was decreased from 53% (w/w) of dry cell mass to 23% due to the nonanoic acid. Similar phenomena were also observed with octanoic acid and its metabolic intermediates, acetic and butyric acids.     

Enzymic alcoholysis of blackcurrant oil

Alcoholysis of blackcurrant oil mediated by Pseudomonas fluorescens lipase performed at 30°C in ethanol (96%, v/v) used both as a solvent and as a reactant. After 16 h, 95% of triacylglycerols present in the oil was converted into a mixture consisting of fatty acid ethyl esters, free fatty acids, monoacylglycerols and diacylglycerols. The highest amount of fatty acid ethyl esters (52%) was achieved after 8 h.     

A comparative analysis of the ultrastructure of hydrocarbon-oxidizing micro-organisms.

The ultrastructure of a variety of micro-organisms was compared after growth on hydrocarbon and non-hydrocarbon substrates. Hydrocarbon-grown organisms were characterized by the presence of intracellular electron-transparent inclusions which in many cases appeared membrane-bound. With one exception, non-hydrocarbon-grown organisms did not contain electron-transparent inclusions. Insignificant amounts of poly-beta-hydroxybutyric acid were produced by the hydrocarbon-grown micro-organisms. After growth on hydrocarbons, all the microorganisms had accumulated varying amounts of the respective unmodified hydrocarbon growth substrate.     

Food reserves of scots pine (Pinus sylvestris L.)

Summary Starch, soluble sugars, triacylglycerols, diacylglycerols and free fatty acids were measured in 30-year-old Scots pine (Pinus sylvestris L.) trees during an annual cycle in the sapwood (youngest ten xylem rings). The radial distribution of carbohydrates and lipids was studied in the trunkwood of 90 -to 150-year-old Scots pine trees collected at the end of the growing season. Determination of the compounds was performed using specific enzymatic assays, capillary gas chromatography and thin layer chromatography. The amounts of glucose, fructose, sucrose, and galactose/arabinose in the sapwood were slightly higher in winter than in summer. Raffinose/stachyose increased up to 5-fold during the cold period. At the beginning of the growing season starch amounts rose, and then decreased in summer and autumn. No concentration changes were observed in the total amounts of diacylglycerols and fatty acids throughout the year. Triacylglycerol levels were slightly higher in February than in summer and autumn. Relative frequencies of individual fatty acids were similar in all lipid fractions. Glucose, fructose, sucrose, starch and triacylglycerols disappeared almost entirely at the transition zone from sapwood to heartwood. In contrast, free fatty acids and galactose/arabinose rose in centripetal direction, and diacylglycerols remained constant across trunk cross-sections. The relative amounts of individual fatty acids changed markedly in the free fatty acid fraction and in the triacylglycerols when crossing the sapwood-heartwood boundary. Concentration changes of reserve materials are discussed in relation to season, mobilization and translocation processes, dormancy, frost resistance, and heartwood formation. The results are compared to those found in needles.     

Ketogenesis in isolated rat liver mitochondria. II. Factors affecting the rate of β-oxidation

1. During fatty acid oxidation by rat liver mitochondria, the rate of β-oxidation is dependent on the relative amounts of substrate and mitochondrial protein, on the energy state of the mitochondria, on the chain length and the number of double bonds of the fatty acid and on the concentration of various compounds in the reaction medium (l-carnitine, CoASH, hexokinase, albumin).2. The rate of β-oxidation of long-chain fatty acids decreases when the ratio of albumin over fatty acid is increased. This effect is most marked in the absence of added carnitine.3. Addition of excess hexokinase decreases the rate of β-oxidation in the presence of added carnitine.4. Maximal rates of β-oxidation are observed with octanoate and decanoate (40–60 nmoles acetyl-CoA/min per mg mitochondrial protein at 25 °C).5. Odd-numbered fatty acids are oxidized at a much lower rate than the even-numbered homologues. In a low-energy state propionyl-CoA accumulates; in a high-energy state in the presence of bicarbonate, Krebs-cycle intermediates accumulate.6. l-Carnitine enhances the rate of β-oxidation of all fatty acids except butyrate. The stimulatory effect is most pronounced with odd-numbered and with long-chain fatty acids.7. In the absence of added carnitine the rate of β-oxidation of long-chain fatty acids decreases with the chain length and increases with the number of double bonds. It is suggested that the solubility of the long-chain fatty acids in the aqueous medium is the rate-limiting factor under these conditions.8. In the presence of carnitine and albumin, palmitate, oleate, linoleate and linolenate are all oxidized at about the same rate (25–30 nmoles/min per mg protein at 25 °C).9. Propionyl-CoA is not formed as an intermediate during oxidation of unsaturated fatty acids.     

Cellular lipid accumulation by Pseudomonas aeruginosa 44T1

Summary Growth of Pseudomonas aeruginosa strain 44T1 on glucose, an n-alkane mixture or olive oil was characterized by the formation of intracellular lipid inclusions and extracellular accumulation of rhamnolipids. Maximum values of cellular lipid accumulation were obtained in olive-oil-grown cells and reached up to 38% w/w of its dry biomass. The principal fatty acids of cellular lipids drived from P. aeruginosa cultures varied with the carbon source employed. The major fatty acids detected were palmitic and trans-oleic acids. Arachidonic acid was only found in medium containing glucose or the n-alkane mixture. Offprint requests to: A. Manresa     

MODULATION OF FATTY ACIDS IN THE MEMBRANES OF ANACYSTIS NIDULANS (CYANOBACTERIA): INCORPORATION OF ODD-NUMBERED CARBON FATTY ACIDS1

Anacystis nidulans Richt., a unicellular cyanobacterium, can incorporate exogenously supplied fatty acids, including odd-numbered carbon fatty acid (OFAs), into the acylglycerols of cell membranes. Data are presented for the uptake of undecanoic acid (11:0) into cells of A. nidulans, the subsequent elongation up to C17, and incorporation of OFA into the four major membrane acylglycerols. The incorporation of OFAs was followed by desaturation of part of the saturated fatty acid to monoenoic fatty acid. Positional analyses of the double bonds of these manoenoic fatty acids suggest that there is one desaturase that inserts a Δ⁹ bond in both odd- and even-numbered fatty acids of varying chain length. Our data also suggest that there is no positional specificity for chain length on the glycerol backbone by the acyltransferases.     

Production of fatty acids and lipid by a Candida sp. growing on a fraction of n-alkanes predominating in tridecane

A Candida sp. was grown on a fraction of n-alkanes (dodecane 22%, tridecane 48%, tetradecane 28%) as sole carbon source. The growth rate was increased most markedly by using high concentrations of n-alkanes (16.7% v/v). When grown in a 5 liter fermentor, the yeast reached its highest yield (60 g. of cell dry wt/l) with a concomitant high yield of fatty acids (21 g of fatty acids/l), by using a nitrogen-deficient medium. To achieve good growth, it was essential to use an inoculum (1 part into 10) of rapidly growing cells and beneficial to increase the agitation rate gradually once growth had begun. After 108 hr maximum conversions of substrate to product were: 71.5% (w/w) for alkanes into cells and 24.8% (w/w) for alkanes into fatty acids. Of the, total fatty acids at the end of the fat-accumulating phase of growth 54% were shorter in chain length than palmitic acid (C₁₆H₃₂O₂). When grown on glucose, as sole carbon source, less than 2% of the total fatty acids were shorter than palmitic acid. When n-alkanes were added to cells growing on glucose, short-chain fatty acids (C₁₀ to C₁₄) were synthesized immediately, indicating a derepressed enzyme system for hydrocarbon assimilation and the absence of diauxie. The production of these acids was at the apparent sacrifice of linoleic acid synthesis. In spite of the high conversion ratios, it is concluded that it would be uneconomical to produce fatty acids, even expensive ones such as lauric acid, by microbial transformation of n-alkanes.     

The Production of γ-Decalactone by Fermentation of Castor Oil

A microbial process for the production of optically-active γ-decalactone from the ricinoleic acid present as triglycerides in castor oil has been developed, γ-decalactone (γDL) is a component of some fruit flavours, being an important organoleptic component of peach flavours. Screening showed two red yeast microorganisms, Rhodotorula glutinis and Sporobolomyces odortts to be especially suitable for this biotransformation. The process involves lipase-mediated hydrolysis of the castor oil to give free ricinoleic acid, uptake of the acid by the cells and aerobic fermentation to achieve abbreviated β-oxidation of the ricinoleic acid (12-hydroxyoleic acid) into 4-hydroxydecanoic acid (4HDA), lactonisation of the acid into γ-DL, followed by solvent extraction and distillation. γ-DL broth concentrations of 0.5-1.2g · 1^-t were obtained after 3-5 days from fermentation media containing 10 g · 1^-1 castor oil, representing an 8.3-20.0% theoretical yield. Intermediates detected were consistent with the operation of the β-oxidation pathway. Appreciable amounts of novel metabolites identified as cis and trans isomers of a tetrahydrofuran (C₁₀) were also produced. Their formation from 4HDA appeared to be non-enzymic and was favoured by anaerobic conditions. Yields of γ-DL were inversely proportional to the concentration of castor oil present in the medium, indicating that substrate inhibition takes place. The highest yields of γ-DL were obtained when castor oil was present from the beginning of the fermentation, rather than when added once the fermentation had become established, demonstrating that the β-oxidation pathway and/or transport system require continual induction. Significant amounts of γ-DL were not produced from other fatty acids, including ricinelaidic acid, the trans isomer of ricinoleic acid. γ-DL formation was dramatically inhibited by antibiotic inhibitors of oxidative phosphorylation, indicating the importance of intact β-oxidation pathways, whereas inhibitors of protein synthesis and cell-wall synthesis had much less marked effects. Selective extraction of 4HDA from the fermentation broths, and of γDL from broth lactonised by heating at low pH, could be achieved by adsorption to Amberlite XAD-1 and XAD-7 resins respectively. Some product could be recovered from the exit gases of the fermenter by passing through propylene glycol traps. This pathway is unusual in that it is a rare example of the truncated β-oxidation of a fatty acid by microorganisms. This effect probably occurs because of partial inhibition of one or more enzymes of the β-oxidation pathway by the C₁₀ hydroxylated fatty acid intermediate(s) allowing intracellular accumulation of the 4HDA, followed by leakage out of the cell; although further metabolism of this C₁₀ intermediate does take place slowly.     

Utilization of mevalonate by aorta for the synthesis of medium-chain n-fatty acids (C8, C10) and acylglycerols

[2-14C]Mevalonate was incorporated into acylglycerols by bovine aorta. The majority of the radioactivity was found in diacylglycerols. Hydrolysis, followed by analysis by thin-layer chromatography, paper chromatography, gas chromatography, and gas chromatography-mass spectrometry showed that the radioactivity was greatest in the medium-chain n-fatty acids, caprylic (C8) and capric (C10) acids. Triacylglycerols and diacylglycerols have important functions in the maintenance of the metabolic integrity of organs and their constituent cells. Therefore, medium-chain fatty acids may play significant roles, such as solubilization and transport of cholesterol and regulation of metabolism, as constituents of diacylglycerols. The results suggest that the trans-methylglutaconate shunt of mevalonate metabolism functions in the aorta.     

Presumptive involvement of methyl ricinoleate β-oxidation in the production of γ-decalactone by the yeast Pichia guilliermondii

Abstract Production of γ-decalactone by yeasts from fatty acids has been reported but little is known about the mechanisms involved in this process. This paper provides information about the mechanisms involved in the production of γ-decalactone by Pichia guilliermondii in the presence of a fatty acid methyl ester. Culturing of P. guilliermondii in media containing methyl ricinoleate (12(R)-hydroxy-9(Z)-octadecenoic acid) revealed a coordinated induction of β-oxidation activities and γ-decalactone production. However, no γ-decalactone synthesis was noted when methyl ricinoleate was changed into methyloleate or methyl linoleate, even though these fatty acid methyl esters are able to induce β-oxidation activities in P. guilliermondii . These observations led us to conclude that methyl ricinoleate is an inducer of β-oxidation and is probably the substrate for γ-decalactone production. The fatty acid ester β-oxidation should be involved, at least in part, in this production.     

Single cell oil production by Yarrowia lipolytica growing on an industrial derivative of animal fat in batch cultures

The growth of an oleaginous strain of Yarrowia lipolytica on an industrial fat composed of saturated free fatty acids (stearin) was studied. Lipid accumulation during primary anabolic growth was critically influenced by the medium pH and the incubation temperature. This process was independent of the nitrogen concentration in the culture medium, but was favored at a high carbon substrate level and at a low aeration rate. At pH 6 and a temperature of 28-33 degrees C, 9-12 g/l of dry biomass was produced, whereas significant quantities of lipids were accumulated inside the yeast cells (0.44-0.54 g of lipid per gram of biomass). The strain showed the tendency to degrade its storage lipids, although significant amounts of substrate fat, rich in stearic acid, remained unconsumed in the culture medium. Y. lipolytica presented a strong fatty acid specificity. The fatty acids C12:0, C14:0, and C16:0 were rapidly incorporated and mainly used for growth needs, while C18:0 was incorporated with reduced rates and was mainly accumulated as storage material. Reserve lipids, principally composed of triacylglycerols (55% w/w of total lipids) and free fatty acids (35% w/w), were rich in stearic acid (80% w/w), while negligible amounts of unsaturated fatty acids were detected. When industrial glycerol was used as co-substrate, together with stearin, unsaturated fatty acid concentration in the reserve lipid increased.     

Degradation of long-chain n-alkanes by the yeast Lodderomyces elongisporus

Summary Cells of the yeast Lodderomyces elongisporus, precultured on glycerol, were incubated with long-chain n-alkanes. The results whow that monoterminal alkane oxidation is the main pathway of alkane degradation in the investigated yeast. The amount of diterminal activity is negligible, while subterminal degradation did not occur at all.Fatty acids were the first detectable intermediates. Using different n-alkanes, in every case the fatty acids with substrate chain length predominated in the cells. The formation of radioactive fatty acids from (1-¹⁴C)-hexadecane was time-dependent and indicated that desaturation elongation and -oxidation occurred.Extracellularly, the fatty acid pattern was similar, except for the additional presence of fatty acid methyl esters and the prevalence of octadecenoic acid after growth of cells on n-hexadecane.     

Lipophilic Metabolites from Five‐Needle Pines,Pinus armandii and Pinus kwangtungensis,Exhibiting Antibacterial Activity

Lipophilic extractive metabolites from needles and defoliated twigs of Pinus armandii and P. kwangtungensis were studied by GC/MS. Needles of P. armandii contained predominantly 15‐O‐functionalized labdane type acids (anticopalic acid), fatty acids, nonacosan‐10‐ol, sterols, nonacosan‐10‐ol and sterol saponifiable esters, and acylglycerols, while P. kwangtungensis needles contained no anticopalic acid, but more trinorlabdane (14,15,16‐trinor‐8(17)‐labdene‐13,19‐dioic acid) and other labdane type acids, nonacosan‐10‐ol and its saponifiable esters. The major compounds in the P. armandii defoliated twig extract were abietane and isopimarane type acids, fatty acids, sterols, labdanoids (cis‐abienol), cembranoids (isocembrol and 4‐epi‐isocembrol), saponifiable sterol esters, and acylglycerols. The same extract of P. kwangtungensis contained larger quantities of fatty acids, caryophyllene oxide, serratanoids, sterols, saponifiable sterol esters, and acylglycerols, but lesser amounts of abietane and isopimarane type acids, cis‐abienol, and lacked cembranoids. Both twig and needle extracts of P. armandii and P. kwangtungensis, as well as the extracts’ fractions, significantly inhibited the growth of Gram‐negative bacteria Serratia marcescens with MIC of 0.1 mg ml^?1, while in most cases they slightly stimulated the growth of Gram‐positive bacteria Bacillus subtilis at the same concentrations. Thus, lipophilic extractive compounds from the needles and defoliated twigs of both pines are prospective for the development of antiseptics against Gram‐negative bacteria.     

Immunohistochemical localization of mitochondrial fatty acid β-oxidation enzymes in Müller cells of the retina

The presence of a mitochondrial fatty acid β-oxidation system in the retina was shown by immunohistochemistry. Fatty acids are considered to serve as a major energy source metabolized by fatty acid β-oxidation together with glucose metabolized by glycolysis in the organs of the entire body, but almost nothing is known about this metabolic system in the retina. Adult rat retinae were subjected to immunofluorescence and immuno-electron microscopy for the localization of fatty acid β-oxidation enzymes, together with western blot analysis for quantitation of the amount of enzyme proteins and DNA microarray analysis for gene expression. All the enzymes examined were shown to be present in the retina, but in small amounts, with the amount of protein and gene expression in the retina being about 1/10 of those in the liver. Immunohistochemistry at light and electron microscopic levels revealed the enzymes to be more preferentially localized to the mitochondria of Müller cells than the retinal neurons. The Müller cells were isolated from the retina and confirmed for the presence of mitochondrial fatty acid β-oxidation enzymes. A mitochondrial fatty acid β-oxidation system was thus shown to be present in the retina heterogeneously.     

Diacylglycerols interfere in normal phase HPLC analysis of lipoxygenase products of docosahexaenoic or arachidonic acids

A methodological problem with the normal phase high performance liquid chromatography (HPLC) of hydroxylated products of docosahexaenoic and arachidonic acids is described. Diacylglycerols present in lipid extracts of rat retina co-elute with monohydroxy derivatives of docosahexaenoic or arachidonic acid, when samples are applied to uPorosil columns and eluted with hexane/isopropanol/acetic acid. Analysis of fatty acid composition of diacylglycerols which were acetone-extracted from the incubation medium showed a profile similar to diacylglycerols extracted from the tissue by hexane/isopropanol, although acetone extraction resulted in extremely variable recovery of diacylglycerols. This co-elution of diacylglycerols with monohydroxy polyunsaturated fatty acids can lead to a significant error in estimation of lipoxygenation activity by conversion of radiolabeled precursors, because the incorporation of fatty acids into diacylglycerols is very active in many tissues. An alternative extraction method and reverse phase HPLC procedures that result in the complete separation of hydroxy fatty acids and diacylglycerols are described.