Response surface modeling of glycerolysis catalyzed by Candida rugosa lipase immobilized in different polyurethane foams for the production of partial glycerides

Monoglycerides (MG) and diglycerides (DG) are the most widely used emulsifiers in food and pharmaceutical industries. In this study, MG and DG were produced by inter-esterification of refined olive residue oil with glycerol (glycerolysis), in n-hexane, catalyzed by Candida rugosa lipase immobilized in different biocompatible hydrophilic polyurethane foams, A and B. These foams, with aquaphilicities of 3.7 and 2.8, were prepared with a toluene diisocyanate (“Hypol FHP 2002™”) and a diphenylmethane diisocyanate (“Hypol FHP X4300^TM”) pre-polymer, respectively.Response surface methodology was used for modeling the reaction, as a function of the molar ratio glycerol/triglycerides (Gly/TG, 0.5–2.0) and the initial water activity (a_w) of the biocatalyst (A, 0.24–0.91; B, 0.37–0.91). Experiments were carried out following a central composite rotatable design. With lipase in foam A, production of MG and DG could be described by first order polynomials. With foam B, MG and DG production could be fitted to concave and flat surfaces, described by a second and a first orders polynomials, respectively.The best productions of MG and DG were achieved with the lipase in the less hydrophilic foam, B: at 24 h reaction time, 32% (w/w) MG and 18% (w/w) DG were obtained, when the initial a_w of the biocatalyst was 0.83, with a Gly/TG of 1.     

Use of mono- and diacylglycerol lipase as immobilized fungal whole cells to convert residual partial glycerides enzymatically into fatty acid methyl esters

The accumulation of partial glycerides such as monoglyceride (MG) and diglyceride (DG) is one of the rate-limiting steps in plant oil methanolysis catalyzed by Rhizopus oryzae producing triacylglycerol lipase. To convert partial glycerides efficiently into their corresponding methyl esters (MEs), we attempted to use a mono- and diacylglycerol lipase (mdlB) derived from Aspergillus oryzae. By considering cost efficiency, R. oryzae and recombinant mdlB-producing A. oryzae were immobilized independently within polyurethane foam biomass support particles and directly utilized as a whole-cell biocatalyst. The mdlB-producing A. oryzae effectively exhibited substrate specificity toward MG and DG and was then used for the methanolysis of intermediate products (approximately 82% ME), which were produced using R. oryzae. In the presence of 5% water, the use of mdlB-producing A. oryzae resulted in less than 0.1% of MG and DG, whereas a considerable amount of triglyceride was present in the final reaction mixture. On the basis of these results, we developed a packed-bed reactor (PBR) system, which consists of the first column with R. oryzae and the second column containing both R. oryzae and mdlB-producing A. oryzae. Ten repeated-batch methanolysis cycles in the PBR maintained a high ME content of over 90% with MG and DG at 0.08–0.69 and 0.22–1.45%, respectively, indicating that the PBR system can be used for long-term repeated-batch methanolysis with partial glycerides at low levels. The proposed method is therefore effective for improving enzymatic biodiesel production.     

Characterization of a partially purified diacylglycerol lipase from bovine aorta

A partially-purified diacylglycerol (DG) lipase from bovine aorta has been characterized with respect to the effects of lipid metabolites and two lipase inhibitors, phenylboronic acid and tetrahydrolipstatin (THL). DG lipase activity was determined by the hydrolysis of the sn-1 position of 1-[1-⁴C]palmitoyl-2-oleoyl-sn-glycerol. The products of the lipase reaction, 2-monoacylglycerol (2-monoolein) and non-esterified fatty acids (oleate, arachidonate) produced a concentration-dependent (20–200 μM) inhibition of DG lipase activity. Oleoyl-CoA and dioleoylphosphatidic acid also inhibited aortic DG lipase activity, but lysophosphatidylcholine had little or no effect. The inhibition of aortic DG lipase by phenylboronic acid was competitive, with a K_i of approx. 4 mM. THL was a very potent inhibitor of aortic DG lipase; the concentration required for inhibition to 50% of control was 2–6 nM. THL was a very potent inhibitor of concentration of substrate in the assay was increased. Attempts to identify the aortic DG lipase by covalent-labelling with [¹⁴C]THL were unsuccessful. Immunoblotting experiments revealed that hormone-sensitive triacylglycerol lipase (HSL) could not be detected in bovine aorta.     

The effect of temperature, water activity and sorbic acid on ketone rancidity produced by Penicillium crustosum Thom in coconut and palm kernel oils

In a model system medium-chain fatty acids (MCFA) C₆–C₁₂ in coconut and palm kernel oil are converted to methyl ketones, one carbon atom less than the parent fatty acids, by two strains of Penicillium crustosum Thom. Conversion rates of up to 32% were seen for decanoic acid at 25°C. The optimum temperature for ketone production was 25°C in liquid suspension culture. Coconut oil contains 3.23 mmol/g MCFAs compared with 2.34 mmol/g for palm kernel oil. Coconut oil is more prone to fungal spoilage (growth and ketone production) than palm kernel oil. The main end product of fermentation was 2-undecanone reflecting the high concentration of dodecanoic acid in the substrates. Ketonic rancidity is fungal engendered. The reaction can be controlled by reducing the temperature (4°C), reducing the water activity (0.91) or by addition of sorbic acid (20 mmol/l).     

Continuous and consecutive conversion of free fatty acid in rice bran oil to triacylglycerol using immobilized lipase

Free fatty acid (FFA), monoacylglycerol (MG) and diacylglycerol (DG) in high-FFA rice bran oil were continuously converted with glycerol (G) to form triacylglycerol (TG), using lipase fromRhizomucor miehei immobilized on anion-exchange resin. The reaction was continued for more than 1 month by a reactor with two circulation loops, each being connected to a fixed-bed reactor and a dehydrator. The reaction of 2 FFA + G DG + 2H₂O appeared to occur until the glycerol was exhausted; the reaction of FFA + DG TG + H₂O then followed. The consecutive esterificaion continued in the presence of 2–8 ppm water and the TG content reached 74%–88%. The industrial feasibility of this process was assessed from the standpoints of enzyme cost and value added by esterification.     

Saponified palm kernel oil and its major free fatty acids as carbon substrates for the production of polyhydroxyalkanoates in Pseudomonas putida PGA1

The synthesis of polyhydroxyalkanoates (PHA) by Pseudomonas putida PGA1, using saponified palm kernel oil (SPKO), was investigated. The PHA produced from SPKO was compared with those produced by the major free fatty acids found in the palm kernel oil. Owing to the absence of lipase activity in P.␣putida, palm kernel oil did not support cell growth. However, SPKO could support cell growth and produced relatively high yield of both dry cells and PHA. The polyester produced was similar in properties to those derived from lauric (C_12:0) and myristic (C_14:0) acids, while oleic acid (C_18:1) gave rise to PHA that was sticky and of broader molecular mass distribution. Nuclear magnetic resonance and gas chromatography showed that these PHA were copolymers consisting mainly of n-alkanoate monomers ranging from C₆ to C₁₄, with C₈ as the predominant component. PHA derived from SPKO and oleic acid also contained a small amount of unsaturated monomers. Received: 25 March 1996 / Received last revision: 30 September 1996 / Accepted: 18 October 1996     

Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora

Mortierella ramanniana var. angulispora accumulates triacylglycerol (TG) in lipid bodies. Studies on lipid transport into lipid bodies are essential for elucidating mechanisms of lipid body formation. We used fluorescent dyes and fluorescent lipid analogs to visualize lipid body formation with a confocal laser scanning microscope. Different sizes of lipid bodies were stained by Nile red, a lipid body marker – one with a diameter of about 1 μm and the other with a diameter of about 2–3 μm. Lipid bodies matured into larger ones with culture. To metabolically monitor lipid bodies, we used 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-phosphatidic acid (C₅-DMB-PA), and C₅-DMB-phosphatidylcholine (C₅-DMB-PC). These were taken up into fungal cells and incorporated into intracellular organelles at 30°C. C₅-DMB-PA was quickly incorporated into lipid bodies while C₅-DMB-PC was initially incorporated into internal membranes, presumably endoplasmic reticulum membranes, and fluorescence was then gradually transported into lipid bodies. The transport of fluorescent lipids accompanied their metabolism into diacylglycerol (DG) and TG, which, taken together with the fluorescence distribution, suggested that conversion to TG was not necessary for transport into lipid bodies. It is likely that the synthesized DG was mainly located in lipid bodies and the conversion to TG took place in lipid bodies. C₅-DMB-PA and C₅-DMB-PC were converted to DG and TG in the membrane and lipid body fractions of this fungus, which agreed with in vivo metabolism of these fluorescent lipids and in vitro enzyme activity related to PA and PC metabolism. These results indicate that transport and metabolism of C₅-DMB-PA and C₅-DMB-PC represent two different routes for lipid body formation in this fungus.     

Absorption of dietary triacylglycerol by lipolysis and lipid resynthesis in the mesenteron of larvalAeshna cyanea (insecta,odonata)

Summary Voluntary uptake of triolein, margarine, and lipid-rich natural food (Tubifex) by fasting dragonfly larvae (Aeshna cyanea) led to heavy accumulations of lipid absorption droplets in the enterocytes within 2 days, while subsequent lipid clearance of the midgut epithelium took several weeks depending on the ingested lipid load. No endocytotic lipid uptake was observed after application of a molecular-dispersed fat dye. The smallest lipid droplets first appeared in the subapical groundplasm of the enterocytes and showed a reversible increase in size on their way towards the base. Lipid droplets were also observed at appropriate intervals after oral administration of oleic acid, after feeding margarine in the cold, and after injection of triolein into the isolated midgut.Comparative biochemical analysis after triolein feeding evidenced release of lipase and hydrolytic liberation of FA from TG in the midgut lumen, as well as time-dependent accumulations of TG in the midgut epithelium and of DG in the hemolymph.Oral injection of [¹⁴C] oleic acid was followed by its rapid absorption into the midgut epithelium, where it was utilized for the synthesis of MG and esterification to DG and TG. Discharge of radioactive lipid into the hemolymph occurred in the form of FA and DG, while the rectal fat body showed approximately equal labeling of the FA, DG, and TG fractions.Abbreviations AG acylglycerol - DG diacylglycerol - ER endoplasmic reticulum - FA fatty acid - MG monoacylglycerol - TG triacylglycerol Dedicated to Prof. Dr. Dr. R.Lehmensick, Bonn, in honor of his 85^th birthday.     

Diversity of Δ12 Fatty Acid Desaturases in Santalaceae and Their Role in Production of Seed Oil Acetylenic Fatty Acids

Plants in the Santalaceae family, including the native cherry Exocarpos cupressiformis and sweet quandong Santalum acuminatum, accumulate ximenynic acid (trans-11-octadecen-9-ynoic acid) in their seed oil and conjugated polyacetylenic fatty acids in root tissue. Twelve full-length genes coding for microsomal Δ12 fatty acid desaturases (FADs) from the two Santalaceae species were identified by degenerate PCR. Phylogenetic analysis of the predicted amino acid sequences placed five Santalaceae FADs with Δ12 FADs, which include Arabidopsis thaliana FAD2. When expressed in yeast, the major activity of these genes was Δ12 desaturation of oleic acid, but unusual activities were also observed: i.e. Δ15 desaturation of linoleic acid as well as trans-Δ12 and trans-Δ11 desaturations of stearolic acid (9-octadecynoic acid). The trans-12-octadecen-9-ynoic acid product was also detected in quandong seed oil. The two other FAD groups (FADX and FADY) were present in both species; in a phylogenetic tree of microsomal FAD enzymes, FADX and FADY formed a unique clade, suggesting that are highly divergent. The FADX group enzymes had no detectable Δ12 FAD activity but instead catalyzed cis-Δ13 desaturation of stearolic acid when expressed in yeast. No products were detected for the FADY group when expressed recombinantly. Quantitative PCR analysis showed that the FADY genes were expressed in leaf rather than developing seed of the native cherry. FADs with promiscuous and unique activities have been identified in Santalaceae and explain the origin of some of the unusual lipids found in this plant family.     

Monoacylglycerol and diacylglycerol acyltransferases and the synthesis of neutral glycerides in Manduca sexta

The insect fat body and the adipose tissue of vertebrates store fatty acids (FA) as triacylglycerols (TG). However, the fat body of most insects has the unique ability to rapidly produce and secrete large amounts of diacylglycerol (DG). Monoacylglycerol acyltransferase (MGAT), which catalyzes the synthesis of DG from MG, and a diacylglycerol acyltransferase (DGAT), which catalyzes the synthesis of TG from DG, are key enzymes in the metabolism of neutral glycerides. However, very little is known about these acyltransferases in insects. In the present study we have cloned two predicted MGATs and a DGAT from Manduca sexta and compared their sequences with predicted MGAT and DGAT homologs from a number of insect species. The comparison suggested that insects may only have a single DGAT gene, DGAT1. The apparent absence of a DGAT2 gene in insects would represent a major difference with vertebrates, which contain DGAT1 and DGAT2 genes. Insects seem to have a single MGAT gene which is similar to the MGAT2 of vertebrates. A number of conserved phosphorylation sites of potential physiological significance were identified among insect proteins and among insect and vertebrate proteins. DGAT1 and MGAT are expressed in fat body, midgut and ovaries. The relative rates of utilization of FAs for the synthesis of DG and TG correlated with the relative expression levels of MGAT and DGAT suggesting that regulation of the expression levels of these acyltransferases could be determining whether the fat body secretes DG or stores fatty acids as TG. The expression patterns of the acyltransferases suggest a role of the monoacylglycerol pathway in the production and mobilization of DG in M. sexta fat body.     

Effect of water on methanolysis of glycerol trioleate catalyzed by immobilized lipase Candida sp. 99–125 in organic solvent system

In this study, hydrolysis and methanolysis of glycerol trioleate (TG) by lipase Candida sp. 99–125 were investigated under different water conditions. Both the reaction rates were relatively low without water, while increasing water content to 5 wt.% (or more, from 10–20%) based on the TG amount caused remarkable higher TG conversion for both reactions. Moreover, comparing the time course curves of the hydrolysis and methanolysis, it could be concluded that the methanolysis reaction catalyzed by this Candida sp. 99–125 appeared to accord with the successive reaction mechanism. TG was first hydrolyzed to partial glycerides and oleic acid (OA), then oleic acid methyl ester (OAME) was produced by esterification of the OA with methanol. This water effect was also confirmed by the experiments that water substitutions such as t-butanol and some surfactants added into the system did not get such high yields as that of the water included system. So these results showed that water took part in the methanolysis reaction, and successive hydrolysis–esterification process might be the catalytic mechanism of this lipase.     

Characterization of 19 Genes Encoding Membrane-Bound Fatty Acid Desaturases and their Expression Profiles in Gossypium raimondii Under Low Temperature

To produce unsaturated fatty acids, membrane-bound fatty acid desaturases (FADs) can be exploited to introduce double bonds into the acyl chains of fatty acids. In this study, 19 membrane-bound FAD genes were identified in Gossypium raimondii through database searches and were classified into four different subfamilies based on phylogenetic analysis. All 19 membrane-bound FAD proteins shared three highly conserved histidine boxes, except for GrFAD2.1, which lost the third histidine box in the C-terminal region. In the G. raimondii genome, tandem duplication might have led to the increasing size of the FAD2 cluster in the Omega Desaturase subfamily, whereas segmental duplication appeared to be the dominant mechanism for the expansion of the Sphingolipid and Front-end Desaturase subfamilies. Gene expression analysis showed that seven membrane-bound FAD genes were significantly up-regulated and that five genes were greatly suppressed in G. raimondii leaves exposed to low temperature conditions.     

Attalea colenda (Arecaceae), a potential lauric oil resource

Attalea colenda, a palm tree native to the coastal plain of western Ecuador, produces from one to four infructescences per tree every year, each with an average of 5065 fruits. The oil content of the seeds is 56.9% dry weight. Kernel oil production per infructescence is 7–16 kg. A hectare with 50 trees can produce between 0.35 and 3.2 tons of oil per year. The kernel oil is chemically similar to coconut oil and and kernel oil from the African oil palm. The increasing demand for lauric oil on the world market makesA. colenda a potential oil source.     

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.

     

Screening of lipases for production of novel structured lipids from single cell oils

Lipids enriched in polyunsaturated fatty acids are very susceptible to oxidation, causing the formation of potentially harmful oxidized products. Hence, it is critical to keep the temperature as low as possible during reaction and storage. In this study, five commercial immobilized lipases were evaluated for their capability to produce novel structured lipids (SLs) enriched with medium-chain fatty acids (MCFAs) through acidolysis of single cell oil (SCO) with capric acid. Among the examined lipases, NS40086 and Lipozyme RM IM showed the highest incorporation degree. The acidolysis reactions resulted in an obvious variation in the fatty acids composition as well as their positional distribution. The obtained SLs contained (33.58 %–34.09 %) capric acid at sn-1, 3 positions with increasing the content of arachidonic acid at the sn-2 position up to (49.82 %–50.25 %). The NS40086 lipase displayed 1, 3 regiospecificity towards the TAG of SCO. The acidolysis reactions using NS40086 lipase resulted in a generation of 23 TAG molecular species containing capric acid. Moreover, the NS40086 lipase was more active than Lipozyme RM IM at relatively low temperatures (35 °C and 40 °C), which could be used effectively as a promising biocatalyst in lipid synthesis.     

Effect of Δ9-stearoyl-ACP-desaturase-C mutants in a high oleic background on soybean seed oil composition

Key message

Two new sources of elevated seed stearic acid were identified and the feasibility of an elevated stearic acid, high oleic acid germplasm was studied.

Abstract

Soybean [Glycine max (L.) Merr.] oil typically contains 2–4 % stearic acid. Oil with at least 20 % stearic acid is desirable because of its improved baking properties and health profile. This study identifies two new sources of high stearic acid and evaluates the interaction of high stearic and oleic acid alleles. TCHM08-1087 and TCHM08-755, high stearic acid ‘Holladay’ mutants, were crossed to FAM94-41-3, a line containing a point mutation in a seed-specific isoform of a Δ9-stearoyl-acyl carrier protein-desaturase (SACPD-C). F₂-derived lines were evaluated for fatty acid content in four field environments. Sequencing of SACPDs in TCHM08-1087 and TCHM08-755 revealed distinct deletions of at least one megabase encompassing SACPD-C in both lines. After genotyping, the additive effect for stearic acid was estimated at +1.8 % for the SACPD-C point mutation and +4.1 % for the SACPD-C deletions. Average stearic acid in lines homozygous for the deletions was 12.2 %. A FAM94-41-3-derived line and TCHM08-1087-11, a selection from TCHM08-1087, were crossed to S09-2902-145, a line containing missense mutations in two fatty acid desaturases (FAD2-1A and FAD2-1B). F₁ plants were grown in a greenhouse and individual F₂ seed were genotyped and phenotyped. No interaction was observed between either FAD2-1A or FAD2-1B and any of the SACPD-C mutant alleles. Seed homozygous mutant for SACPD-C/FAD2-1A/FAD2-1B contained 12.7 % stearic acid and 65.5 % oleic acid while seed homozygous for the SACPD-C deletion and mutant for FAD2-1A and FAD2-1B averaged 10.4 % stearic acid and 75.9 % oleic acid.     

Partial characterization of a crude cold-active lipase from Rhodococcus cercidiphylli BZ22

Cold-active lipase production by the psychrophilic strain Rhodococcus cercidiphylli BZ22 isolated from hydrocarbon-contaminated alpine soil was investigated. Depending on the medium composition, high cell densities were observed at a temperature range of 1–10 °C in Luria–Bertani (LB) broth or 1–30 °C in Reasoner’s 2A (R2A). Maximum enzyme production was achieved at a cultivation temperature of 1–10 °C in LB medium. About 70–80 % of the secreted enzyme was bound to the cell and was highly active as a cell-immobilized lipase which exhibited good reusability; more than 60 % of the initial lipase activity was retained after five-fold reuse. The properties of the lipase produced by the investigated strain were compared with those of a mesophilic porcine pancreatic lipase (PPL). The thermal stability of the cell-immobilized bacterial lipase was higher than that of the extracellular enzyme. Highest activity was detected at 30 °C for the cell-immobilized enzyme and for PPL, while the extracellular enzyme displayed highest activity at 10–20 °C. The bacterial lipase hydrolyzed p-nitrophenyl (p-NP) esters with different acyl chain lengths (C₂–C₁₈). The highest hydrolytic activity was obtained with p-NP-butyrate (C₄) as substrate, while the highest substrate affinity was obtained with p-NP-dodecanoate (C₁₂) as substrate, indicating a clear preference of the enzyme for medium acyl chain lengths.     

Effects of growth phase and nitrogen starvation on expression of fatty acid desaturases and fatty acid composition of Isochrysis aff. galbana (TISO)

Very long-chain polyunsaturated fatty acids (VLC-PUFAs) are important dietary requirements for maintaining human health. Many marine microalgae are naturally high in ω − 3 VLC-PUFAs, however, the molecular mechanisms underpinning fatty acid (FA) desaturation and elongation in algae are poorly understood. An advanced molecular understanding would facilitate improvements of this nascent industry. We aimed to investigate expression responses of four front-end fatty acid desaturase genes and downstream effects on FA profiles to nitrogen limitation and cultivation growth stage in Isochrysis aff. galbana (TISO). Cultures were grown in nitrogen-replete and -deplete medium; samples were harvested during logarithmic, late logarithmic and stationary growth phases to analyse FA content/composition and gene expression of ?⁶-, ?⁸-, ?⁵- and ?⁴-desaturases (d6FAD (putative), d8FAD, d5FAD and d4FAD, respectively). d6FAD (putative) exhibited no differential expression, while d8FAD, d5FAD and d4FAD were significantly upregulated during logarithmic growth of nutrient-replete cultures, coinciding with rapid cell division. In conclusion, it is demonstrated that expression of some FADs in I. aff. galbana varies with culture age and nitrogen status which has downstream consequences on FA desaturation levels. This has implications for the commercial production of VLC-PUFAs where a trade-off between total lipid yield and VLC-PUFAs has to be made.