Recombinant Escherichia coli cells expressing linoleate 13‐hydratase from Lactobacillus acidophilus were permeabilized by treating with 0.2 M NaCl. The optimal conditions for the production of 13‐hydroxy‐9,15(Z,Z)‐octadecadienoic acid (13‐HODE) from α‐linolenic acid by permeabilized recombinant cells were pH 6.0, 40 °C, 7.5 % (v/v) methanol, 60 g/l permeabilized cells, and 15 g/l α‐linolenic acid. Under these conditions, permeabilized cells produced 7.5 g/l 13‐HODE after 6 h, with a conversion yield of 50 % (w/w) and a volumetric productivity of 1.25 g/l/h. These values were 161 and 160 % of those obtained by nonpermeabilized cells, respectively. To the best of our knowledge, this is the first report on the process optimization for the biotechnological production of 13‐HODE. 相似文献
Whole cells of recombinant Escherichia coli expressing diol synthase from Aspergillus nidulans produced 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid from α‐linolenic acid via 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid as an intermediate. The optimal conditions for 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid production using whole recombinant cells were exhibited at pH 7.0, 40 °C, and 250 rpm with 40 g/L cells, 12 g/L, α‐linolenic acid, and 5 % (v/v) dimethyl sulfoxide in a 250‐mL baffled flask containing 50 mL reaction solution. Under these conditions, whole recombinant cells produced 9.1 g/L 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid for 100 min, with a conversion yield of 75 % (w/w), a volumetric productivity of 5.5 g/L/h, and specific productivity of 137 mg/g‐cells/h. As an intermediate, 8‐hydroperoxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid was observed at approximately 1.4 g/L after 100 min. With regard to dihydroxy fatty acid production, this is the highest reported volumetric and specific productivities thus far. This is the first report on the biotechnological production of 5,8‐dihydroxy‐9,12,15(Z,Z,Z)‐octadecatrienoic acid. 相似文献
Macrophage apoptosis, a key process in atherogenesis, is regulated by oxidation products, including hydroxyoctadecadienoic acids (HODEs). These stable oxidation products of linoleic acid (LA) are abundant in atherosclerotic plaque and activate PPARγ and GPR132. We investigated the mechanisms through which HODEs regulate apoptosis. The effect of HODEs on THP‐1 monocytes and adherent THP‐1 cells were compared with other C18 fatty acids, LA and α‐linolenic acid (ALA). The number of cells was reduced within 24 hours following treatment with 9‐HODE (p < 0.01, 30 μM) and 13 HODE (p < 0.01, 30 μM), and the equivalent cell viability was also decreased (p < 0.001). Both 9‐HODE and 13‐HODE (but not LA or ALA) markedly increased caspase‐3/7 activity (p < 0.001) in both monocytes and adherent THP‐1 cells, with 9‐HODE the more potent. In addition, 9‐HODE and 13‐HODE both increased Annexin‐V labelling of cells (p < 0.001). There was no effect of LA, ALA, or the PPARγ agonist rosiglitazone (1μM), but the effect of HODEs was replicated with apoptosis‐inducer camptothecin (10μM). Only 9‐HODE increased DNA fragmentation. The pro‐apoptotic effect of HODEs was blocked by the caspase inhibitor DEVD‐CHO. The PPARγ antagonist T0070907 further increased apoptosis, suggestive of the PPARγ‐regulated apoptotic effects induced by 9‐HODE. The use of siRNA for GPR132 showed no evidence that the effect of HODEs was mediated through this receptor. 9‐HODE and 13‐HODE are potent—and specific—regulators of apoptosis in THP‐1 cells. Their action is PPARγ‐dependent and independent of GPR132. Further studies to identify the signalling pathways through which HODEs increase apoptosis in macrophages may reveal novel therapeutic targets for atherosclerosis. 相似文献
An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13‐hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo‐product, 13‐oxo‐9(Z),11(E)tridecadienoic acid (13‐OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13‐hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13‐hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13‐OTA along with the 13‐ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13‐ and 9‐hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH4)2 SO4 precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0. 相似文献
Dietary trans monoenes have been associated with an increased risk of heart disease in some studies and this has caused much concern. Trans polyenes are also present in the diet, for example, trans α‐linolenic acid is formed during the deodorisation of α‐linolenic acid‐rich oils such as rapeseed oil. One would expect the intake of trans α‐linolenic acid to be on the increase since the consumption of rapeseed oil in the western diet is increasing. There are no data on trans α‐linolenic acid consumption and its effects. We therefore carried out a comprehensive study to examine whether trans isomers of this polyunsaturated fatty acid increased the risk of coronary heart disease. Since inhibition of Δ6‐desaturase had also been linked to heart disease, the effect of trans α‐linolenic acid on the conversion of [U‐13C]‐labelled linoleic acid to dihomo‐γ‐linolenic and arachidonic acid was studied in 7 healthy men recruited from the staff and students of the University of Edinburgh. Thirty percent of the habitual fat was replaced using a trans ‘free’‐ or ‘high’ trans α‐linolenic acid fat. After at least 6 weeks on the experimental diets, the men received 3‐oleyl, 1,2‐[U‐13C]‐linoleyl glycerol (15 mg twice daily for ten days). The fatty acid composition of plasma phospholipids and the incorporation of 13C‐label into n‐6 fatty acids were determined at day 8, 9 and 10 and after a 6‐week washout period by gas chromatography‐combustion‐isotope ratio mass spectrometry. Trans α‐linolenic acid of plasma phospholipids increased from 0.04 ? 0.01 to 0.17 ? 0.02 and cis ? ‐linolenic acid decreased from 0.42 ? 0.07 to 0.29 ? 0.08 g/100 g of fatty acids on the high trans diet. The composition of the other plasma phospholipid fatty acids did not change. The enrichment of phosphatidyl 13C‐linoleic acid reached a plateau at day 10 and the average of the last 3 days did not differ between the low and high trans period. Both dihomo‐γ‐linolenic and arachidonic acid in phospholipids were enriched in 13C, both in absolute and relative terms (with respect to 13C‐linoleic acid). The enrichment was slightly and significantly higher during the high trans period (P<0.05). Our data suggest that a diet rich in trans α‐linolenic acid (0.6% of energy) does not inhibit the conversion of linoleic acid to dihomo‐γ‐linolenic and arachidonic acid in healthy middle‐aged men consuming a diet rich in linoleic acid. 相似文献
The asymmetric Sharpless epoxidation of methyl 13S‐hydroxy‐9Z, 11E‐octadeca‐dienoate (13S‐HODE, 1 ) with tert‐butyl hydroperoxide (TBHP) catalysed by titanium tetraisopropoxide {Ti(iOPr)4} in the presence of L(+)‐diisopropyl tartrate (L‐DIPT) gave methyl 13S‐hydroxy‐11S, 12S‐epoxy‐9Z‐octadecenoate 2 (erythro isomer) in 84% diastereomeric excess (de). The epoxidation of 1 with TBHP catalysed by Ti(iOPr)4 in the presence of D(‐)‐DIPT yielded methyl 13S‐hydroxy‐11RR12R‐epoxy‐9Z‐octadecenoate (threo isomer) 3 in 76% de. 相似文献
The seed oil of Arum maculatum has been found to contain 13‐phenyltridec‐9‐enoic (0.4%) and 15‐phenyl‐pentadec‐9‐enoic (1%) acids, detected by gas chromatographymass spectrometry of the picolinyl ester and related derivatives. 相似文献
Linoleate 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82 was identified as arachidonate (ARA) 11R-LOX by the determination of the product obtained from the conversion of ARA. The specific activity and catalytic efficiency (kcat/Km) of the enzyme for C20 and C22 polyunsaturated fatty acids followed the order ARA > eicosapentaenoic acid > docosahexaenoic acid. The production of the lipid mediator 11R-hydroxyeicosatetraenoic acid (11R-HETE) was performed using Escherichia coli cells expressing ARA 11R-LOX from Nostoc sp. The reaction conditions, such as pH, temperature, solvent and its concentration, and substrate and cell concentrations, were optimized using the recombinant cells, and the optimal conditions for the production of 11R-HETE from ARA were pH 7.0, 25°C, 10 g L−1 cells, 5.0 mM ARA, 4% (v/v) ethanol, and 10 mM cysteine as a reducing agent. Under these optimized conditions, E. coli cells expressing 11R-LOX converted 5.0 mM ARA into 4.74 mM 11R-HETE in 60 min, with a molar conversion yield of 95% a volumetric productivity of 79 μM min−1 and a specific productivity of 7.9 μM min−1 g−1. To the best of our knowledge, this is the first report on the quantitative biotechnological production of 11R-HETE. 相似文献
Δ6‐desaturase is located in a pivotal position in the metabolism of essential fatty acids (EFA). Various methods have been used to estimate Δ6‐desaturase activity, including the assessment of: (i) tissue fatty acid compositions (and associated product/precursor ratios), (ii) Δ6‐desaturase activities ex vivo, and (iii) isotopically labelled linoleic acid metabolism in vivo. This review critically examines these methods and considers their appropriateness and reliability in assessing linoleic acid metabolism in diabetes and cardiovascular disease. In general, there was a good agreement between the three methods and the effect of experimental diabetes on linoleic acid metabolism. In humans, however, the effect of diabetes on tissue fatty acid composition was inconsistent, and there was a paucity of data on linoleic acid metabolism ex vivo and in vivo. The inconsistency in human fatty acid compositional data may relate to variable and uncontrolled intakes of linoleic acid and its n‐6 metabolites, but also to a less extreme insulin deficiency as studied in animals. Risk markers for cardiovascular disease generally reduced rat liver Δ6‐desaturase activity ex vivo. This was not, however, reflected in tissue fatty acid compositions in these controlled studies. Linoleic acid metabolism, as determined by tissue fatty acid composition in humans, is reduced in cardiovascular disease; however, the omnivorous dietary patterns and decreased linoleic acid intakes make this conclusion potentially unreliable. Few stable‐isotope studies have been conducted on the effect of cardiovascular risk markers on linoleic acid metabolism, and there is a requirement for this type of work to be standardised to facilitate inter‐study comparisons. These studies may eventually help optimise EFA intake in health and disease conditions. 相似文献
A novel enzymatic production system of optically pure β‐hydroxy α‐amino acids was developed. Two enzymes were used for the system: an N‐succinyl L ‐amino acid β‐hydroxylase (SadA) belonging to the iron(II)/α‐ketoglutarate‐dependent dioxygenase superfamily and an N‐succinyl L ‐amino acid desuccinylase (LasA). The genes encoding the two enzymes are part of a gene set responsible for the biosynthesis of peptidyl compounds found in the Burkholderia ambifaria AMMD genome. SadA stereoselectively hydroxylated several N‐succinyl aliphatic L ‐amino acids and produced N‐succinyl β‐hydroxy L ‐amino acids, such as N‐succinyl‐L ‐β‐hydroxyvaline, N‐succinyl‐L ‐threonine, (2S,3R)‐N‐succinyl‐L ‐β‐hydroxyisoleucine, and N‐succinyl‐L ‐threo‐β‐hydroxyleucine. LasA catalyzed the desuccinylation of various N‐succinyl‐L ‐amino acids. Surprisingly, LasA is the first amide bond‐forming enzyme belonging to the amidohydrolase superfamily, and has succinylation activity towards the amino group of L ‐leucine. By combining SadA and LasA in a preparative scale production using N‐succinyl‐L ‐leucine as substrate, 2.3 mmol of L ‐threo‐β‐hydroxyleucine were successfully produced with 93% conversion and over 99% of diastereomeric excess. Consequently, the new production system described in this study has advantages in optical purity and reaction efficiency for application in the mass production of several β‐hydroxy α‐amino acids.
Linoleic acid isomerases (LAI) are enzymes responsible for conversion of linoleic acid (LA) to conjugated linoleic acids (CLA) in different isomeric forms. CLAs are well known for numerous beneficial effects as functional foods. Despite identification of several LAI producing‐bacteria and release of their LAI nucleotide sequences to Bio‐Banks, no related bioinformatics study on these important enzymes is addressed yet. To gain insights into the structural/functional and phylogentic relations of LAIs as well as recombinant production of the desired enzyme, we employed several bioinformatical tools to analyze their primary structure and physicochemical properties. The results indicated that LAIs produced in different bacterial strains might be divided in two distinct families (Propionibacterium acnes and myosin cross‐reactive antigen (MCRA)‐like LAIs) with specific isomerase activities. In another part of the study, physicochemical properties, solubility upon over expression in E. coli, disulfide bond formation, and potential glycosylation sites in LAI sequences were predicted using bioinformatics tools and the most appropriate strategy for recombinant production of each LAI was determined. Our predicted data may be useful for further experimental studies toward production of the desired LAI. 相似文献
N‐Acetyl‐D ‐neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N‐acetyl‐D ‐glucosamine (GlcNAc) and N‐acetyl‐D ‐mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32±0.56 g/liter Neu5Ac were obtained from 65.61±2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4±0.4 % purity, 80.87±0.79 % recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes. 相似文献
Polyunsaturated fatty acids (PUFA) are important ingredients of human diet because of their prominent role in the function of human brain, eye and kidney. α‐Linolenic acid (ALA), a C18, n‐3 PUFA is a precursor of long chain PUFA in humans. Commercial lipases of Candida rugosa, Pseudomonas cepacea, Pseudomonas fluorescens, and Rhizomucor miehei were used for hydrolysis of flax seed oil. Reversed phase high performance liquid chromatography followed by gas chromatography showed that the purified oil contained 12 triacylglycerols (TAGs) with differences in fatty acid compositions. Flax seed oil TAGs contained α‐linolenic acid (50%) as a major fatty acid while palmitic, oleic, linoleic made up rest of the portion. Among the four commercial lipases C. rugosa has preference for ALA, and that ALA was enriched in free fatty acids. C. rugosa lipase mediated hydrolysis of the TAGs resulted in a fatty acid mixture that was enriched in α‐linolenic to about 72% yield that could be further enriched to 80% yield by selective removal of saturated fatty acids by urea complexation. Such purified ALA can be used for preparation of ALA‐enriched glycerides. Practical applications : This methodology allows purifying ALA from fatty acid mixture obtained from flax seed oil by urea complexation. 相似文献
4‐Hydroxy‐2‐trans‐nonenal (HNE) is a toxic aldehyde produced mostly in oils containing polyunsaturated fatty acid due to heat‐induced lipid peroxidation. The present study examined the effects of the heating time, the degree of unsaturation, and the antioxidant potential on the formation of HNE in two light olive oils (LOO) and two sunflower oils (one high oleic and one regular) at frying temperature. HNE concentrations in these oil samples heated for 0, 1, 3, and 5 hours at 185 °C were measured using high‐performance liquid chromatography. The fatty‐acid distribution and the antioxidant capacity of these four oils were also analyzed. The results showed that all oils had very low HNE concentrations (<0.5 μg g?1 oil) before heating. After 5 hours of heating at 185 °C, HNE concentrations were increased to 17.98, 25.00, 12.51, and 40.00 μg g?1 in the two LOO, high‐oleic sunflower oil (HOSO), and regular sunflower oil (RSO), respectively. Extending the heating time increased HNE formation in all oils tested. It is related to their fatty‐acid distributions and antioxidant capacities. RSO, which contained high levels of linoleic acid (59.60%), a precursor for HNE, was more susceptible to degradation and HNE formation than HOSO and LOO, which contained only 6–8% linoleic acid. 相似文献