Synthesis activity-based zymography for detection of lipases and esterases

A new zymography method for lipases and esterases was developed on the basis of the esterification reaction between fatty acids and alcohols. The enzymes were separated by SDS-PAGE and native PAGE. The gel was washed and then incubated in an aqueous solution containing fatty acids (oleic acid 18:1 or caprylic acid 8:0) and dodecanol. Synthesis was visualized by in situ precipitation of water-insoluble and non-diffusible fatty acid esters, such as dodecyl oleate and dodecyl octanoate. The synthesis activity-based zymography was confirmed with different enzyme samples, including commercial lipase preparations, purified recombinant lipase and cutinase, and crude culture supernatants of lipolytic enzyme-producing soil bacteria.     

Rotein Composition Cf Flasmin Preparation “Hcmclysin”

A commercial preparation of human plasmin (Homolysin), capable of catalyzing the transformation of human growth hormone (hGH) into biologically activated species, was analyzed by electrophoresis and electrofocusing on polyacrylamide gel. Each major component of the preparation was characterized with regard to molecular size (retardation coefficient, K_R). molecular net charge (y-intercept on the Ferguson plot, Y₀), apparent isoelectric point (pi') and enzyme activity. The multiple components of Homolysln revealed by staining corresponded to various aggregation states of plasmin and exhibited full serine protease activity. Pclyacrylamlde gel electrophoresis of Homolysln in the presence of sodium dodecylsulfate (SDS) yielded 2 sub units which corresponded in molecular weight to the known plasmin subunits.     

Yeast engineering to express sex pheromone gland genes of the oriental fruit moth,Grapholita molesta

Mating disruption by using sex pheromone is an ecofriendly alternative way to control insect pests. To be effective, large amounts of sex pheromone are needed, leading to a relatively high production cost. To reduce the cost for chemical synthesis of sex pheromone, yeast engineering technology has been devised. This study used a baker's yeast, Saccharomyces cerevisiae, to express genes associated with sex pheromone biosynthesis of the Oriental fruit moth, Grapholita molesta. Compared to other fatty acid biosynthetic pathways, two steps that are unique to pheromone gland of G. molesta are proposed: desaturation at even number catalyzed by desaturase (Gm-DES) and terminal reduction catalyzed by fatty acyl reductase (Gm-FAR). Gm-DES and Gm-FAR were cloned into a yeast expression vector, pYES2.1. They were used to transform S. cerevisiae by a double transfection method. The transformed yeast was induced with 2% galactose to over-express these two exogenous genes. Their expression was confirmed by RT-PCR and western blotting. To facilitate pheromone production, transformed yeasts were supplied with myristic acid during over-expression. Resulting fatty acid composition was analyzed by GC-MS after fatty acid methyl ester derivatization. Control yeast produced mostly saturated fatty acids. However, a single gene (Gm-DES)-transformed yeast produced unsaturated fatty acids at ∆9 such as Z9-tetradecenoic acid (Z9-14:1), palmitoleic acid (Z9-16:1), and oleic acid (Z9-18:1) in addition to saturated fatty acids. The double-transformed yeast produced an additional component, alcohol form of oleic acid (Z9-18:OH). These results suggest that Gm-DES can catalyze desaturation of fatty acids at ∆9 and Gm-FAR can reduce terminal carboxylic acid into alcohol.     

Isolation,purification, and properties of mammalian and avian liver and yeast fatty acid synthetase acyl carrier proteins

Acyl carrier proteins were isolated from rat, human, pigeon, and chicken liver and yeast fatty acid synthetase complexes. These proteins were separated from the other proteins of subunit I of each complex by ultrafiltration after dialysis of subunit I for 3 h against low ionic strength buffer [Qureshi et al. (1974) Biochem. Biophys. Res. Commun.60, 158–165]. Subunit I of each fatty acid synthetase was previously separated from subunit II by affinity chromatography on Sepharose ?-aminocaproyl pantetheine and subsequent sucrose density gradient centrifugation. The separated acyl carrier proteins were then subjected to gel filtration on a Sephadex G-50 column. The proteins obtained from each fatty acid synthetase were homogeneous with respect to size and charge on gel filtration, paper and disc gel electrophoresis, and chromatography on diethylaminoethyl-cellulose. The physical properties and the ability to accept acetyl and malonyl groups from acetyl- and malonyl-CoA in the presence of transacylase were similar to those of Escherichia coli acyl carrier protein. These proteins ranged in molecular weight from 7500 to 10,000. Each of the acyl carrier proteins showed the presence of β-alanine and each yielded acetyl- and malonyl-A₁ and A₂ peptic peptides, thus indicating the presence of a 4′-phosphopantetheine prosthetic group in each. They differed somewhat from each other in amino acid composition, but each had a high number of negatively charged (aspartate and glutamate) amino acid residues.     

Characterization of multiple charge isomers of the inhibitor protein of the cyclic AMP-dependent protein kinase from bovine heart and rabbit skeletal muscle

The presence of multiple forms of the inhibitor of the cyclic AMP-dependent protein kinase has been investigated in rabbit skeletal and bovine cardiac muscle. These tissues have each been fractionated by two separate procedures, one involving mild techniques, the other utilizing trichloroacetic acid precipitation and heating to 95 °C. The proteins in partially purified preparations have been characterized by DEAE-cellulose chromatography, gel filtration, isoelectric focusing, and sodium dodecyl sulfate and nondenaturing gel electrophoresis. In each tissue three charge isomers have been detected. Each charge isomer may exist in the two apparent molecular size forms designated I and I′ (J. M. McPherson, S. Whitehouse, and D. A. Walsh, 1979, Biochemistry18, 4835–4845). The distribution of inhibitor between the multiple forms depends on the purification procedure used and potentially only one form may exist in tissues.     

Hypolipidemic constituents from the aerial portion of Sibiraea angustata

Two new monoterpene acylglucosides (1–2) and one new aromatic glycoside (3), together with five known compounds (4–8), were isolated from 95% ethanol extract of Sibiraea angustata. The structures of these compounds were characterized by 2D-NMR and mass spectrometry. Compounds were evaluated for their hypolipidemic activity using oleic acid-induced lipid accumulation in HepG2 cells. RT-PCR analysis revealed that compound 5 could decrease the expression level of fatty acid synthase (FASN). Lipidomics analysis indicated that compound 5 significantly decreased the levels of 11 lipids in oleic acid-induced lipid accumulation, including triglycerides (TG), diglycerides (DG), phosphatidylcholines (PC) and 1-acyl-sn-glycero-3-phosphocholines (lysoPC). These data demonstrated that terpene acylglucosides are the major active constituents in Sibiraea angustata.     

Significant increase of oleic acid level in the wild species <Emphasis Type="Italic">Lepidium campestre</Emphasis> through direct gene silencing

Key message

Simultaneous RNAi silencing of the FAD2 and FAE1 genes in the wild species Lepidium campestre improved the oil quality with 80 % oleic acid content compared to 11 % in wildtype.

Abstract

Field cress (Lepidium campestre) is a wild biennial species within the Brassicaceae family with desirable agronomic traits, thus being a good candidate for domestication into a new oilseed and catch crop. However, it has agronomic traits that need to be improved before it can become an economically viable species. One of such traits is the seed oil composition, which is not desirable either for food use or for industrial applications. In this study, we have, through metabolic engineering, altered the seed oil composition in field cress into a premium oil for food processing, industrial, or chemical industrial applications. Through seed-specific RNAi silencing of the field cress fatty acid desaturase 2 (FAD2) and fatty acid elongase 1 (FAE1) genes, we have obtained transgenic lines with an oleic acid content increased from 11 % in the wildtype to over 80 %. Moreover, the oxidatively unstable linolenic acid was decreased from 40.4 to 2.6 %, and the unhealthy erucic acid was reduced from 20.3 to 0.1 %. The high oleic acid trait has been kept stable for three generations. This shows the possibility to use field cress as a platform for genetic engineering of oil compositions tailor-made for its end uses.

     

The enzymatic acyl exchange of phospholipids with lipases

The acyl exchange of phospholipids with lipases was investigated. The lipase from Rhizopus delemar catalyzed the acyl exchange reaction between various phospholipids and fatty acids. When we incubated 1,2-dipalmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) and oleic acid with lipase from R. delemar, the yield of diacyl phosphatidyl choline (PC) was 25% and the fatty acid composition of the converted PC was an oleic acid content of 25% and a palmitic acid content of 75%. This reaction exhibited 1-positional specificity. Three industrial lipases from Rhizopus sp., Mucor javanicus, and Candida cylindracea had the activity of the acyl exchange of phosphatidyl choline. The lipase from R. sp. gave the best result.     

Rapid, multisample isoelectric focusing in sucrose density gradients using conventional polyacrylamide electrophoresis equpiment: a two-peak transient in the approach-to-equilibrium.

Using a semiporous plug of agar gel to support a sucrose density gradient column without restricting electrical conductivity, Massey and Deal [J. Biol. Chem.248, 56 (1973)] were able to use a conventional polyacrylamide gel electrophoresis apparatus to carry out single tube isoelectric focusing experiments in density gradients in only 2 hr using minute amounts (50 μg) of sample and very little ampholyte (0.18 ml); no cooling apparatus was required. In this work we report that 1) polyacrylamide provides a superior gel plug and 2) that ten isoelectric focusing tubes can easily be run simultaneously in a conventional polyacrylamide gel electrophoresis apparatus. In addition, the isoelectric points of eight proteins, with pI values ranging from 5.1 to 8.8 have been determined and the kinetics of the approach-to-isoelectric-focusing-equilibrium have been analyzed. Of special interest is the discovery that in the initial stages of focusing, in these sucrose density gradients, a major peak is formed at each end of the column; these two peaks migrate toward each other and finally coalesce into a single peak. Similar, although less pronounced, effects were previously observed by Catsimpoolas and Wang [Anal. Biochem.39, 141 (1971)] in focusing experiments in polyacrylamide gels. With all other conditions constant, the time required to reach equilibrium is 1) less in broad range (e.g., 3–10) pH gradients than it is in narrow range (e.g., 5–8) pH gradients and 2) generally greater with higher molecular weight substances than with lower molecular weight substances. Explanations are given for all of these kinetic phenomena.     

Purification and primary structure of glucagon from ostrich pancreas splenic lobes

Glucagon is a highly conserved polypeptide hormone which appears to play a more important role in regulation of glycaemia in birds than insulin. Ostrich glucagon was isolated and purified from ostrich pancreas splenic lobes using an adapted acid ethanol extraction procedure, gel filtration, ion exchanges, and HPLC steps. The purified glucagon fraction appeared to contain small quantities of a more acidic contaminant (polyacrylamide gel isoelectric focussing, PAGE) but appeared homogeneous on SDS-PAGE. Amino acid analysis and sequence analysis showed identity with the duck hormone. Identity with the duck hormone was confirmed by liquid phase as well as gas phase sequencing. The ostrich glucagon preparation seemed to have a higher Km than the porcine homologue in stimulating glycerol release from isolated chicken adipocytes.     

Alterations of the fatty acid composition and lipid metabolome of breast muscle in chickens exposed to dietary mixed edible oils

The fatty acid composition of chicken’s meat is largely influenced by dietary lipids, which are often used as supplements to increase dietary caloric density. The underlying key metabolites and pathways influenced by dietary oils remain poorly known in chickens. The objective of this study was to explore the underlying metabolic mechanisms of how diets supplemented with mixed or a single oil with distinct fatty acid composition influence the fatty acid profile in breast muscle of Qingyuan chickens. Birds were fed a corn-soybean meal diet supplemented with either soybean oil (control, CON) or equal amounts of mixed edible oils (MEO; soybean oil : lard : fish oil : coconut oil = 1 : 1 : 0.5 : 0.5) from 1 to 120 days of age. Growth performance and fatty acid composition of muscle lipids were analysed. LC-MS was applied to investigate the effects of CON v. MEO diets on lipid-related metabolites in the muscle of chickens at day 120. Compared with the CON diet, chickens fed the MEO diet had a lower feed conversion ratio (P < 0.05), higher proportions of lauric acid (C12:0), myristic acid (C14:0), palmitoleic acid (C16:1n-7), oleic acid (C18:1n-9), EPA (C20:5n-3) and DHA (C22:6n-3), and a lower linoleic acid (C18:2n-6) content in breast muscle (P < 0.05). Muscle metabolome profiling showed that the most differentially abundant metabolites are phospholipids, including phosphatidylcholines (PC) and phosphatidylethanolamines (PE), which enriched the glycerophospholipid metabolism (P < 0.05). These key differentially abundant metabolites – PC (14:0/20:4), PC (18:1/14:1), PC (18:0/14:1), PC (18:0/18:4), PC (20:0/18:4), PE (22:0/P-16:0), PE (24:0/20:5), PE (22:2/P-18:1), PE (24:0/18:4) – were closely associated with the contents of C12:0, C14:0, DHA and C18:2n-6 in muscle lipids (P < 0.05). The content of glutathione metabolite was higher with MEO than CON diet (P < 0.05). Based on these results, it can be concluded that the diet supplemented with MEO reduced the feed conversion ratio, enriched the content of n-3 fatty acids and modified the related metabolites (including PC, PE and glutathione) in breast muscle of chickens.     

Isolation of human growth hormone isohormones D and E in milligram amounts (II), using isoelectric focusing on polyacrylamide gel

Human growth hormone (hGH) isohormones D and E were prepared from a partial enzymatic hydrolyzate catalyzed by plasmin, using isoelectric focusing on polyacrylamide gel to fractionate the digest. Separation between the two isohormones species was optimized by employing a maximally flattened pH gradient between pH's 4.3 and 5.6. Gel slices containing isohormones D and E were extracted and concentrated by Steady-State Stacking on polyacrylamide gel. The extract was purified by gel filtration and lyophilized. Overall yields of lyophilized isohormones D and E were 10.9 mg and represented a recovery of 51% relative to the densitometrically estimated isohormone concentrations in the hGH digest. Purity of the products, based on Lowry analysis, U.V. absorbance and radioimmunoassay (RIA) was 60-70%. The isolated isohormone species were active in the rat tibia-line assay and in the specific RIA for hGH.     

Characterization of intact and trypsin-digested biosynthetic human growth hormone by high-pressure liquid chromatography

The structural properties of purified human growth hormone (hGH) produced by Escherichia coli K-12 into which the hGH gene has been inserted have been fully characterized by high-pressure liquid chromatography of native hGH and tryptic digests of hGH. All of the tryptic peptides have been separated by high-pressure liquid chromatography and their sequence determined. Comparison of the primary structure with that of the purified pituitary-derived hGH has established the integrity of the biosynthetic hGH disulfide arrangement and amino acid sequence with the presence of an extra NH₂-terminal methionine.     

Macrolide (cucujolide) biosynthesis in the rusty grain beetle,Cryptolestes ferrugineusI

Males of the cucujid grain beetles, Cryptolestes ferrugineus (Stephens), C. pusillus (Schönhen), C. turcicus (Grouvelle), Oryzaephilus mercator (Fauval) and O. surinamensis (L.), produce seven structurally related macrolides (“cucujolides”). Species-specific combinations of these macrocyclic lactones function as aggregation pheromones. To study the biosynthesis of these cucujolides, mixed sex cultures of the rusty grain beetle, C. ferrugineus, were fed oats impregnated with radiolabelled substrates. Culture volatiles were trapped on Porapak Q, eluted by back-flushing with solvent, concentrated and purified to constant specific activity by high performance liquid chromatography and preparative gas chromatography. The incorporation of radiolabelled acetate and mevalonate into cucujolide I [4(E),8(E)4,8-dimethyldecadien-10-olide] provided evidence that this pheromone is of terpenoid origin. The efficient incorporation of radiolabelled fatty acid substrates into cucujolides II 3(Z)dodecen-11-olide], III 5(Z)tetradecen-13-olide], IV [3(Z),6(Z)dodecadien-11-olide] and V [5(Z),8(Z)tetradecadien-13-olide] provided evidence that these cucujolides are of fatty acid origin. Radiolabelled palmitic acid was incorporated into cucujolides II, III, IV and V; radiolabelled oleic acid was incorporated into II and III; and radiolabelled linoleic acid was incorporated into IV. Radiolabelled acetate was incorporated into cucujolides II, III and V, indicating that C. ferrugineus can produce these cucujolides de novo.     

Enzymatic nitration of the oleic acid derivatives: Synthesis,isolation and characterization

Unsaturated fatty acids are nitrated endogenously to produce nitrated lipids. Recent studies have shown that these nitrated lipids have high chemical reactivity and profound biological implications. We report an efficient, enzymatic synthesis of nitrated derivatives of the oleic acid. The methyl oleate could react with NO and horseradish peroxidase (HRP)/H₂O₂/NO based nitrating systems to give various nitration products which could be isolated by silica gel column and TLC fractionation, respectively. As reacting directly with NO, the obtained products contain (E)-methyl 9-nitrooctadec-9-enoate (1), (E)-methyl 10-nitrooctadec-9-enoate (2), (E)-methyl 9-nitrooctadec-10-enoate (3) and (E)-methyl 10-nitrooctadec-8-enoate (4), characterized by extensive IR, NMR and GC–MS analysis. Whereas the products of the reaction between the methyl oleate and NO with the presence of HRP/H₂O₂ were mainly composed of (E)-methyl 9-nitrooctadec-9-enoate and (E)-methyl 10-nitrooctadec-9-enoate. The improving selectivity of the products is attributed to the HRP catalysis system.     

Heterologous expression of stearoyl-acyl carrier protein desaturase (S-ACP-DES) from Arabidopsis thaliana in Escherichia coli

Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains, among which stearoyl-acyl carrier protein desaturase (S-ACP-DES) was widely distributed in the plant kingdom. We cloned the cDNA coding for fab2/ssi2, an S-ACP-DES from Arabidopsis thaliana, into the vector pET30a and heterologously expressed this fatty acid desaturase in Escherichia coli BL21 (DE3). After being induced with IPTG, the fusion protein was efficiently expressed in a soluble form. The SSI2 desaturase was purified by nickel ion affinity chromatography and the product obtained showed a single band by SDS–PAGE analysis. The expression of ssi2 modified the fatty acid composition of the recombinant strain. The ratio of palmitic acid (16:0) decreased from 45.2% (the control strain) to 35.2% while palmitoleate (16:1Δ9) and cis-vaccenate (18:1Δ11) levels were enhanced to some extent. The desaturase enzymatic activity was measured in vivo when the enzyme substrate stearic acid was provided in the culture medium. A new fatty acid, oleic acid (18:1Δ9) was found in the recombinant strain which did not exist in wild-type E. coli. These results demonstrated that the cofactors of the host system can complement the requirement of the SSI2 desaturase.     

Synthesis of long-chain fatty acid derivatives as a novel anti-Alzheimer’s agent

In order to develop new drugs for Alzheimer’s disease, we prepared 17 fatty acid derivatives with different chain lengths and different numbers and positions of double bonds by using Wittig reaction and stereospecific hydrogenation of triple bonds as key reactions. Among them, (4Z,15Z)-octadecadienoic acid (10) and (23Z,34Z)-heptatriacontadienoic acid (16) showed the most potent neurite outgrowth activities on Aβ(25–35)-treated rat cortical neurons, which activities were comparable to that of a positive control, NGF. Both fatty acids 10 and 16 possess two (Z)-double bonds at the n-3 and n-14 positions, which might be important for the neurite outgrowth activity.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Design,synthesis and biological evaluation of novel thiazole derivatives as potent FabH inhibitors

Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram positive and negative bacteria. Three series of Schiff bases containing thiazole template were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 11 and 18 were potent inhibitors of E. coli FabH.     

Overexpression of stearoyl-ACP desaturase enhances accumulations of oleic acid in the green alga Chlamydomonas reinhardtii

FAB2, which encodes stearoyl-acyl carrier protein desaturase, catalyzes the conversion of stearic acid (18:0) to oleic acid (18:1) in fatty acid biosynthesis. In this study, we isolated FAB2 from Chlamydomonas reinhardtii, named CrFAB2, and generated CrFAB2-overexpressing transgenic lines to identify a major role of CrFAB2 in fatty acid biosynthesis of C. reinhardtii. In CrFAB2-overexpressing lines, oleic acid (18:1) content was increased by approximately 2.4-fold compared to the wild-type control plants. Interestingly, CrFAB2 overexpression resulted in the induction of CrFAD2 expression. Consistent with this result, the induction of linoleic acid (18:2) was also detected in CrFAB2-overexpressing lines, and total fatty acid content in these lines was induced by approximately 28 % by CrFAB2 overexpression compared to the wild-type control. Our results indicate that CrFAB2 overexpression enhances the synthesis of oleic acid (18:1) and that CrFAB2 may also play a key role in regulating total fatty acid content in the green alga C. reinhardtii.