Optimized enzymatic synthesis of ascorbyl esters from lard using Novozym 435 in co-solvent mixtures

A mild and efficient method for the conversion of fatty acid methyl esters from lard into ascorbyl esters via lipase-catalyzed transesterification in co-solvent mixture is described. A solvent engineering strategy was firstly applied to improve fatty acid ascorbyl esters production. The co-solvent mixture of 30% t-pentanol:70% isooctane (v/v) was optimal. Response surface methodology (RSM) and central composite design (CCD) were employed to estimate the effects of reaction parameters, such as reaction time (12–36 h), temperature (45–65 °C), enzyme amount (10–20%, w/w, of fat acid methyl esters), and substrate molar ratio of fatty acid methyl esters to ascorbic acid (8:1–12:1) for the synthesis of fatty acid ascorbyl esters in co-solvent mixture. Based on the RSM analysis, the optimal reaction conditions were determined as follows: reaction time 34.32 h, temperature 54.6 °C, enzyme amount 12.5%, substrate molar ratio 10.22:1 and the maximum conversion of fatty acid ascorbyl esters was 69.18%. The method proved to be applicable for the synthesis of ascorbyl esters using Novozym 435 in solvent.     

Studies on Moss Spores. IV. Mass Spectrometric Identification of Saturated and Monoenoic Long Chain Fatty Acids in the Triglyceride Fraction of Polytrichum commune Spores

The saturated long chain fatty acid methyl esters of the triglyceride fraction of Polytrichum commune spores were separated by silver nitrate TLC and identified by a combination of gas chromatographic-mass spectrometric technique. The saturated fatty acid methyl esters were straight-chained, and even-numbered with carbon numbers ranging from 12 to 26 or odd-numbered with carbon numbers ranging from 13 to 25. The major components of the fraction containing saturated fatty acid methyl esters were methyl palmitate and methyl stearate. The fatty acid methyl esters of the monoenoic fraction isolated by silver nitrate TLC were converted to TMSO derivates which were analysed by gas chromatography-mass spectrometry. The analysis gave evidence of positional isomers. The fraction contained the following straight chain monoenoic fatty acid methyl ester isomers: methyl 7-cis-hexadecenoate, methyl 9-cis-hexadecenoate, methyl 9-cis-heptadecenoate, methyl 9-cis-octadecenoate, methyl 11-cis-octadecenoate, and methyl 11-cis-eicosenoate. The major components were methyl 9-cis-octadecenoate and methyl 7-cis-hexadecenoate.     

The Collection and Elution of Radioactive Fatty Acid Methyl Esters During Quantitative Gas-Liquid Chromatography

Abstract

An improved procedure is described for the collection and elution of low levels of radioactive fatty acid methyl esters separated by gas-liquid chromatography. A gas chromatographic effluent splitter was employed to partition fatty acid methyl ester samples in the column effluent, Condensation of a portion of the eluted fatty acids was accomplished in borosilicate glass tubing collectors maintained at ?70°C, Quantitation of nanomolar levels of fatty acid methyl esters was accomplished by calibrating the gas chromatographic flame ionization detectors with the splitters opened or closed. The elution of condensed radioactive fatty acid methyl esters from the glass collectors was complete when benzene followed by a toluene based scintillation fluid were employed as solvents. The method described may be applicable to the analysis of cis-trans isomers of unsaturated fatty acids.     

Properties and Substrate Specificities of Four Esterases from Aspergillus niger NRRL 337

Properties and substrate specificities of four esterases (Esterase-I, -II, -III, -IV) from Aspergillus niger were studied. Esterase-I and Esterase-II were found to be markedly stable to heat. When Esterase-I was assayed at 35°C using methylacetylsalicylate as a substrate, even after heating at 100°C for 15 min 60% of its activity remained. However, Esterase-I scarcely hydrolyzed the substrate at 70°C or over, because of a reversible change in conformation by heating as found by CD measurement. The maximum activity of Esterase-I was found at 55°C at 20 min of reaction time. Esterase-II was stable up to 80°C and had an optimum temperature for reaction at 80°C, but was irreversively inactivated by heating for 15 min at 90°C.

The four esterases hydrolyzed aliphatic esters of short chain fatty acids and acetyl esters of phenols, but neither methyl esters of aromatic carboxylic acids nor acetyl esters of aromatic alcohols.     

Quantitative release of fatty acids from lipids by a simple hydrolysis procedure

Glycerolipids and sphingolipids are hydrolyzed with 0.5 M HCl in acetonitrile-water 9:1 (by vol) for 45 min at 100 degrees C or 4 hr at 70 degrees C. After hydrolysis, free fatty acids (FFA) are recovered in chloroform and separated by thin-layer chromatography. More than 95% of the radioactivity from labeled phospholipids is recovered as FFA, and more than 97% of the lipid phosphorus is recovered as water-soluble phosphate. The yields of FFA, including polyunsaturated acids, after hydrolysis are as good as or better than those obtained for methyl esters using methanolysis catalyzed by acid, alkali, or BF3. High recoveries of FFA from glycerophospholipids, sphingomyelin, and neutral glycerides are attained. The procedure is quantitative, simple, inexpensive, and produces no methyl esters as secondary products.     

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 μmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g?L^?1 with fatty acid methyl esters of 11.5 mg?L^?1. Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 μmol photons per square meter per second lipid content reached 10.37 mg?L^?1 with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.     

Feeding stimulants eliciting the probing behavior for Peregrinator blannulipes Montrouzier et Signore (Hemiptera: Ruduviidae) from Tribolium confusum (Jacquelin du Val)

Four fatty acid methyl esters identified in the solvent extract of Tribolium confusum (Jacquelin du Val) larvae as kairomones were individually and collectively tested for probing behavior of Peregrinator biannulipes Montrouzier et Signoret. All identified fatty acid methyl eaters, methyl palmitate, methyl linolate, methyl oleate and methyl stearate, exhibited characterisitic kairomonal probing behavior of P. biannulipe toward the lure. These fatty acid methyl ester were active at 0.2 microg/lure but a synergistic effect was not observed among them. Commercially available C8-C14 even-numbered fatty acid methyl esters that were not detected in the extract of T. confusum larvae also elicited a probing behavior but their activities were weaker than those of four fatty acid methyl ester (C16:0, C18:0, C18:1 and C18:2) identified in the extract. On the other hand, C17 and C19 odd-numbered fatty acid methyl esters did not show any activity at all.     

Lipase-catalyzed simultaneous biosynthesis of biodiesel and glycerol carbonate from corn oil in dimethyl carbonate

Biodiesel [fatty acid methyl esters (FAMEs)] and glycerol carbonate were synthesized from corn oil and dimethyl carbonate (DMC) via transesterification using lipase (Novozyme 435) in solvent-free reaction in which excess DMC was used as the substrate and reaction medium. Glycerol carbonate was also simultaneously formed from DMC and glycerol. Conversions of FAMEs and glycerol carbonate were examined in batch reactions. The FAMEs and glycerol carbonate reached 94 and 62.5% from oil and DMC (molar ratio of 1:10) with 0.2% (v/v) water and 10% (w/w) Novozyme 435 (based on oil weight) at 60°C. When Novozyme 435 was washed with acetone after each reaction, more than 80% activity still remained after seven recycling.     

Estimating and improving cold filter plugging points by blending biodiesels with different fatty acid contents

Biodiesels are alkyl esters produced by transesterification of higher fatty acids (aliphatic chains composed of 14 to 22 carbon units) from animal fats and/or vegetable oils. The cold filter plugging points (CFPP) of biodiesels are not only higher than that of petro-diesel, but they also differ from the melting point of the raw (unesterified) materials. In this study, we empirically derived equations that estimated the CFPP of a biodiesel based on its fatty acid content, using various biodiesel blends containing four methyl esters with different fatty acid compositions: soybean (SME), palm (PME), rapeseed (RME), and lard (LME). These blending ratio experiments yielded three equations that described the correlation between CFPP and fatty acid content: Y (CFPP, °C) = −3.1X (blending ratio) − 12.7 (PME/SME); Y = 2.2X − 10.7 (LME/SME); and Y = −4.0X − 13.0 (PME/RME). We also obtained the correlation between CFPP and total saturated fatty acid methyl ester content in the biodiesels: Y (CFPP, °C) = 0.449X (total saturated fatty acid methyl ester content, wt%) − 9.198. These empirical equations accurately predicted CFPP values of biodiesel compounds with known fatty acid compositions, facilitating the use of diverse biodiesels in industrial fields. The first two authors equally contributed to this work.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Stereochemical aspects of synthetic and naturally occurring 2-hydroxy fatty acids. Their absolute configurations and assays of optical purity

d- and l-Cerebronic acid⁵, ⁶ methyl esters were obtained from a dl-mixture by converting the racemic ester to the l-acetylmandelates and separating the diastereomers by thin-layer chromatography; the diasteromers were then decomposed with mild acid methanolysis. The optical purities of the d- and l-enantiomers were determined as described below and found to be 82.5% and 87.1%, respectively. Absolute configurations of the enantiomers were confirmed by their ORD spectra; specific rotations at the peak of the anomalous curves at 223 nm of the d- and l-isomers were ?989 ° and +1120 °, respectively. Considering their optical purities, the actual maximum rotations of these enantiomers were calculated as ?1520 ° and +1509 °, respectively. The conditions for the reaction of l-acetylmandelyl chloride and 2-hydroxy fatty acid esters were modified to be suitable for the optical assay of 2-hydroxy fatty acid methyl esters. The method was also shown to be applicable to the assay of 3-hydroxy isomers and possibly to other positional isomers. The absolute configuration of 2-hydroxy fatty acid methyl esters obtained from calf brain cerebrosides and from yeast cerebrins were determined to be d(?), confirming previous assignments. Methyl cerebronate obtained from its less soluble strychnine salt was found to be the l(+)-enantiomer, contrary to a previous assignment. Methyl d-cerebronate was hydrolyzed with tetrahydrofuran-HCl to obtain D-cerebronic acid without racemization.     

Continuous lipase-catalyzed esterification of soybean fatty acids under ultrasound irradiation

This work investigates the continuous production of alkyl esters from soybean fatty acid (FA) charges using immobilized Novozym 435 as catalyst. The experiments were performed in a packed-bed bioreactor evaluating the effects of FA charge to alcohol (methanol and ethanol) molar ratio, from 1:1 to 1:6, substrate flow rate in the range of 0.5–2.5 mL/min and output irradiation power up to 154 W, at fixed temperature of 65 °C, on the reaction conversion. Results showed that almost complete conversions to fatty acids ethyl esters were achieved at mild ultrasonic power (61.6 W), FA to ethanol molar ratio of 1:6, operating temperature (65 °C) and remained nearly constant for long-term reactions without negligible enzyme activity losses.     

Free ceramide, sphingomyelin, and glucosylceramide of isolated rat intestinal cells.

Free ceramide, glucosylceramide, and sphingomyelin were isolated from mature cells of adult rat small intestine. Free ceramide and ceramide cleaved from sphingomyelin by enzymatic hydrolysis were fractionated by thin-layer chromatography on borate-impregnated silica gel plates. Sphingoid bases were characterized by gas-liquid chromatography of aldehydes formed upon periodate oxidation. Fatty acids were quantified as methyl esters. Ceramide structures were confirmed by direct-inlet mass spectrometry. Free ceramide was found to contain two major long-chain bases in nearly equal quantity: sphingosine, mainly linked to palmitic acid, and 4D-hydroxysphinganine associated with C20 to C24 fatty acids, 22% being hydroxylated. Sphinganine occurred as a minor component linked to nonhydroxy fatty acids. Sphingomyelin contained the three long-chain bases and 63% of its ceramide was N-palmitoyl-sphingosine. Mass spectrometry of glucosylceramide confirmed 4D-hydroxyshingamine as the major sphingoid base associated preferentially with longer chain hydroxy fatty acids.     

High pressure liquid chromatography methods for separation of omega- and (omega-1)-hydroxy fatty acids: their applications to microsomal fatty acid omega-oxidation

Fatty acids (C12-C18) and their omega- and (omega-1)-hydroxy derivatives, when converted to p-bromophenacyl (PBP) esters, can be completely separated from one another by high pressure liquid chromatography (HPLC) on a silicic acid column using 0.5% (v/v) isopropanol in n-hexane. In this system, fatty acid PBP esters are eluted at the solvent front, whereas the retention times of the omega- and (omega-1)-hydroxy derivatives are 14-20 and 24-29 min, respectively. The PBP esters can also be separated by reverse phase HPLC on a muBondapak C18 column, a method which has been developed by Fan et al. (Fan, L. L., Masters, B. S. S., and Prough, R. A. (1976) Anal. Biochem. 71, 265-272) for separation of methyl esters of fatty acids and their omega- and (omega-1)-hydroxy derivatives. In the latter method, however, the retention times of omega- and (omega-1)-hydroxy derivatives are only about 2 min apart and an increase in the solvent polarity is needed for elution of the esters of unmodified fatty acids. Fatty acid PBP esters, however, can be obtained as independent peaks which are not disturbed by the solvent front. An application of the former method to measure fatty acid omega oxidation by liver microsomes and by a reconstituted monooxygenase system containing purified cytochrome P-450 is described.     

6-acyl galactosyl ceramides of pig brain: structure and fatty acid composition

Two glycolipids were isolated from pig brain and were shown to be the fatty acid esters of kerasin and cerebron in which the second fatty acid moiety is attached to the 6-position of the galactose. The point of attachment was shown in two ways: by permethylation and by cleavage with periodate. Methanolysis of the permethylated cerebroside esters yielded O-methyl sphingosines, methyl esters of nonhydroxy or 2-methoxy acids, and methyl 2,3,4-trimethyl galactoside. Cleavage of the cerebron ester with periodate, followed by treatment with sodium borohydride and dilute HCl, yielded ceramide plus 1-monoglyceride. The ester-linked fatty acids were primarily 16:0, 18:0, and 18:1, while the amide-linked fatty acids showed the wide assortment of chain lengths typical of brain cerebrosides. The methylation step, with silver oxide and methyl iodide, yielded two derivatives with the cerebroside esters, but the structural explanation for the difference was not elucidated. The galactose in the cerebron ester was shown to exist in the beta-pyranoside form.     

Dimethyl carbonate as potential reactant in non-catalytic biodiesel production by supercritical method

In this study, the non-catalytic supercritical method has been studied in utilizing dimethyl carbonate. It was demonstrated that, the supercritical dimethyl carbonate process without any catalysts applied, converted triglycerides to fatty acid methyl esters with glycerol carbonate and citramalic acid as by-products, while free fatty acids were converted to fatty acid methyl esters with glyoxal. After 12 min of reaction at 350 degrees C/20 MPa, rapeseed oil treated with supercritical dimethyl carbonate reached 94% (w/w) yield of fatty acid methyl ester. The by-products from this process which are glycerol carbonate and citramalic acid are much higher in value than glycerol produced by the conventional process. In addition, the yield of the fatty acid methyl esters as biodiesel was almost at par with supercritical methanol method. Therefore, supercritical dimethyl carbonate process can be a good candidate as an alternative biodiesel production process.     

Transesterification of phospholipids or triglycerides to fatty acid benzyl esters with simultaneous methylation of free fatty acids for gas-liquid chromatographic analysis

A novel method is presented for transesterification of fatty acid esters in phospholipids and triglycerides to benzyl esters while simultaneously recovering free fatty acids as methyl esters. Transesterification is catalyzed by 0.2 M (m-trifluoromethyl phenyl)trimethyl ammonium hydroxide in methylene chloride, 10% (v/v) benzyl alcohol, and 1% (w/v) potassium tert-butoxide, and is complete in 30 min at room temperature. Methyl esters of all common fatty acids separate from the benzyl esters formed from phospholipids. This method has broad utility and is applicable to the formation of esters optimized for detection by absorbance or fluorescence (high performance liquid chromatography), electron capture (gas-liquid chromatography), or negative ion chemical ionization (gas-liquid chromatography-mass spectrometry).     

In Vitro Metabolism of Mevalonic Acid in the Bovine Retina

Bovine retinas were incubated with 3RS-[5-3H]-mevalonic acid under conditions similar to those previously shown to support opsin biosynthesis in vitro. TLC of the total lipids indicated the formation of numerous radiolabeled components, including sterols, hydrocarbons, and "fatty acid-like material." The nonsaponifiable lipids were analyzed by TLC, GLC, and chromatography on columns of silicic acid-Super Cel, silica gel G-Super Cel-silver nitrate, and alumina-Super Cel-silver nitrate. The major nonsaponifiable components had the chromatographic properties of squalene and "methylated sterols" (i.e., C30, C29, and C28 monohydroxy sterols). Cholesterol represented no more than 1% of the total radioactivity in the nonsaponifiable lipid fraction. The "fatty acid-like material" was derivatized with diazomethane, and the resulting methyl esters were analyzed by GLC before and after catalytic hydrogenation. The radioactivity did not correspond to the normal fatty acids endogenous to the retina, but rather had the chromatographic properties of C15 and C20 isoprenoid acids. These results obtained with intact retinas are consistent with our previous observations concerning mevalonic acid metabolism in cell-free homogenates of bovine retinas.     

Determination of monoenoic fatty acid double bond position by permanganate-periodate oxidation followed by high-performance liquid chromatography of carboxylic acid phenacyl esters

This investigation was carried out to develop methods for a reverse-phase, high-performance liquid chromatography analysis of the monocarboxylic and dicarboxylic acids produced by permanganate-periodate oxidation of monoenoic fatty acids. Oxidation reactions were performed using [U-14C]oleic acid and [U-14C]oleic acid methyl ester in order to measure reaction yields and product distributions. The 14C-labeled oxidation products consisted of nearly equal amounts of monocarboxylic and dicarboxylic acid (or dicarboxylic acid monomethyl ester), with few side products (yield greater than 98%). Conversion of the carboxylic acids to phenacyl esters proceeded to completion. HPLC of carboxylic acid phenacyl esters was performed using a C18 column with a linear solvent gradient beginning with acetonitrile/water (1/1) and ending with 100% acetonitrile. Excellent resolution was achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid phenacyl esters. Resolution was also achieved for all components of a mixture of C5 through C12 monocarboxylic acid phenacyl esters and C6 through C11 dicarboxylic acid monomethyl, monophenacyl esters. The resolution obtained by HPLC demonstrates that, for a wide range of monoenoic fatty acids, both products of a permanganate-periodate oxidation can be identified on a single chromatogram. Free fatty acids and fatty acid methyl esters were analyzed with equal success. Neither the oxidation nor the esterification reaction caused detectable hydrolysis of methyl ester. The method is illustrated for free acids and methyl esters of 14:1 (cis-9), 16:1 (cis-9), 18:1 (cis-6), 18:1 (cis-9), and 18:1 (cis-11).     

Arachidonic acid and related methyl ester mediate protein kinase C activation in intact platelets through the arachidonate metabolism pathways

Unlike unsaturated fatty acids, which almost fully activated purified brain protein kinase C in a phosphatidylserine- and Ca2(+)-free reaction, related methyl esters were poorly active in vitro. In contrast, methyl arachidonate was revealed to be as potent as arachidonic acid in activating protein kinase C in intact platelets. Arachidonic acid-mediated activation peaked at 20 s while methyl arachidonate-mediated activation plateaued at 2 min when both lipids were added at 50 microM. At concentrations higher than 0.3 mM, all tested unsaturated fatty acids and related methyl esters were weak activators of the enzyme, with the exception of linolenic acid and methyl linolenate which evoked strong enzyme activation. However, inhibitors of arachidonate metabolism blocked both arachidonic-acid and methyl-arachidonate-induced responses. At 5 microM arachidonic acid and methyl arachidonate, protein kinase C activation was due to a cyclooxygenase product(s) whereas at 50 microM the lipoxygenase pathway was mostly involved in the reaction. Therefore, arachidonic acid and its methyl ester activate protein kinase C in platelets mainly through action of their metabolites and eicosanoid synthesis. It is suggested that such indirect protein kinase C activation may account for the tumor-promoting activity of unsaturated fatty acids and related methyl esters.