A method for the estimation of thiol esters.

1. A method is described for the estimation of thiol ester groups. The thiol ester is converted into the corresponding thiol by reaction with ammonia; the thiol is then titrated amperometrically with mercuric chloride. 2. The method may be used in the presence of SH and S.S groups. The SH groups are titrated at pH3 in the presence of excess of chloride; under these conditions thiol esters do not react with mercuric chloride. Thiol ester plus thiol is then estimated by titration after reaction with ammonia. Finally, titration after reaction with ammonia and sulphite gives the thiol ester plus thiol plus disulphide. 3. The procedure has been applied to glyceraldehyde phosphate dehydrogenase. The enzyme was found to contain 15-16 SH groups/mol. and no S.S groups. After reaction with acetyl phosphate 1.8-3.5 thiol ester groups were detected, the number depending on the conditions of acetylation. In the absence of bound NAD, the number of thiol ester groups formed was 1.8/mol., although a value of 2.9 labile acetyl groups/mol. was given by the method of Lipmann & Tuttle (1945). The presence of thiol ester groups in the S-(d-3-phosphoglyceryl)-enzyme was also demonstrated.     

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.     

An enzymatic method for the histochemical localization of free and esterified cholesterol separately

Synopsis An enzymatic method for the histochemical localization of cholesterol is presented. It makes possible the localization of free cholesterol, cholesterol esters, or both and is compatible with routine histological staining procedures. The method is based on the production of H₂O₂ from free cholesterol by cholesterol oxidase. Sites of peroxide production are visualized by a brown reaction product formed in a peroxidase-catalysed reaction between diaminobenzidine and H₂O₂. cholesterol esters can be demonstrated as cholesterol after hydrolysis by cholesterol ester hydrolase. Some examples of the application of the method are given.     

A rapid method for the preparation of amino acid resin esters for Merrifield solid-phase peptide synthesis

An improved method for the preparation of Merrifield resin esters is presented. This method is rapid, is free of racemization, and is not complicated by a quaternization side reaction. Chloromethylated resin beads, t-butoxycarbonyl amino acid, and potassium t-butoxide are heated at 80 °C in dimethylsulfoxide for one-half hour to yield resin esters of suitable substitution for solid-phase petide synthesis. All twenty of the BOC protected common amino acids were esterified to the resin by this method. Resin substitution values lie in the range of 0.13 meq/g (BOC-Glu (NH₂)) to 0.66 meq/g (BOC/Pro), with most of the amino acids yielding 0.3–0.4 meq/g (on a resin containing 0.8 meq Cl/g).     

An improved method for the synthesis of 2-arachidonoylglycerol

2-Arachidonoyl glycerol (2-AG) is an endogenous agonist for cannabinoid receptors and has exhibited various biological activities. In this study, we reported an improved method for the synthesis of 2-AG by enzymatic ethanolysis of arachidonic acid-rich oil. The effects of solvent type, addition amounts of solvent and lipase, and reaction time on the content of 2-monoacylglycerols (2-MAGs) in the crude reaction mixture were investigated. Under the optimal conditions, 34.1% (area/area) 2-MAGs were produced in the crude mixture. 2-MAGs were obtained at 98–99% purity and 85.3% 2-MAGs yield (mol/mol) after solvent fractionation to fully remove impurities, including diacylglycerols (DAGs), triacylglycerols (TAGs) and fatty acid ethyl esters. Subsequently, pure 2-MAGs were further purified by crystallization in hexane to remove all saturated and partially unsaturated 2-MAGs to enrich 2-AG. After a two-step purification, 79.4% 2-AG was obtained at 42.9% 2-MAGs yield. Compared to previous methods for the synthesis of 2-AG, the method reported herein is easier to scale, greener, simpler and more economical.     

Improvement of a process for purification of tocopherols and sterols from soybean oil deodorizer distillate

Soybean oil deodorizer distillate (SODD) is a useful material for purification of tocopherols and phytosterols (referred to as sterols). The SODD was first distilled, and the two substances were enriched. The preparation, which mainly contained free fatty acids (FFAs), sterols, and tocopherols, was named SODD tocopherols/sterols concentrate (SODDTSC). If sterols are converted to steryl esters and FFAs are converted to fatty acid methyl esters (FAMEs), relatively easy purification of tocopherols and steryl esters can be achieved because the boiling points of FAMEs, tocopherols, and steryl esters are different significantly. Hence, the development of a new two-step in situ reaction system was tried out for esterification of sterols with FFAs (first step) and esterification of FFAs with methanol (MeOH) (second step). A mixture of SODDTSC/water (95:5, w/w) and 250 units (U)/g-mixture of Candida rugosa lipase was prepared beforehand for the first-step reaction, and was agitated at 40 °C for 24 h with dehydration at 20 mmHg. Sterols were efficiently esterified, and the degree of esterification reached 95%. To the reaction mixture were added 7 M amounts of MeOH against unreacted FFAs, 20 wt.% water, and 25 U/g-mixture of Alcaligenes sp. lipase. The second-step reaction was then conducted at 30 °C for 20 h. Consequently, 95% FFAs were converted to FAME, and steryl esters synthesized by the first-step reaction were not reconverted to free sterols. Finally, SODDTSC (1.5 kg) was subjected to this two-step in situ reaction, and tocopherols and steryl esters were purified from the reaction mixture by short-path distillation. Tocopherols were purified to 72% (yield, 88%) and steryl esters were purified to 97% (yield, 97%).     

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.     

A novel lipase/acyltransferase from the yeast Candida albicans: expression and characterisation of the recombinant enzyme

A gene encoding an extracellular lipase (CaLIP4) from Candida albicans was successfully expressed in Saccharomyces cerevisiae after mutagenesis of its unusual CUG serine codon into a universal one. The ability of this lipase, which shares 60% sequence homology with the lipase/acyltransferase from Candida parapsilosis, to synthesise esters was investigated. CaLIP4 behaved as a true lipase, displaying activity towards insoluble triglycerides and having no activity in the presence of short-chain fatty acid (FA) esters and phosphatidylcholine. Methyl, ethyl and propyl esters were efficiently used. The lipase exhibited highest selectivity for unsaturated FA. With saturated FAs, C14–C16 acyl chains were preferred. In a biphasic aqueous/lipid system, CaLIP4 displayed a high alcoholysis activity with a range of alcohols (e.g. methanol, ethanol, propanol and isopropanol) as acyl acceptor. During the course of the alcoholysis reaction, new esters are produced at concentrations above the thermodynamic equilibrium of the esterification reaction, indicating that ester synthesis does not proceed by esterification but mainly by direct acyltransfer. Ester synthesis is under kinetic control due to the high rate of alcoholysis. Unwanted hydrolysis is limited by competition between the acyl acceptor (alcohol) and water for the acyltransfer reaction, favouring the alcohol.     

A new method for improving the enantioselectivity of lipase-catalyzed hydrolysis in organic solvent containing a small amount of water in the presence of metal ions

The hydrolysis of racemic butyl 2-(4-substituted phenoxy)propionates having various substituents catalyzed by lipase MY from Candida rugosa was achieved in di-isopropyl ether containing 0.75% (v/v) of 2.4 M LiCl or 1.2 M MgCl₂ aqueous solution. Water molecules hydrated to the metal ion in isopropyl ether acted as a nucleophile to cause the hydrolysis of these esters as with water alone. Metal ions used significantly enhanced their enantioselectivities by 100-fold or above, as compared with the ordinary reaction media.     

Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein

In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

Racemization in the Coupling Reaction,Pro-Val+Pro,with the Activated Ester Methods

The degree of racemization in the several activated ester methods of the peptide synthesis was measured in using the critical racemization test, Pro-Val+Pro, with help of gas chromatography. The results were compared with that in the coupling reaction, Leu-Phe+Val, in which no racemization had been reported in the corresponding reaction conditions by F. Weygand et al., when the activated dipeptide esters had been prepared from Z-Leu+Phe-activated esters. The significantly higher racemization was observed in the methods of N-hydroxypiperidine ester and thiophenyl ester, even when the activated dipeptide esters were prepared from Z-Pro+Val-activated esters. On the other hand, almost no racemization was observed in the N-hydroxysuccinimide ester and p-nitrophenyl ester methods. A great extent of the racemization was detected when the activated dipeptide esters were prepared directly from Z-Pro-Val-OH.     

Optimization of alkali-catalyzed transesterification of Pongamia pinnata oil for production of biodiesel

Studies were carried out on transesterification of Karanja oil with methanol for the production of biodiesel. The reaction parameters such as catalyst concentration, alcohol/oil molar ratio, temperature, and rate of mixing were optimized for production of Karanja oil methyl ester (KOME). The fatty acid methyl esters content in the reaction mixture were quantified by HPLC and 1H NMR method. The yield of methyl esters from Karanja oil under the optimal condition was 97-98%.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

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.     

Investigation of the chiral recognition ability of human carboxylesterase 1 using indomethacin esters

Human carboxylesterase 1 (hCES1) is an enzyme that plays an important role in hydrolysis of pharmaceuticals in the human liver. In this study, elucidation of the chiral recognition ability of hCES1 was attempted using indomethacin esters in which various chiral alcohols were introduced. Indomethacin was condensed with various chiral alcohols to synthesize indomethacin esters. The synthesized esters were hydrolyzed with a human liver microsome (HLM) solution and a human intestine microsome (HIM) solution. High hydrolytic rate and high stereoselectivity were confirmed in the hydrolysis reaction in the HLM solution but not in the HIM solution, and these indomethacin esters were thought to be hydrolyzed by hCES1. Next, these indomethacin esters were hydrolyzed in recombinant hCES1 solution and the hydrolysis rates of the esters were calculated. The stereoselectivity confirmed in HLM solution was also confirmed in the hCES1 solution. In the hydrolysis reaction of esters in which a phenyl group is bonded next to the ester, the V_max value of the (R) form was 10 times larger than that of the (S) form.     

Glycerol-derived ester oligomers from cork suberin

The cork suberin polyester was partially depolymerized by a methanolysis reaction catalyzed by calcium hydroxide. The methanolisate was analysed by ESI-MS/MS in the form of [M+Li](+) adduct-ions. This reaction solubilized a mixture of monomers and oligomers, including a set of glycerol-derived dimeric and trimeric esters. Four types of glycerol esters were identified: monoacylglycerols of alpha,omega-diacids, of omega-hydroxyacids and of monoacids; diglycerol diesters of alpha,omega-diacids; diacylglycerols of alpha,omega-diacids; monoacylglycerols of linear dimeric esters of alpha,omega-diacids and omega-hydroxyacids. The alpha,omega-diacids and omega-hydroxyacids found as monomer residues in the glycerol esters are the main ones found as cork suberin monomers. It is concluded that suberin is a glycerol-derived lipid of polymeric dimensions. Due to the protective and insulating role that it plays in plants, suberin should be considered together with the other known glycerolipids that build up biological membranes.     

A gas chromatographic method for the quantitative detemination of hexuronic acids in alginic acid

A method has been developed for the quantitative determination of the relative proportions of d-mannuronic and l-guluronic acids in alginic acid. To obtain homogeneous reaction conditions the viscosity of the alginic acid sample was first decreased by limited hydrolysis with mineral acid. The carboxyl groups were then esterified by reaction with 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide, and reduced with sodium borohydride. The resulting hexosans were converted by acid hydrolysis to d-mannose and an equilibrium mixture of l-gulose and 1,6-anhydro-l-gulose. These were treated with sodium borohydride; the 1,6-anhydro-l-gulose was not reduced whereas d-mannose and l-gulose were converted to d-mannitol and d-glucitol. The hexitols were estimated by gas-liquid chromatography as the n-butane boronic acid esters, and the relative proportions of the uronic acids in the alginic acid were calculated by taking into account the equilibrium ratio of l-gulose and 1,6-anhydro-l-gulose. The method can be used to analyze as little as 2 mg of alginic acid.     

Use of the excluded protecting group (EPG) method for peptide synthesis.

The excluded protecting group (EPG) method has been used for the solution synthesis of several peptides including Merrifield's Model Tetrapeptide, linear antamanide and an analogue of magainin-1, [Ala(19), Asn(22)]magainin-1. In the approach reported, the C-terminal amino acid is esterified to the 2-position of cholestane as the [2s,3s]iodohydrin ester and the penultimate amino acid added to the aminoacyl-steroid as the Fmoc-pentafluorophenyl-ester. The Fmoc group is removed with Et(2)NH/DMF ( approximately 15% v/v) and, after evaporation to approximately 10 mL, the solution chromatographed on Sephadex LH-20 in DMF. The dipeptidyl-steroid elutes as the free amine well separated from other reaction mixture components. Fractions containing the dipeptide, as determined by counting and TLC, are pooled and reacted with the next Fmoc-amino acid-pentafluorophenyl ester in the sequence. Repetition of the deprotection/purification/reaction cycle yields the fully protected peptide.On completion of the synthesis, the cholestane iodohydrin ester is selectively removed by treatment with Zn degrees /AcOH to yield the peptide with intact alpha-amino and side chain protecting groups. Global deprotection is achieved with HF. All intermediates from the syntheses reported were characterized. The magainin analogue was shown to have full biologic activity. The Fmoc iodohydrin esters of 16 of the 20 proteogenic amino acids have been prepared and characterized for use as the C-terminal amino acids in other EPG syntheses.     

Substrate selectivity of imidazole-appended dimethyl-β-cyclodextrin

The substrate selectivity of the hydrolysis reaction by imidazole-appended dimethyl-β-cyclodextrin (2) was studied using different kinds of p-nitrophenyl esters of amino acids. The tendency for the hydrolysis reaction of the amino acid ester by 2 reflected a difference in K_m rather than in k_cat. 2 had the ability to undergo a stereo-selective hydrolysis reaction.