Mutagen Formation in the Reaction of Nitrite with the Food Components Analogous to Sorbic Acid

Mutagen formation by the reaction of sodium nitrite with some sorbic acid analogs was investigated by using the microbial mutagenicity tests (rec-assay and Ames test) together with chemical examination using TLC, and it became clear that the conjugated dienoic carbonyl structure is essential for mutagen formation by the reaction with nitrite By a large scale reaction of sodium nitrite with sorbic acid methyl ester, 5-nitro-2,4-hexadienoic acid methyl ester and ethylnitrohc acid were isolated and identified as the mam mutagens The results led to the assumption that a nitro group adjacent to the double bond is an important factor to develop mutagenicity     

Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of reduced nicotinamide-adenine dinucleotide in the presence of formylphenylacetic acid ethyl ester.

The oxidase-peroxidase from Datura innoxia which catalyses the oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid was also found to catalyse the oxidation of NADH in the presence of Mn2+ and formylphenylacetic acid ethyl ester. NADH was not oxidized in the absence of formylphenylacetic acid ethyl ester, although formylphenylacetonitrile or phenylacetaldehyde could replace it in the reaction. The reaction appeared to be complex and for every mol of NADH oxidized 3-4 g-atoms of oxygen were utilized, with a concomitant formation of approx. 0.8 mol of H2O2, the latter being identified by the starch-iodide test and decomposition by catalase. Benzoylformic acid ethyl ester was also formed in the reaction, but in a nonlinear fashion, indicating a lag phase. In the absence of Mn2+, NADH oxidation was not only very low, but itself inhibited the formation of benzoylformic acid ethyl ester from formylphenylacetic acid ethyl ester. A reaction mechanism for the oxidation of NADH in the presence of formylphenylacetic acid ethyl ester is proposed.     

Formate ester formation in amide solutions

Simple aliphatic alcohols, deoxynucleosides and nucleosides undergo reaction with formamide yielding formate esters. Formate ester formation was observed to occur slowly at 100°C and more rapidly at 130°C. As expected, formate esters were hydrolyzed to the alcohol and formic acid upon heating in aqueous solution. It was proposed to study the possibility that formate esters are formed initially in amide solvents, followed by displacement of formate by dihydrogen phosphate ion to form monophosphate esters. Experiments are described which demonstrate the formation and hydrolysis of formate esters, as well as their lack of reaction with hydrogen phosphate ion. Formate esters are not intermediates in the phosphorylation of nucleosides in formamide. Their formation has been observed and such an esterification is a side reaction during the phosphorylation of nucleosides in formamide.     

Linear and cyclic ester Oligomers of succinic acid and 1,4-butanediol: Biocatalytic synthesis and characterization

Abstract

The lipase-catalyzed synthesis of cyclic ester oligomers from non-activated succinic acid (A) and 1,4-butanediol (B) in the presence of immobilized Candida antarctica lipase B was investigated. Batch and pulse fed-batch systems were implemented to increase the formation of cyclic ester products. The substrate conversions after 24 h were 86% and 95% under batch and fed-batch operation, respectively and the product of the reaction was, for both systems, a mixture of cyclic (CEOs) and linear (LEOs) ester oligomers. Fed-batch operation afforded a product containing 71% cyclic ester oligomers (CEOs) as compared with only 52% CEOs in batch operation. Cyclic ester oligomers accumulated as the reaction progressed, with the dimer CEO₁ the most predominant product (i.e. 50% of the total products formed in fed-batch operation). The pulse fed-batch operation system was superior to the batch operation not only because higher substrate conversion and more CEOs were obtained, but also because it resulted in products with a higher degree of polymerization (DP up to 7). Cyclic ester oligomers are produced from the early stage of the reaction simultaneously with the linear ester oligomers by a ring-closure reaction on the active site of the enzyme, and not as a result of ring-chain equilibria.     

Enhancement by catechols of hydroxyl-radical formation in the presence of ferric ions and hydrogen peroxide

The effect of caffeic acid, a kind of catechol, on the Fenton reaction was examined by using the ESR spin trapping technique. Caffeic acid enhanced the formation of hydroxyl radicals in the reaction mixture, which contained caffeic acid, hydrogen peroxide, ferric chloride, EDTA, and potassium phosphate buffer. Chlorogenic acid, which is an ester of caffeic acid with quinic acid, also stimulated the formation of the hydroxyl radicals. Quinic acid did not stimulate the reaction, suggesting that the catechol moiety in chlorogenic acid is essential to the enhancement of the hydroxyl-radical formation. Indeed, other catechols and related compounds such as pyrocatechol, gallic acid, dopamine, and noradrenaline effectively stimulated the formation of the hydroxyl radicals. The above results confirm the idea that the catechol moiety is essential to the enhancement. Ferulic acid, 4-hydroxy-3-methoxybenzoic acid, and salicylic acid had no effect on the formation of the hydroxyl radicals. The results indicate that the enhancement by the catechols of the formation of hydroxyl radicals is diminished if a methyl ester is formed at the position of the hydroxyl group of the catechol. In the absence of iron chelators such as EDTA, DETAPAC, desferrioxamine, citrate, and ADP, formation of hydroxyl radicals was not detected, suggesting that chelators are essential to the reaction. The enhancement of the formation of hydroxyl radicals is presumably due to the reduction of ferric ions by the catechols. Thus, the catechols may exert deleterious effects on biological systems if chelators such as EDTA, DETAPAC, desferrioxamine, citrate, and ADP are present.     

Tryptophanamide formation by Escherichia coli tryptophanyl-tRNA synthetase

When tryptophanyl-tRNA synthetase from Escherichia coli is allowed to react with L-tryptophan and ATP-Mg in the presence of inorganic pyrophosphatase, the fluorescence change of the reaction mixture reveals three or four sequential processes, depending on the buffer used. Quenched-flow and stopped-flow experiments show that the first two processes, which occur in the 0.001-1.0-s time scale, can be correlated to the formation of two moles of tryptophanyl-adenylate per mole of dimeric enzyme. These two processes are reversible by adding PPi, as seen in the fluorimeter. The third process leads to a reaction product that can no longer reform ATP after addition of PPi and that represents tryptophanyl-ATP ester, as demonstrated by thin-layer chromatography. This compound has been previously shown to be formed by tryptophanyl-tRNA synthetase from E. coli [K. H. Muench (1969) Biochemistry 8, 4872-4879]. Its formation is accompanied by a fluorescence decrease which reaches a minimum in about 30 min. The nature of the fourth process depends on the reaction conditions employed. In sodium bicarbonate or potassium phosphate buffer, the fourth process corresponds to the non-enzymatic hydrolysis of tryptophanyl-ATP ester. This spontaneous hydrolysis competes with formation of the ester and limits its concentration. Eventually, the progressive exhaustion of ATP brings the fluorescence intensity of the reaction mixture back to its initial value. In contrast, in ammonium bicarbonate buffer the previous third process is no longer visible, as evidenced by the absence of a fluorescence decrease beyond the fast initial quenching linked to the formation of tryptophanyl-adenylate. Instead, a fluorescence increase is observed. However, unlike the fourth process seen in sodium bicarbonate buffer, the fluorescence increase in ammonium bicarbonate is much larger than the initial fluorescence decrease linked to adenylate formation, the final fluorescence greatly surpassing the starting fluorescence signal. The reaction product of this process is tryptophanamide, as evidenced by high-performance liquid chromatography. Tryptophanamide formation is faster than that of tryptophanyl-ATP ester and is enzyme-catalyzed with a Km of 1 mM for ammonia and a rate constant of 5.7 min-1 at pH 8.3, 25 degrees C. The affinity of tryptophanamide for the protein is too weak to allow the formation of a significant concentration of enzyme-product complex. Tryptophanamide is therefore released in the reaction medium and its concentration reaches that of the limiting substrate.     

Oxidation of the substituted catechols dihydroxyphenylalanine methyl ester and trihydroxyphenylalanine by lactoperoxidase and its compounds

The reactions of native lactoperoxidase and its compound II with two substituted catechols have been investigated by ESR spin stabilization and spin trapping and by rapid scan and conventional spectrophotometric techniques. The catechols are Dopa methyl ester (dihydroxyphenylalanine methyl ester) and 6-hydroxy-Dopa (trihydroxyphenylalanine). o-Semiquinone radicals are formed in the anaerobic reaction of Dopa methyl ester with hydrogen peroxide catalyzed by native lactoperoxidase. The comparable anaerobic reaction of 6-hydroxy-Dopa appears to produce hydroxyl radicals in an unusual reaction. Compound II is reduced back to native lactoperoxidase by both catechols. The reaction between Dopa methyl ester and compound II undergoes an oscillation. The results on the overall lactoperoxidase cycle indicate two successive one-electron reductions of the peroxidase intermediates back to the native enzyme. The resulting free radical formation of o- and p-semiquinones and subsequent formation of stable quinones and Dopachromes is dependent upon the stereochemical arrangement of the catechol hydroxyl groups.     

Enzymatic transesterification in a solvent-free system: synthesis of sn-2 docosahexaenoyl monoacylglycerol

Abstract

The enzymatic transesterification of docosahexaenoic acid (DHA) ethyl ester with glycerol was carried out by using several immobilized lipases in a solvent-free system. This reaction involves the initial formation of sn-2 docosahexaenyl monoacylglycerol. This DHA derivative is highly relevant for improving the bioavailability of DHA and it has received increasing interest in the field of nutrition. Three commercial lipases, from Rhizomucor miehei (RML), Alcaligenes sp. (AQ) and Candida antarctica-fraction B (CALB) were immobilized by interfacial adsorption on a commercial hydrophobic support (a methacrylate resin containing octadecyl groups, Sepabeads C-18) and tested for glycerolysis of DHA ethyl ester. In certain cases (e.g. immobilized CALB), the transesterification reaction continues to the formation of triacylglycerol (80%) by using a very high excess of DHA ethyl ester ((115 mmols versus 1.24 mmols of glycerol and high temperatures (50?°C). However, the same biocatalyst working at lower temperatures, 37?°C, synthetizes a 90% of sn-2 monoacylglycerol even in the presence of that a high excess of DHA ethyl ester. Interestingly, immobilized RML derivative synthesizes a 98% of sn-2 monoacylglyceride (2-MG) in 15?min at 37?°C with a 4% of immobilized biocatalyst. These high activity and regioselectivity under very mild reaction conditions are very interesting for the thermal oxidative stability of the omega-3 fatty acid as well as for the thermal stability of the biocatalyst. Using Normal Phase HPLC-ELSD and accurate commercial markers, the formation of the 2-MG was confirmed.     

Nucleophilic addition to the 9 position of 9-phenylcarboxylate-10-methylacridinium protects against hydrolysis of the ester

The chemiluminescent reaction of an acridinium ester (AE) requires addition of peroxide to the 9 position of the acridinium ring. The addition of a hydroxide ion to the 9 position of an acridinium ester to form the carbinol adduct has also been well documented. We have observed a similar addition of other nucleophiles to the acridinium ring to form an acridan adduct. The adduct formed with bisulphite has been particularly well-characterized for rate of formation, rate of reversion, and reaction equilibrium. The formation of an adduct (other than H₂O₂) has been demonstrated to decrease significantly the reactivity of the adjacent ester bond to alkaline hydrolysis. The resulting, more stable adduct is very useful when the acridinium ester is used as a label in DNA probe-based assays. The adduct is highly resistant to hydrolysis under the conditions often desired for DNA probe-based assays (high temperature, elevated pH, extended storage).     

Modulating aroma compounds during wine fermentation by manipulating carnitine acetyltransferases in Saccharomyces cerevisiae

The wine yeast Saccharomyces cerevisiae is central in the production of aroma compounds during fermentation. Some of the most important yeast-derived aroma compounds produced are esters. The esters ethyl acetate and isoamyl acetate are formed from alcohols and acetyl-CoA in a reaction catalysed by alcohol acetyltransferases. The pool of acetyl-CoA available in yeast cells could play a key role in the development of ester aromas. Carnitine acetyltransferases catalyse the reversible reaction between carnitine and acetyl-CoA to form acetylcarnitine and free CoA. This reaction is important in transferring activated acetyl groups to the mitochondria and in regulating the acetyl-CoA/CoA pools within the cell. We investigated the effect of overexpressing CAT2, which encodes the major mitochondrial and peroxisomal carnitine acetyltransferase, on the formation of esters and other flavour compounds during fermentation. We also overexpressed a modified CAT2 that results in a protein that localizes to the cytosol. In general, the overexpression of both forms of CAT2 resulted in a reduction in ester concentrations, especially in ethyl acetate and isoamyl acetate. We hypothesize that overproduction of Cat2p favours the formation of acetylcarnitine and CoA and therefore limits the precursor for ester production. Carnitine acetyltransferase expression could potentially to be used successfully in order to modulate wine flavour.     

Enzymatic synthesis of amide surfactants from ethanolamine

The condensation of a primary amine with fatty acids has been studied to determine optimum conditions for selective formation of amide surfactants via enzymatic amidification. Monoacylated ethanolamide and the diacylated amide-ester can be isolated from the reaction mixture, but the monoacylated ester cannot be isolated. The selectivity of the reaction depends on the solubility of the intermediate amide. Continuous precipitation of this product decreases the amount of amide-ester produced. Solubility values of the desired product (amide) are reported for different conditions.In acetonitrile, the ethyl ester of the corresponding fatty acid has been used successfully to avoid formation/precipitation of the ion-pair of the precursor reagents. In this medium, use of the transacylation reaction permits one to accelerate the reaction without producing a significant change in the selectivity toward the intermediate amide. This strategy is not successful in n-hexane where the solubilities of both ethanolamine and its ion-pair with lauric acid are similar.Results obtained for high loadings of substrates have been analyzed. In n-hexane and acetonitrile, the kinetics of the direct acylation reactions are controlled by the limited solubility of the ion pair formed by the two precursor reagents For the transacylation reaction in acetonitrile, at a sustrate loading of 2 mol l(-1,) selective production of as much as 92 mole percent N-acyl ethanolamine was observed in only 1.5 h.     

Quinone methide as a new intermediate in eumelanin biosynthesis

The conversion of dopachrome to dihydroxyindole(s), a key reaction in eumelanin biosynthetic pathway, has been shown to be under the control of dopachrome conversion factor. Dopachrome conversion factor isolated from the hemolymph of Manduca sexta larvae, which is devoid of any tyrosinase activity, exhibits a narrow substrate specificity and readily bleaches the iminochromes derived from the oxidation of L-dopa, L-dopa methyl ester, and alpha-methyl-L-dopa, but failed to attack the corresponding D-isomers. The product formed in the case of L-dopachrome was identified to be 5,6-dihydroxyindole. Therefore, aromatization of dopachrome seems to accompany its decarboxylation as well. However, the enzyme also converts L-dopachrome methyl ester to an indole derivative indicating that it can deprotonate the alpha-hydrogen when the carboxyl group is blocked. These results are accounted for by the transient formation and further transformation of a reactive quinone methide intermediate during the dopachrome conversion factor-catalyzed reaction. The fact that the enzyme-catalyzed conversion of alpha-methyl dopachrome methyl ester (where both decarboxylation and deprotonation are blocked) resulted in the generation of a stable quinone methide in the reaction mixture confirms this contention and supports our recent proposal that quinone methide and not indolenine is the key transient intermediate in the conversion of dopachrome to dihydroxyindole observed during melanogenesis.     

Full model for reversible kinetics of lipase-catalyzed sugar-ester synthesis in 2-methyl 2-butanol

A kinetic model derived from the ping-pong bi-bi reversible mechanism is proposed to described the acylation of glucose by lauric acid in 2-methyl 2-butanol mediated by Candida antarctica lipase at 60 degrees C. The model accounts for the effect of all four compounds in the reaction mixture, namely lauric acid, glucose, water, and lauroyl glucose ester. A supersaturated glucose solution was used to avoid limitations by glucose dissolution rate. Experiments with varied initial water content were performed to determine the effect of water on the initial reaction rate. The full time course of ester formation is described by five parameters: (a) three parameters evaluated from initial rate measurements; (b) the equilibrium constant, independently evaluated; and (c) one extra parameter fitted to the progress curve of ester formation. This reduced form of a full reversible kinetic model based on the ping-pong bi-bi mechanism is able to describe the complete course of lauroyl glucose ester synthesis. The proposed model provides a good fit for the experimental results.     

The reduction of CrVI to CrIII by the alpha and beta anomers of D-glucose in dimethyl sulfoxide. A comparative kinetic and mechanistic study

The reduction of CrVI by alpha-D-glucose and beta-D-glucose was studied in dimethyl sulfoxide in the presence of pyridinium p-toluensulfonate, a medium where mutarotation is slower than the redox reaction. The two anomers reduce CrVI by formation of an intermediate CrVI ester precursor of the slow redox step. The equilibrium constant for the formation of the intermediate chromic ester and the rate of the redox steps are different for each anomer. alpha-D-Glucose forms the CrVI-Glc ester with a higher equilibrium constant than beta-D-glucose, but the electron transfer within this complex is slower than for the beta anomer. The difference is attributed to the better chelating ability of the 1,2-cis-diolate moiety of the alpha anomer. The CrV species, generated in the reaction mixture, reacts with the two anomers at a rate comparable with that of CrVI. The EPR spectra show that the alpha anomer forms several linkage isomers of the five-coordinate CrV bis-chelate, while beta-D-glucose affords a mixture of six-coordinate CrV monochelate and five-coordinate CrV bis-chelate. The conversion of the CrV mono- to bis-chelate is discussed in terms of the ability of the 1,2-cis- versus 1,2-trans-diolate moieties of the glucose anomers to bind CrV.     

Initial water content and lipase-mediated ester formation in hexane

Lipase biocatalysis was investigated as a tool for the production of esters by two model reactions, esterification of 1-butanol with 2-methyl-1-pentanoic acid and irreversible transesterification between 2-methyl-1-pentanol and vinyl acetate. The reactions were carried out in hexane using lipases from Candida cylindracea and porcine pancreas. The initial water content influenced both the yield of the ester and the enantioselectivity of the reaction (esterifica-tions) or the ester formation only (transesterifications).     

Biotinylated proteins as molecular weight standards on Western blots

Protein molecular weight standards were biotinylated by reaction with biotinyl-N-hydroxysuccinimide ester. The biotinylated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose paper. The resolved protein bands were detected by formation of a streptavidin-biotin/horseradish peroxidase complex and reaction with 4-chloro-1-naphthol and hydrogen peroxide. The biotinylated proteins are easy to prepare and are useful as molecular weight standards with most procedures employing immunodetection of proteins following transfer to nitrocellulose paper.     

Enzymatic reactions in aqueous-organic media. VI. Peptide synthesis by α-chymotrypsin in hydrophilic organic solvents

The peptide synthesis from N-acetyl-L-tyrosine ethyl ester and amino acid amides was realized using α-chymotrypsin as a catalyst in ethanol or acetonitrile containing small amounts of water. In these reaction systems, the precipitates of phosphate salt, which was used as a component of buffer solution, are considered to act as carriers of chymotrypsin. It was found that peptide formation is competitive with hydrolysis of the substrate ester, but the secondary synthesis of the peptide from the hydrolysate was also considered to proceed. The yield of the peptide after 24 h reaction was strongly dependent on the water concentration; maximum yields of the peptide were obtained at water concentrations below 10% (v/v). The addition of tertiary amines, such as triethyl amine, markedly increased the peptide yield, probably due to the increase in the concentration of the nucleophilic amine components by neutralization of hydrohalides of amino acid amides. The effect of reaction temperature and the reactions with CT immobilized on PVA, chitosan, or TEAE-cellulose are also described.     

Application of polyethylene glycol-modified enzymes in biotechnological processes: organic solvent-soluble enzymes

A new approach in biotechnological processes is to use enzymes modified with polyethylene glycol which has both hydrophilic and hydrophobic properties. The modified enzymes are soluble in organic solvents such as benzene, toluene and chlorinated hydrocarbons and exhibit high enzymic activities in these organic solvents. Modified hydrolytic enzymes catalysed the reverse reaction of hydrolysis in organic solvents: formation of acid—amide bonds by modified chymotrypsin, and ester synthesis and ester exchange reactions by modified lipase. Modified catalase and modified peroxidase efficiently catalyse their respective reactions in organic solvents. The results of this research indicate great potential for applications in the fields of biotechnology and enzymology.     

Kinetics of alpha-chymotrypsin catalyzed hydrolysis in equilibrium. II. Comparison of ester and amide substrates

Kinetic of the alpha-chymotrypsin catalyzed reversible hydrolytic reaction of methyl N-acetyl-L-phenylalaninate and N-acetyl-L-phenylalanylglycinamide at pH 5.5 and equilibrium conditions has been studied. The rates of the labeled reaction products incorporated into the substrate a different methanol concentrations shows that the reaction proceeds by a compulsory mechanism with the formation of N-acetyl-L-phenylalanine-alpha-chymotrypsin complex. For the amide substrate the data obtained are also in agreement with the compulsory mechanism of its hydrolysis. Equilibrium kinetics of ester and amide substrates hydrolysis has been compared.     

The structures of thymidine kinase from herpes simplex virus type 1 in complex with substrates and a substrate analogue.

Thymidine kinase from Herpes simplex virus type 1 (TK) was crystallized in an N-terminally truncated but fully active form. The structures of TK complexed with ADP at the ATP-site and deoxythymidine-5'-monophosphate (dTMP), deoxythymidine (dT), or idoxuridine-5'-phosphate (5-iodo-dUMP) at the substrate-site were refined to 2.75 A, 2.8 A, and 3.0 A resolution, respectively. TK catalyzes the phosphorylation of dT resulting in an ester, and the phosphorylation of dTMP giving rise to an anhydride. The presented TK structures indicate that there are only small differences between these two modes of action. Glu83 serves as a general base in the ester reaction. Arg163 parks at an internal aspartate during ester formation and binds the alpha-phosphate of dTMP during anhydride formation. The bound deoxythymidine leaves a 35 A3 cavity at position 5 of the base and two sequestered water molecules at position 2. Cavity and water molecules reduce the substrate specificity to such an extent that TK can phosphorylate various substrate analogues useful in pharmaceutical applications. TK is structurally homologous to the well-known nucleoside monophosphate kinases but contains large additional peptide segments.