Arginine increases the solubility of alkyl gallates through interaction with the aromatic ring

We have recently proposed the application of solubilizing effects of arginine to poorly soluble aromatic compounds for drug discovery research. In this study, we compared the solubilizing effects of arginine with those of other amino acids, salts and a surfactant using alkyl gallates as model drug substances of low aqueous solubility. The solubilizing effects of arginine on the alkyl gallates were distinct compared with those of other amino acids and salts; the effects were even greater than those achieved using a strongly chaotropic guanidinium ion. Transfer free energy of the alkyl gallates from water to arginine solution depended weakly on their dissolution free energy in water, which is in contrast to sodium dodecyl sulphate that showed strong dependence. The present results suggest that arginine solubilizes alkyl gallates through interaction with the aromatic moiety and sodium dodecyl sulphate does so by interacting with alkyl groups.     

Oxidation of benzyl alcohol by whole cells of Pichia pastoris and by alcohol oxidase in aqueous and nonaqueous reaction media

The low substrate specificity of alcohol oxidase from Pichia pastoris makes this enzyme system of potential biotechnological interest. Whole cells of Pichia pastoris are able to oxidize benzyl alcohol to benzaldehyde in aqueous reaction media. The low water solubility of the reactant and product of this bioconversion, combined with the ability of both to strongly inhibit the reaction, favor the use of nonaqueous reaction fluids. Purified alcohol oxidase was shown to function in a number of 2-phase reaction systems of varied aqueous to organic phase ratios (0.01-0.05 v/v). The apparent V(max) and K(m) were 5.26 g/Lh and 7.41 g/L respectively, for the oxidation of benzyl alcohol to benzaldehyde in hexane containing 3% aqueous phase. The volume of the aqueous phase had a strong effect on the reaction, with an aqueous: organic ratio of 3-5% found to be optimum. The enzyme could be firmly immobilized on DEAE-Biogel (Biorad) to enhance stability and biocatalyst recovery.     

Production of a perillyl alcohol dehydrogenase by site-directed mutagenesis of a benzyl alcohol dehydrogenase

Benzyl alcohol dehydrogenase from Acinetobacter calcoaceticus oxidises a wide range of aromatic and other cyclic alcohols and it has high specificity constants for these substrates, but it does not oxidise short- or long-chain aliphatic alcohols. Mutation of an active-site arginine to a histidine can switch the substrate specificity of the enzyme so that it has a very much greater preference for perillyl alcohol than for benzyl alcohol. © Rapid Science Ltd. 1998     

Arginine-aromatic interactions and their effects on arginine-induced solubilization of aromatic solutes and suppression of protein aggregation

We examine the interaction of aromatic residues of proteins with arginine, an additive commonly used to suppress protein aggregation, using experiments and molecular dynamics simulations. An aromatic-rich peptide, FFYTP (a segment of insulin), and lysozyme and insulin are used as model systems. Mass spectrometry shows that arginine increases the solubility of FFYTP by binding to the peptide, with the simulations revealing the predominant association of arginine to be with the aromatic residues. The calculations further show a positive preferential interaction coefficient, Γ(XP), contrary to conventional thinking that positive Γ(XP)'s indicate aggregation rather than suppression of aggregation. Simulations with lysozyme and insulin also show arginine's preference for aromatic residues, in addition to acidic residues. We use these observations and earlier results reported by us and others to discuss the possible implications of arginine's interactions with aromatic residues on the solubilization of aromatic moieties and proteins. Our results also highlight the fact that explanations based purely on Γ(XP), which measures average affinity of an additive to a protein, could obscure or misinterpret the underlying molecular mechanisms behind additive-induced suppression of protein aggregation.     

Binding of aromatic compounds to cornstarch polysaccharides

Sorption of aromatic compounds from aqueous solutions by cryotextures and suspensions of native cornstarches was studied by capillary gas chromatography. Acetophenone and benzyl alcohol were not sorbed by cryotropic-cornstarch gel and native-cornstarch suspension. A linear concentration dependence was found for aldehydes. Phenylethyl alcohol was characterized by a nonlinear concentration dependence. The presence of a benzene ring contributed to decreased binding (relative to the level characteristic of aliphatic compounds). The degree of binding depended considerably on the type of functional group in the aromatic compounds. Cryotextures were more potent than granules of native cornstarch in binding aromatic compounds.     

Binding of aromatic compounds to cornstarch polysaccharides

Sorption of aromatic compounds from aqueous solutions by cryotextures and suspensions of native cornstarches was studied by capillary gas chromatography. Acetophenone and benzyl alcohol were not sorbed by cryotropic-cornstarch gel and native-cornstarch suspension. A linear concentration dependence was found for aldehydes. Phenylethyl alcohol was characterized by a nonlinear concentration dependence. The presence of a benzene ring contributed to decreased binding (relative to the level characteristic of aliphatic compounds). The degree of binding depended considerably on the type of functional group in the aromatic compounds. Cryotextures were more potent than granules of native cornstarch in binding aromatic compounds.     

Reductive biotransformation by wild type and mutant strains of Saccharomyces cerevisiae in aqueous-organic solvent biphasic systems

Whole cells of Saccharomyces cerevisiae analyzed the conversion of benzaldehyde to benzyl alcohol in aqueous-organic biphasic media. Reaction rate increased dramatically as moisture content of the solvent was increased in the range 0% to 2%. The highest biotransformation rates were observed when hexane was used as organic solvent. Benzaldehyde was also converted to benzyl alcohol by a cell-free crude extract in biphasic systems containing hexane, although the rate of product formation was much lower. Mutant strains of S. cerevisiae lacking some or all of the ADH isoenzymes, ADH I, II, and III, manifested similar rates for bioconversion of benzaldehyde to benzyl alcohol in both aqueous and two-phase systems. In general, conversion rates observed in aqueous media were 2 to 3 times higher than those observed in hexane containing 2% moisture.     

Benzyl alcohol penetration into micelles, dielectric constant of the binding site, partition coefficient and high-pressure squeeze-out

The absorbance maximum, lambda max, of a local anesthetic, benzyl alcohol, is shifted to longer wavelengths when solvent polarity is decreased. The shift was approximately a linear function of the dielectric constant of the solvent. This transition in electronic spectra according to the microenvironmental polarity is used to analyze benzyl alcohol binding to surfactant micelles. A facile method is devised to estimate the micelle/water partition coefficient from the dependence of lambda max of benzyl alcohol on surfactant concentrations. The effective dielectric constants of the sodium decyl sulfate, dodecyl sulfate and tetradecyl sulfate micelles were 29, 31 and 33, respectively. The partition coefficient of benzyl alcohol between the micelles and the aqueous phase was 417, 610 and 1089, respectively, in the mole fraction unit. The pressure dependence of the partition coefficient was estimated from the depression of the critical micelle concentration of sodium dodecyl sulfate by benzyl alcohol under high pressure up to 200 MPa. High pressure squeezed out benzyl alcohol molecules from the micelle until about 120 MPa, then started to squeeze in when the pressure was further increased. The volume change of benzyl alcohol by transfer from the aqueous to the micellar phase was calculated from the pressure dependence of the partition coefficient. The volume change, estimated from the thermodynamic argument, was 3.5 +/- 1.1 cm3.mol-1 at 298.15 K, which was in reasonable agreement with the partial molal volume change determined directly from the solution density measurements, 3.1 +/- 0.2 cm3.mol-1. Benzyl alcohol apparently solvates into the micelles close to surface without losing contact with the aqueous phase.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Substrate specificities and inhibition studies.

The apparent Km and maximum velocity values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus were determined for a range of alcohols and aldehydes and the corresponding turnover numbers and specificity constants were calculated. Benzyl alcohol was the most effective alcohol substrate for benzyl alcohol dehydrogenase. Perillyl alcohol was the second most effective substrate, and was the only non-aromatic alcohol oxidized. The other substrates of benzyl alcohol dehydrogenase were all aromatic in nature, with para-substituted derivatives of benzyl alcohol being better substrates than other derivatives. Coniferyl alcohol and cinnamyl alcohol were also substrates. Benzaldehyde was much the most effective substrate for benzaldehyde dehydrogenase II. Benzaldehydes with a single small substituent group in the meta or para position were better substrates than any other benzaldehyde derivatives. Benzaldehyde dehydrogenase II could also oxidize the aliphatic aldehydes hexan-1-al and octan-1-al, although poorly. Benzaldehyde dehydrogenase II was substrate-inhibited by benzaldehyde when the assay concentration exceeded approx. 10 microM. Benzaldehyde dehydrogenase II, but not benzyl alcohol dehydrogenase, exhibited esterase activity with 4-nitrophenyl acetate as substrate. Both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were inhibited by the thiol-blocking reagents iodoacetate, iodoacetamide, 4-chloromercuribenzoate and N-ethylmaleimide. Benzyl alcohol or benzaldehyde respectively protected against these inhibitions. NAD+ also gave some protection. Neither benzyl alcohol dehydrogenase nor benzaldehyde dehydrogenase II was inhibited by the metal-ion-chelating agents EDTA, 2,2'-bipyridyl, pyrazole or 2-phenanthroline. Neither enzyme was inhibited by a range of plausible metabolic inhibitors such as mandelate, phenylglyoxylate, benzoate, succinate, acetyl-CoA, ATP or ADP. Benzaldehyde dehydrogenase II was sensitive to inhibition by several aromatic aldehydes; in particular, ortho-substituted benzaldehydes such as 2-bromo-, 2-chloro- and 2-fluoro-benzaldehydes were potent inhibitors of the enzyme.     

Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

Photocatabolism of Aromatic Compounds by the Phototrophic Purple Bacterium Rhodomicrobium vannielii

The phototrophic purple non-sulfur bacterium Rhodomicrobium vannielii grew phototrophically (illuminated anaerobic conditions) on a variety of aromatic compounds (in the presence of CO₂). Benzoate was universally photocatabolized by all five strains of R. vannielii examined, and benzyl alcohol was photocatabolized by four of the five strains. Catabolism of benzyl alcohol by phototrophic bacteria has not been previously reported. Other aromatic substrates supporting reasonably good growth of R. vannielii strains were the methoxylated benzoate derivatives vanillate (4-hydroxy-3-methoxybenzoate) and syringate (4-hydroxy-3,5-dimethoxybenzoate). However, catabolism of vanillate and syringate led to significant inhibition of bacteriochlorophyll synthesis in R. vannielii cells, eventually causing cultures to cease growing. No such effect on photopigment synthesis in cells grown on benzoate or benzyl alcohol was observed. Along with a handful of other species of anoxygenic phototrophic bacteria, the ability of the species R. vannielii to photocatabolize aromatic compounds indicates that this organism may also be ecologically significant as a consumer of aromatic derivatives in illuminated anaerobic habitats in nature.     

Solution properties of sulfated monohydroxy bile salts. Relative insolubility of the disodium salt of glycolithocholate sulfate

Physical-chemical properties of the major sulfated monohydroxy bile salts of man are described. In general, the sulfates are significantly more water-soluble than the non-sulfated species as a result of lower critical micellar temperatures, high aqueous monomeric solubilities and critical micellar concentrations. Nevertheless, at 37 degrees C the disodium salt of glycolithocholate sulfate, the major monohydroxy bile salt of man is not more soluble than its non-sulfated form. Since aqueous solubility correlates inversely with the cholestatic potential of bile salts, our results suggest that this sulfate may be potentially hepatoxic. Micellar solubility of phosphatidylcholine and cholesterol by the majority of non-sulfated and sulfated monohydroxy bile salts is slight. Nonetheless, phosphatidylcholine is very well solubilized by taurolithocholate sulfate but cholesterol solubility is not increased appreciably. Cholesterol saturation in model bile systems of taurochenodeoxycholate and phosphatidylcholine is impaired by the addition of sulfated lithocholate conjugates but with physiological bile salt compositions this reduction is not significant.     

Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization.

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.     

Anaerobic oxidation of toluene (analogues) to benzoate (analogues) by whole cells and by cell extracts of a denitrifying Thauera sp.

Toluene and related aromatic compounds can be mineralized to CO₂ under anoxic conditions. Oxidation requires new dehydrogenase-type enzymes and water as oxygen source, as opposed to the aerobic enzymatic attack by oxygenases, which depends on molecular oxygen. We studied the anaerobic process in the denitrifying bacterium Thauera sp. strain K172. Toluene and a number of its fluoro-, chloro- and methyl-analogues were transformed to benzoate and the respective analogues by whole cells and by cell extracts. The transformation of xylene isomers to methylbenzoate isomers suggests that xylene degradation is similarly initiated by oxidation of one of the methyl groups. Toluene oxidation was strongly, but reversibly inhibited by benzyl alcohol. The in vitro oxidation of the methyl group was coupled to the reduction of nitrate, required glycerol for activity, and was inhibited by oxygen. Cells also contained benzyl alcohol dehydrogenase (NAD⁺), benzaldehyde dehydrogenase (NADP⁺), benzoate-CoA ligase (AMP-forming), and benzoyl-CoA reductase (dearomatizing). The toluene-oxidizing activity was induced when cells were grown anaerobically with toluene and also with benzyl alcohol or benzaldehyde, suggesting that benzyl alcohol or benzaldehyde acts as inducer. The other enzymes were similarly active in cells grown with toluene, benzyl alcohol, benzaldehyde, or benzoate. This is the first in vitro study of anaerobic oxidation of an aromatic hydrocarbon and of the whole-cell regulation of the toluene-oxidizing enzyme.Dedicated to Prof. Achim Trebst     

Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

Summary A simple method for the synthesis of several amino acid benzyl esterp-toluenesulfonate salts from the corresponding amino acid and benzyl alcohol in presence ofp-toluenesulfonic acid accelerated with microwave irradiation is described. Under similar condition, the amino acid benzyl ester hydrochloride salts have also been obtained by using thionyl chloride instead ofp-toluenesulfonic acid in good yield and purity.     

Metabolism of benzyl alcohol via catechol ortho-pathway in methylnaphthalene-degrading Pseudomonas putida CSV86

Pseudomonas putida CSV86 metabolizes 1- and 2-methylnaphthalene through distinct catabolic and detoxification pathways. In spite of the similarity in the steps involved in the methylnaphthalene detoxification and the toluene side-chain hydroxylation pathways, the strain failed to utilize toluene or xylenes. However, it could grow on benzyl alcohol, 2- and 4-hydroxybenzyl alcohol. Metabolic studies suggest that the benzyl alcohol metabolism proceeds via the benzaldehyde, benzoate, and catechol ortho-cleavage pathway, in contrast to the well established catechol meta-cleavage pathway. Carbon source-dependent enzyme activity studies suggest that the degradation of aromatic alcohol involves two regulons. Aromatic alcohol induces the upper regulon, which codes for aromatic alcohol- and aromatic aldehyde-dehydrogenase and converts alcohol into acid. The aromatic acid so generated induces the specific lower regulon and is metabolized via either the ortho- or the meta-cleavage pathway. CSV86 cells transform 1- and 2-methylnaphthalene to 1- and 2-hydroxymethyl naphthalene, which are further converted to the respective naphthoic acids due to the basal level expression and broad substrate specificity of the upper regulon enzymes.     

Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

A simple method for the synthesis of several amino acidbenzyl ester p-toluenesulfonate salts from thecorresponding amino acid and benzyl alcohol in presence of p-toluenesulfonic acid accelerated with microwave irradiation isdescribed. Under similar condition, the amino acid benzyl esterhydrochloride salts have also been obtained by using thionylchloride instead of p-toluenesulfonic acid in good yieldand purity.     

Bioreversible phosphate protective groups: Synthesis and stability of model acyloxymethyl phosphates

Six bis(acyloxymethyl) esters of phenyl phosphate and benzyl phosphate and three acyloxymethylbenzylphenyl phosphates were prepared. Debenzylation of benzyl analogs gave respective free acids which were isolated as cyclohexylammonium salts. Stability of bis(acyloxymethyl)phenyl phosphates were studied in various aqueous buffers, hog liver esterase, and mice plasma.     

Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenylboron in organic solvents

1. The method is based on the observation that choline esters and sodium tetraphenylboron (Kalignost) form complexes that are insoluble in water but soluble in organic solvents such as nitriles, higher ketones and benzyl alcohol. 2. The extraction procedure is an example of liquid cation exchange where tetraphenylboron is the cation-exchange group. 3. The proportion of choline esters extracted depends on the type and total amount of cation in the aqueous phase and the amount of sodium tetraphenylboron in the organic solvent. 4. The proportion of choline esters extracted is independent of the choline ester concentration, the pH (between 8 and 3) and the relative volumes of the two phases. 5. The affinity of sodium tetraphenylboron for choline esters increases with an increase in the size of the acyl group. 6. The choline ester extracted can be released into an aqueous solution by treatment with strong acids, silver salts and anion-exchange resins.     

Solubilization of cholesterol and polycyclic aromatic compounds into sodium bile salt micelles (part 2)

The aqueous solubility of cholesterol was determined over the temperature range from 288.2 to 318.2 K with intervals of 5 K by the enzymatic method. The solubility was (3.7+/-0.3)x10(-8) mol dm(-3) (average +/- S.D.) at 308.2 K. The maximum additive concentrations of cholesterol into the aqueous micellar solutions of sodium deoxycholate (NaDC), sodium ursodeoxycholate (NaUDC), and sodium cholate (NaC) were spectrophotometrically determined at different temperatures. The cholesterol solubility increased in the order of NaUDC相似文献