Mobile phase compositions for ceramide III by normal phase high performance liquid chromatography

Ceramide III was prepared by the cultivation ofSaccharomyces cerevisiae. Ceramide III was partitioned from the cell extracts by solvent extraction and analyzed by Normal Phase High Performance Liquid Chromatography (NP-HPLC) using Evaporative Light Scattering Detector (ELSD). We experimentally determined the mobile phase composition to separate ceramide III with NP-HPLC. Three binary mobile phases of n-hexane/ethanol,n-hexane/Isoprophyl Alcohol (IPA) andn-hexane/n-butanol and one ternary mobile phase ofn-hexane/IPA/methanol were demonstrated. For the binary mobile phase ofn-hexane/ethanol, the first mobile phase composition, 95/5 (v/v), was step-increased to 72/23 (v/v) at 3 min. In the binary mobile phase, the retention time of ceramide III was 7.87 min, while it was 4.11 min respectively in the ternary system, where the mobile phase composition ofn-hexane/IPA/methanol, 85/7/8 (v/v/v), was step-increased to 75/10/15 (v/v/v) at 3 min. However, in the ternary mobile phase, the more peak area of ceramide III was observed.     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Repeated batch and continuous syntheses of N-(benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester with immobilized thermolysin

Summary N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine methyl ester was synthesized from N-(benzyloxycarbonyl)-l-phenylalanine and l-phenylalanine methyl ester in an aqueous solution (aqueous phasic reaction), in an aqueous/organic biphasic system (biphasic reaction), and in an organic solvent (organic phasic reaction) with immobilized thermolysin. In the aqueous phasic reaction with thermolysin immobilized on Amberlite XAD-7, the whole product was trapped inside the support; extraction with ethyl acetate was needed to recover the product, and the equilibrium yield was low (about 65%). With the biphasic and organic phasic reactions with ethyl acetate as an organic solvent, the yield was around 95%. Because of the high yield and feasibility of operation, repeated batch and continuous reactions were done in the biphasic and organic phasic systems, respectively. The half-lives of the activity for the immobilized enzyme used in the biphasic system at 40°C by repeated batch operation and in a plug flow reactor fed with substrate dissolved in ethyl acetate at 40°C and 30°C were estimated to be about 200 h (67 batches), 420 h, and 1100 h, respectively.     

Kinetics of lipase-catalyzed hydrolysis of palm oil in lecithin/izooctane reversed micelles

Candida rugosa lipase has been used to investigate the hydrolysis of palm oil in a lecithin/isooctane reversed micellar system. The reaction obeys Michaelis-Menten kinetics for the initial conditions. Kinetic parameters such as maximum rate and Michaelis constant (K _m) were determined for lipase-catalyzed hydrolysis in n-hexane and isooctane. According to the K _m values, the enzyme affinity towards the substrate was increased in isooctane. The maximum degree of hydrolysis was generally decreased as the initial substrate concentration was increased. This may suggest that the hydrolysis in lecithin reversed micelles should be regarded as a one-substrate first-order reversible reaction. It is shown in this study that the proposed one-substrate first-order kinetic model can serve for the precise prediction of the degree of hydrolysis for a known reaction time or vice versa, when the initial substrate concentration is less than 0.325 mol/dm³. A disagreement with this model was found when the initial substrate concentration was higher than approximately 0.3 mol/dm³. This may be due to the effects of the products on lipase activity or even to the conversion of the reversed micellar system to other systems. Received: 16 May 1997 / Received revision: 22 October 1997 / Accepted: 24 October 1997     

Microbial Oxidation of Tetradecanols and Related Substances in Organic Solvents

Microbial oxidations of n-tetradecane, tetradecanols and tetradecanoic acid were investigated by using intact cells of Corynebacterium equi, a hydrocarbon-assimilating bacterium, in an aqueous phase and organic solvents. The bacterial cells were hydrophobic and could be well dispersed in all organic solvents employed to give homogeneous reaction mixtures, and among them, isooctane was found to be the best for the reaction. n-Tetradecane and tetradecanoic acid were completely oxidized in the aqueous phase, but not in isooctane. In contrast, 1-tetradecanol was oxidized much more readily in isooctane than in the aqueous phase, and an oxidation product identified as myristyl myristate was accumulated in isooctane at the conversion rate of 80%. 2-Tetradecanol was also readily oxidized in isooctane, and 2-tetradecanone was obtained at the conversion rate of nearly 100%. Similar results were obtained when toluene and n-hexane were used as the solvent in place of isooctane, while no reaction was observed when chloroform was employed.     

A Study on Low-boiling Chemical Constituents of Cryptotaenia japonica Hassk

Low-boiling compounds escaping during steam distillation of Cryptotaenia japonica Hassk were collected and examined with chromatographies and by the preparation of their derivatives.

The following compounds were identified. Six paraffin hydrocarbons: n-pentane, n-hexane, n-heptane, n-octane, isooctane, n-nonane; seven carbonyl compounds: formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, isovaleraldehyde, acetone, 3-methyl-2-pentanone; three esters: n-propyl and isopropyl formates, n-propyl acetate; eight alcohols: methanol, ethanol, n-propanol, isopropanol, isobutanol, sec-butanol, isopentanol, n-octanol; two sulfides: dimethyl and methyl ethyl sulfides; six monoterpene hydrocarbons: α-pinene, camphene, β-pinene, sabinene, myrcene, limonene.     

Hautriwaic acid as one of the hepatoprotective constituent of Dodonaea viscosa

It is widely known that hepatitis and its complications such as cirrhosis or hepatocellular carcinoma are one of the major health problems of the world especially since no specific treatment is available. In the present study we investigated the hepatoprotective potential of the methanolic extract of the whole plant of Dodonaea viscosa and its ethyl acetate, aqueous, butanol and n-hexane fractions against carbon tetrachloride (CCl₄) induced hepatoxicity in rats. Hepatoprotection was assessed in terms of reduction in serum enzymes (ALT, AST, and ALP) that occur after CCl₄ injury, and by histopathology and immunohistochemistry. The methanolic extract reduced the serum enzyme level (ALT, AST, and ALP) down to control levels despite CCl₄ treatment. It also reduced the CCl₄-induced damaged area to 0% as assessed by histopathology. The CD68+ macrophages were also reduced in number around the central vein area by the methanolic extract. These hepatoprotective effects were better than the positive control silymarin. Similar hepatoprotective activities were found with the ethyl acetate, and aqueous fractions of the methanolic extract. The butanol and n-hexane fractions showed elevated levels of ALT, AST and ALP as compared to the positive control silymarin. Histopathology showed ∼30% damage to the liver cells with the butanol and n-hexane fractions which still showed some protective activity compared to the CCl₄ treated control. HPLC fingerprinting suggested that hautriwaic acid present in the methanolic extract and its ethyl acetate, and aqueous fractions may be responsible for this hepatoprotective activity of Dodonaea viscosa which was confirmed by in vivo experiments.     

The Quantitative Yield in Purification of Cytokinins. Model-experiments with Kinetin, 6-Furfuryl-amino-purine

Model-experiments with kinetin, 6-furfuryl-amino-purine, to determine the quantitative yield in purification of cytokinin extracts have been performed. If kinetin in acidic water solution is partitioned three times with equal volumes of ethyl ether, about 50 per cent of the kinetin passes into the ether phases. In similar experiments with ethyl acetate more than 90 per cent of the kinetin goes over to the ethyl acetate phases. Accordingly, use of these two solvents in purification of cytokinin extracts leads to very large losses. Use of petroleum ether or n-hexane on the other hand leads to none or very small losses. Partitioning of alkaline kinetin solutions three times with equal volumes of 1-butanol results in an almost quantitatitve extraction of the kinetin from the water solution. About 7 per cent of the original amount of kinetin follows the acid auxin, when a saturated ether solution of kinetin is extracted according to a common method for acid auxins. Kinetin may thus interfere in later analyses for auxins. The dangers involved when cytokinins are “purified” according to normal practice are pointed out.     

Investigation on<Emphasis Type="Italic"> Semecarpus Lehyam</Emphasis>—a<Emphasis Type="Italic"> Siddha</Emphasis> medicine for breast cancer

The use of complementary and alternative medicines for breast cancer patients has been increasing every year. Traditional Indian systems of medicine, such as Siddha, have been reported to benefit patients in India through herbal interventions for cancer. One such herbal medicine is Semecarpus Lehyam (SL), and this study aims at providing a scientific basis for the anti-tumor property of SL with respect to breast cancer. SL was subjected to serial extraction with four organic solvents of increasing polarity (n-hexane, chloroform, ethyl acetate, n-butanol and water). The solvents from all fractions were removed, dried and dissolved in dimethyl sulfoxide for testing their anti-tumor activity against two breast cancer cell lines, MCF-7 [estrogen receptor (ER)-positive] and MDA 231 (ER-negative) using cell viability and apoptosis assays. The most potent SL fractions were also combined with radiation and doxorubicin to determine the radio- and chemo-sensitizing effects of SL on these breast cancer cell lines. In terms of cytotoxicity as well as induction of apoptosis, the n-hexane and chloroform fractions of SL were more significantly active against MDA 231 cells than MCF-7 cells. The n-butanol fraction of SL showed some activity against MCF-7 cells. When combined with radiation or doxorubicin, the n-hexane and chloroform fractions enhanced the radio-sensitivity (11.8-fold) and chemo-sensitivity (6.5-fold) of MDA 231 cells. This study demonstrated SL to be a potent anti-tumor agent against the ER-negative breast cancer cell line. The study is also the first step in the scientific validation of SL for use against breast cancer, particularly the ER-negative type.     

Efficient bioreduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydrobutanoate by whole cells ofCandida magnoliae in water/n-butyl acetate two-phase system

The asymmetric biosynthesis of ethyl (S)-4-chloro-3-hydrobutanoate from ethyl 4-chloro-3-oxobutanoate was investigated by using whole cells ofCandida magnoliae JX120-3 without the addition of glucose dehydrogenase or NADP⁺/NADPH. In a one-phase system, the bioconversion yield was seriously affected on the addition of 12.1 g/L ethyl 4-chloro-3-oxobutanoate. In order to reduce this substrate inhibition, a water/n-butyl acetate two-phase system was developed, and the bioreduction conditions optimized with regard to the yield and product enantiometric excess value. The optimal conditions were as following: water ton-butyl acetate volume ratio of 1∶1, 4.0 g DCW/L active cells, 50 g/L glucose and 35°C. By adopting a dropwise substrate feeding strategy, high concentration of ethyl 4-chloro-3-oxobutanoate (60 g/L) could be asymmetrically reduced to ethyl (S)-4-chloro-3-hydrobutanoate with high yield (93.8%) and high enantiometric excess value (92.7%).     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Alpha-tocopheryl acetate hydrolase in chicken liver. Characterisation and properties

An enzyme catalysing the hydrolysis ofa-tocopheryl acetate was characterised in chicken liver. The enzyme was localised in the microsomes, had an optimum pH 8.6 and aK _m value of 0.5 mM. The enzyme did not hydrolyse retinyl acetate, cholesteryl acetate and ethyl acetate, thus indicating a high degree of specificity.a-Tocopheryl acetate hydrolase required bile salts as a specific co-factor. The results suggested a role for this enzyme in the absorption of vitamin E.     

Synthesis of dipeptide precursors with an immobilized thermolysin in ethyl acetate

N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), a precursor of the aspartame, and N-(benzyloxycarbonyl)-L-phenylalanyl-Lphenylalanine methyl ester (Z-PhePheOMe) were synthesized from the respective amino acid derivatives with an immobilized thermolysin (EC 3.4.24.4) in ethyl acetate. Various factors affecting the synthesis of these dipeptide precursors were clarified. The initial synthetic rate was the highest at the water content of 3.5% for both reactions. The substrate concentration dependencies of the initial synthetic rate of Z-AspkPheOMe and Z-PhePheOMe with the immobilized enzyme in ethyl acetate were different from those in an aqueous buffer solution saturated with ethyl acetate but similar to those in the aqueous/organic biphasic system using the free enzyme. Particularly, the initial synthetic rate of Z-AspPhOMe increased in order higher than first order with respect to the concentration of L-phenylalanine methyl ester (PheOMe), whereas it decreased sharply with the concentration of N-(benzyloxycarbonyl)-L-aspartic acid (Z-Asp). Such kinetic behavior could be explained by regarding the inside of the immobilized enzyme as being a biphasic mode composed from the organic phase and aqueous phase where the enzymatic reaction takes place. The reaction in the aqueous/organic biphasic system using the free enzyme could be simulated by taking into consideration the partition of the substrate and the initial rate of synthesis in the aqueous buffer saturated with ethyl acetate. Based on this analysis, the rate of reaction with the immobilized enzyme in ethyl acetate could also be predicted. Z-AsPheOMe and Z-PhePheOMe were synthesized by the fed-batch method where the acid component of the substrate was intermittently added during the course of reaction and by the batch method. In the synthesis of Z-AspPheOMe, the synthetic rate and maximum yield of reaction as well as the stability of the immobilized enzyme were higher in the fed-batch reaction than those in the batch reaction. In the synthesis of Z-PhePheOMe, the results obtained by both methods were similar. (c) 1994 John Wiley & Sons, Inc.     

The Role of Metabolism in Establishing Chemical-Specific Adjustment Factors for n-Hexane and Methyl n-Butyl Ketone

This paper focuses on the derivation of chemical-specific adjustment factors (CSAFs) for two neurotoxic solvents, n-hexane and methyl n-butyl ketone (MBK). Workers are exposed to the chemicals mainly via inhalation when they are used, for example, as solvents in adhesives. In order to derive CSAFs for n-hexane and MBK, research data were used from animal studies conducted in our laboratory. Also, MEDLINE and TOXLINE databases were searched for references containing animal and human toxicokinetic and toxicodynamic data. International Programme on Chemical Safety (IPCS) guidelines were followed. From the available data, the composite factor (CF) for n-hexane was calculated to be 18.2. Because n-hexane and MBK are most often used in combination with other solvents, e.g., methyl ethyl ketone (MEK), the metabolic induction of the toxicity of n-hexane or MBK by MEK should be considered in the derivation of CSAFs. However, in the presence of MEK the calculated CF for both n-hexane and MBK is 13. As more human data become available, the CFs for the mixtures may be reduced compared to the individual chemicals.     

Modelling and experimental studies on lipase-catalyzed isoamyl acetate synthesis in a microreactor

A lipase-catalyzed synthesis of isoamyl acetate was studied in a continuously operated pressure-driven microreactor. The esterification of isoamyl alcohol and acetic acid occurred at the interface between n-hexane and an aqueous phase with dissolved lipase B from Candida antarctica. By adjusting flow rates of both phases, a parallel laminar flow with liquid–liquid boundary in the middle of the microchannel could be reestablished and a separation of phases was achieved at the y-shaped exit of the microreactor. Since product remained in the organic phase, this also enabled its continuous separation from the aqueous phase with the enzyme. A three-dimensional mathematical model was developed, considering the velocity profile developed for steady-state conditions between two immiscible fluids. The model contained convection, diffusion, and enzyme reaction terms, where esterification rate was described with a Ping-Pong Bi-Bi mechanism and inhibition by both substrates. Experimental data, which were in good agreement with model simulations, have demonstrated 35% conversion at residence time 36.5 s at 45 °C and at 0.5 M acetic acid and isoamyl alcohol inlet concentrations, which is much faster as in any literature reported so far. According to model simulations, obtained by non-equidistant finite differences numerical solutions of complex non-linear equations system, further microreactor design and process optimization are feasible.     

A comparison of lipase-catalyzed ester hydrolysis in reverse micelles, organic solvents, and biphasic systems

The performance of lipases from Candida rugosa and wheat germ have been investigated in three reaction media using three acetate hydrolyses as model reactions (ethyl acetate, allyl acetate, and prenyl acetate). The effect of substrate properties and water content were studied for each system (organic solvent, biphasic system, and reverse micelles). Not unexpectedly, the effect of water content is distinct for each system, and the optimal water content for enzyme activity is not always the same as that for productivity. A theoretical model has been used to simulate and predict enzyme performance in reverse micelles, and a proposed partitioning model for biphasic systems agrees well with experimental results. While the highest activities observed were in the micellar system, productivity in microemulsions is limited by low enzyme concentrations. Biphasic systems, however, support relatively good activity and productivity. The addition of water to dry organic solvents, combined with the dispersion of lyophilized enzyme powders in the solvent, resulted in significant enzyme aggregation, which not surprisingly limits the applicability of the "anhydrous" enzyme suspension approach. (c) 1995 John Wiley & Sons, Inc.     

n-Dodecane as a substrate for nitrogen fixation by an alkane-utilizingAzospirillum sp.

Azospirillum sp. ANK-BI-11 was isolated from petroliferous soil. Glucose, nutrient broth, and sugar acids showed better growth thann-alkanes under aerobic conditions. The utilization of glucose was inhibited in the presence ofn-hexane. Microaerobically, succinic acid, pyruvic acid, and lactic acid were the best C-sources for acetylene reduction, whereas glucose was the best source for growth.n-Dodecane, a nonconventional C-source, also showed good response towards acetylene reduction, although growth was not so pronounced here as with glucose but was equal to that of Na-succinate. Optimum pH and temperature for acetylene reduction were between 7.0 and 8.0 and 30°C, respectively. Scanning electron microscopic studies revealed structural alteration in the shapes and sizes of the cells ofAzospirillum sp. when grown onn-hexane andn-dodecane compared with the cells grown on glucose.     

Efficient release of ferulic acid from sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis>) stems by chemical hydrolysis

An efficient method for release of ferulic acid from sweet potato stems was developed. Ferulic acid along with phenolic compounds were released from stems by acid and alkaline treatments. The base hydrolysis with 0.1 N NaOH yielded the highest quantity of total extracts (471.1 mg/g). The stems released more phenolic compounds when 0.0125∼0.025 N NaOH was employed. Where as ferulic acid release was maximal with 0.05 N H₂SO₄ (0.32 mg/g). Ferulic acid was separated from phenolics by column chromatography. Among the elution solvents, ethyl acetate fractions (80%) contained ferulic acid. Ethyl acetate eluants were further fractionated with n-hexane/ethyl acetate/formic acid (100/50/0.5, v/v/v). All fractions showed ferulic acid and phenolic compounds. Fraction V among them was ascribed to ferulic acid with an yield of 5.41 mg/g of dry sweet potato tissue.     

Thermal stability enhancement of Candida rugosa lipase using ionic liquids

The thermal stability of Candida rugosa (C. rugosa) lipase was investigated and compared in n-hexane, benzene, dibutyl-ether as well as [bmim]PF₆ and [omim]PF₆ ionic liquids and the effect of solvent polarity and water activity were evaluated. Deactivation of the enzyme followed a series-type kinetic model. First order deactivation rate constants and the ratios of specific activities were determined and the kinetics of deactivation were studied. Among the organic solvents, the best stability was observed in n-hexane with a half-life of 6.5?h at water activity of 0.51. In ionic liquids, however, even longer half lives were obtained, and the enzyme was stable in these solvents at 50°C. The highest half-life times were obtained in [bmim]PF₆ (12.3?h) and [omim]PF₆ (10.6?h). A direct correlation was found between solvent polarity and thermal stability since the higher the polarity of the solvent, the lower was the stability decrease at 50°C comparing to that at 30°C.     

Three Systems Used for Biocatalysis in Organic Solvents a Comparative Study

The activity and operational stability of horse liver alcohol dehydrogenase (HLADH) and α-chymotrypsin were investigated in three systems commonly used for biocatalysis in organic solvents:

1. enzyme adsorbed on a solid support (celite) and added to the organic solvent (isooctane)

2. enzyme powder directly added to the organic solvent (isooctane).

3. enzyme dissolved in a microemulsion (AOT/isooctane).

The activity and the operational stability in all systems were strongly dependent on the water content. The initial reaction rate was high in both the microemulsion and the celite system, but was much lower when adding the enzymes directly to the organic solvent. HLADH was observed to be more stable when added directly to the organic solvent or dissolved in the microemulsion than when adsorbed on celite, whereas for α-chymotrypsin stability was higher when adsorbed on celite or added directly to the organic solvent. For a hydrolytic reaction, a microemulsion was preferred due to the high water content. When adding the enzymes directly to the organic solvent both HLADH and chymotrypsin were adsorbed strongly to the glass walls of the reaction vessel. None of the systems were superior in all respects for the two enzymes studied.