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1.
Optically active cyanohydrin esters such as (S)-α-cyano-3-phenoxybenzyl (CPB) acetate can be obtained with enzymatic transesterification of racemic cyanohydrins in organic media. The influence of internal diffusion limitation on transesterification of CPB alcohol with vinyl acetate catalyzed by immobilized lipase was studied. Internal diffusion limitation could not be ignored since the immobilized lipase granule was not small enough. In order to express the effect of internal diffusion limitation quantitatively, a method was proposed which calculates the effective diffusion coefficient (De) first and then calculates apparent Thiele modulus (Φ) and finally obtains internal diffusion effectiveness factor, η value (0.55). De was calculated to be 3.1 × 10−10 m2 s−1 by supposing that the immobilized lipase was a sphere granule and the diffusion only existed in one dimension. Φ was calculated to be 2.2 by presuming that transport of substrate through the catalyst could be described by Fick's law. Using King–Altman method, a kinetic model of the transesterification of CPB alcohol with vinyl acetate in organic media in the presence of internal diffusion limitation was proposed based on ping-pong bi–bi mechanism. The relative error of the model was 11.18%. A practicable method to evaluate η value and relatively simple model for such kind of reaction system was proposed in this paper.  相似文献   

2.
Employing high-performance liquid chromatography–electrospray mass spectrometry, we describe a new assay for monitoring 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase activity. Incubations were carried out with HMG-CoA reductase (rat liver), HMG-CoA and NADPH, and terminated by the addition of HCl. The reaction product, mevalonolactone, and internal standard, were extracted with ethyl acetate, dissolved in methanol, and analyzed by LC–MS. Using an isocratic mobile phase of 10% acetonitrile and 0.1% formic acid (flow-rate, 0.2 ml/min), the protonated molecules of mevalonolactone at m/z 131 and internal standard, β,β-dimethyl-γ-(hydroxymethyl)-γ-butyrolactone, at m/z 145, were detected using selected ion monitoring. The limit of detection was approximately 6.5 pg, and the limit of quantitation was approximately 16.3 pg. Extraction recovery was >90%. The relative standard deviations for intra- and inter-day assays were approximately 4.1±2.7 and 9.4±3.4%, respectively. Mevalonolactone was examined over a period of 3 days and found to be stable. Using this assay, lovastatin and mevastatin inhibited HMG-CoA reductase activity with IC50 values 0.24±0.02 and 2.16±0.31 μM, respectively. These methods offer some advantages over those reported previously which employ radiolabeled substrate and products, and should be useful in searching for compounds that could lower serum cholesterol or alter cell growth and differentiation.  相似文献   

3.
Lipase-catalyzed enantioselective hydrolysis of the acetic ester of racemic α-cyano-3-phenoxybenzyl alcohol (CPBA) was examined to prepare (S)-CPBA. Most of the lipases tested hydrolyzed (S)-CPBA acetate preferentially, while Candida cylindracea lipase favored (R)-CPBA acetate. Enantioselective hydrolysis by Arthrobacter lipase gave the optically pure (S)-CPBA in the reaction mixture of pH 4.0. The kinetic studies showed that (R)-CPBA acetate reacted as a competitive inhibitor. The Arthrobacter lipase solution in the water/oil biphasic reaction system could be used repeatedly. The lipase immobilized to resins had insufficient activity or low operational stability for the repeated batch reaction. The unhydrolyzed (R)-CPBA acetate was racemized by heating with triethylamine and could be reused as the substrate of the enzymatic hydrolysis. A chemico-enzymatic process for the preparation of (S)-CPBA was developed based on these studies.  相似文献   

4.
The application of unusual high pH-values within enzymatic cyanohydrin synthesis has been investigated. Usually enzymatic cyanohydrin synthesis in two-phase systems requires low pH-values within the aqueous phase to suppress the non-enzymatic side reaction. In contrast, we investigated the usage of pH-values above pH 6 by using the highly enantioselective (S)-selective hydroxynitrile lyase from Manihot esculenta. With these unusual reaction conditions also the unfavorable substrate 3-phenoxy-benzaldehyde can be converted by the wild type enzyme with excellent conversion and enantiomeric excess yielding pure (S)-3-phenoxy-benzaldehyde cyanohydrin with an enantiomeric excess of 97%. Although the variant MeHNL–W128A shows a higher activity with respect to this reaction, the enantioselectivity was reduced (85% e.e.(S)). Additionally, a new continuous spectroscopic cyanohydrin assay monitoring the formation of 3-phenoxy-benzaldehyde cyanohydrin was developed. Dedicated to Prof. Dr. Christian Wandrey on the occasion of his 65th birthday.  相似文献   

5.
(R)-Mandelonitrile was successfully synthesized by an enzymatic transcyanation reaction of benzaldehyde and acetone cyanohydrin catalyzed by a hydroxynitrile lyase from Eriobotrya japonica (EjHNL) in an aqueous-organic biphasic system. The effects of pH, temperature, organic solvent, substrate concentration and enzyme concentration on the initial activity and enantioselectivity of the enzyme were studied. Both pH and temperature had a large effect on the initial velocity and enantiomeric excess (e.e.) of the product, (R)-mandelonitrile. High enantiomeric purity of the product was observed at low pH and temperature because the non-enzymatic reaction producing racemates of mandelonitrile was almost suppressed. The optimum pH and temperature to obtain high e.e. were pH 4.0 and 10 °C, respectively. Surprisingly, the organic solvents had a significant influence on the initial velocity of the reaction but less influence on the enantiomeric purity of product. The EjHNL was very stable in ethyl acetate, diethyl ether, methyl-t-butyl ether, diisopropyl ether, dibutyl ether and hexane for 12 h. The best solvent for the highest initial velocity and e.e. was diethyl ether with an optimum aqueous phase content of 50% (v/v). The initial reaction rate increase as the aqueous phase content rose, but when the content was more than 50%, a reduction of e.e. was observed. Increasing the concentration of the substrates accelerated the initial velocity, but caused a slight decrease in the e.e. of the product. Under the optimized conditions, the conversion and e.e. of (R)-mandelonitrile for 3 h were 40 and 99%, respectively. The aqueous phase containing the enzyme also showed considerably efficient reusability for 4 batch reactions.  相似文献   

6.
Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.Abbreviations CPBA m-Chloro-peroxy benzoic acid - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - F-1,6-P2 frnctose-1,6-bisphosphate - DAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - 2PGA 2-phosphoglycerate - PEP phosphoenol pyruvate - Pyr pyruvate - EtOH ethanol - PFK phosphofructokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - ADH alcohol dehydrogenase Dedicated to Prof. Dr. Wolfgang Gerok at the occasion of his 60th birthday  相似文献   

7.
Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).  相似文献   

8.
Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35 produce extracellular β-glycosidases that are active on the cyanogenic glycoside amygdalin. From the culture broths of M. circinelloides, only one β-glycosidase could be identified, while two different enzymes – both having amygdalase activity – were found in culture broths of P. aurantiogriseum. The study of the mechanism of hydrolysis of the β-bis-glycoside amygdalin with purified enzymes from the two organisms indicated a possible sequential (two-step) reaction. In all cases, the first step of hydrolysis from amygdalin to prunasin was very rapid, while the second step from prunasin to cyanohydrin was much slower. No cyanohydrin lyase activity was found in the culture broths of either fungus. Received: 16 May 1997 / Accepted: 11 September 1997  相似文献   

9.
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%).  相似文献   

10.
Cytotoxic, phytotoxic, antimicrobial and antioxidant effects of quercetin 3-O-glucoside (Q3G) isolated by HPLC from aerial parts of Prangos ferulaceae was studied by MTT assay, lettuce germination assay, disk diffusion and DPPH method. Our results showed that Q3G exhibits high antioxidant effect with RC50 of 22 μg/mL, it has low cytotoxicity and no antibacterial effects. Q3G exhibits high phytotoxic effect with IC50 value of 282.7 μg/ml, as well. It is assumed that Q3G does not play a defense role in plants and it may act as an allelopatic agent. The article is published in the original.  相似文献   

11.
β-Glucosidase-catalyzed synthesis ofn-octyl β-d-glucoside through condensation was performed by mixing glucose and octanol saturated with acetate buffer at 50°C. The highest yield of synthesis, 28% based on glucose, was obtained at the total glucose concentration of 0.1 mol/1. At higher glucose concentrations, the product concentration increased but the yield decreased. The stability of the enzyme in the system was so high that, even in the free form, it could be used repeatedly for synthesis without a significant loss in its activity. The overall yield of the four consecutive batch syntheses was 58% when the initial glucose concentration was adjusted to 0.1 mol/1 for each batch.  相似文献   

12.
Optimal growth and PHB accumulation in Bacillus megaterium occurred with 5% (w/v) date syrup or beet molasses supplemented with NH4Cl. When date syrup and beet molasses were used alone without an additional nitrogen source, a cell density of about 3gl–1 with a PHB content of the cells of 50% (w/w) was achieved. NH4NO3 followed by ammonium acetate and then NH4Cl supported cell growth up to 4.8gl–1, whereas PHB accumulation was increased with NH4Cl followed by ammonium acetate, NH4NO3 and then (NH4)2SO4 to a PHB content of nearly 42% (w/w). Cultivation of B.megaterium at 30l scale gave a PHB content of 25% (w/w) of the cells and a cell density of 3.4gl–1 after 14h growth.  相似文献   

13.
A cataluminescence (CTL) sensor using Y2O3 nanoparticles as the sensing materials was proposed for the determination of ethyl acetate. This ethyl acetate sensor showed high sensitivity and specificity at the optimal temperature of 264°C. Quantitative analysis was performed at a wavelength of 425 nm. The linear ranges of CTL intensity vs ethyl acetate concentrations were 2.0–250 ppm (r = 0.9965) and 250–6500 ppm (r = 0.9997) with a detection limit (3σ) of 0.5 ppm. There was no response or weak response when foreign substances such as formic acid, n‐hexane, toluene, acetic acid, benzene, and formaldehyde passing through the surface of Y2O3 nanoparticles. The sensor had a long lifetime more than 80 h with 3600 ppm ethyl acetate. It had been applied successfully to determine ethyl acetate in artificial air samples. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

14.
Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998  相似文献   

15.
(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.  相似文献   

16.
Antimicrobial activity of methanolic extracts obtained from the aerial parts of Evax pygmaea was tested against five bacteria and two strains of phytopathogenic fungi using the agar diffusion and broth microdilution methods. Antioxidant properties were evaluated through the ability of the different fractions to scavenge the stable ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and DPPH (1,1-diphenyl-2-picrylhydrazyl) radicals. The TEAC (Trolox Equivalent Antioxidant Capacity) and IC50 values of the fractions were calculated and compared. The experimental data indicated that all fractions exhibit moderate to appreciable antibacterial activities against all Gram-positive cocci and Gram-negative rods except Pseudomonas aeruginosa, but no antifungal activity was observed. Ethyl acetate and methanol fractions were found to cause significant free radical-scavenging effects in both assays. These results may suggest that E. pygmaea could be used as a natural preservative ingredient in the food and/or pharmaceutical industries.  相似文献   

17.
Measurement of three-dimensional (3D) knee joint angle outside a laboratory is of benefit in clinical examination and therapeutic treatment comparison. Although several motion capture devices exist, there is a need for an ambulatory system that could be used in routine practice. Up-to-date, inertial measurement units (IMUs) have proven to be suitable for unconstrained measurement of knee joint differential orientation. Nevertheless, this differential orientation should be converted into three reliable and clinically interpretable angles. Thus, the aim of this study was to propose a new calibration procedure adapted for the joint coordinate system (JCS), which required only IMUs data. The repeatability of the calibration procedure, as well as the errors in the measurement of 3D knee angle during gait in comparison to a reference system were assessed on eight healthy subjects. The new procedure relying on active and passive movements reported a high repeatability of the mean values (offset<1°) and angular patterns (SD<0.3° and CMC>0.9). In comparison to the reference system, this functional procedure showed high precision (SD<2° and CC>0.75) and moderate accuracy (between 4.0° and 8.1°) for the three knee angle. The combination of the inertial-based system with the functional calibration procedure proposed here resulted in a promising tool for the measurement of 3D knee joint angle. Moreover, this method could be adapted to measure other complex joint, such as ankle or elbow.  相似文献   

18.
Previous studies suggested that methanol and acetate were the likely methanogenic precursors in the cold Zoige wetland. In this study, the contribution of the two substances to methanogenesis and the conversion in Zoige wetland were analyzed. It was determined that methanol supported the highest CH4 formation rate in the enrichments of the soil grown with Eleocharis valleculosa, and even higher at 15°C than at 30°C; while hydrogenotrophic methanogenesis was higher at 30°C. Both methanol- and acetate-using methanogens were counted at the highest (107 g−1) in the soil, whereas methanol-using acetogens (108 g−1) were ten times more abundant than either methanol- or acetate-using methanogens. Both methanol and acetate were detected in the methanogenesis-inhibited soil samples, so that both could be the primary methanogenic precursors in E. valleculosa soil. However, the levels of methanol and acetate accumulated in 2-bromoethane-sulfonate (BES)- and CHCl3-treated soils were in reverse, i.e., higher methanol in CHCl3- and higher acetate in BES-treated soil, so that methanol-derived methanogenesis could be underestimated due to the consumption by acetogens. Analysis of the soil 16S rRNA genes revealed Acetobacterum bakii and Trichococcus pasteurii to be the dominant methanol-using acetogens in the soil, and a strain of T. pasteurii was isolated, which showed the high conversion of methanol to acetate at 15°C.  相似文献   

19.
In this work we showed that living cells of Leishmania chagasi was able to hydrolyse 3′AMP 10 times more than 5′AMP. When parasites were grown in a low phosphate concentration (2 mM) the cellular proliferation decreased by 50% compared to cells grown in the presence of a high phosphate concentration (80 mM). However, the ecto-3′nucleotidase activity was 2-fold higher when L. chagasi was grown in a low phosphate concentration. This modulation observed on ecto-3′nucleotidase activity was not observed on ecto-5′nucleotidase activity. These results suggest that low concentration of Pi in the culture medium modulates ecto-3′nucleotidase activity that may lead to modulation of important processes for the cell. Interestingly, the macrophage–parasite interaction increased by 45% when L. chagasi were grown at low phosphate concentration compared to the parasites grown in the presence of high phosphate source. Altogether, the results described here suggest that 3′nucleotidase activity modulated by external stimuli, Pi concentration, could be involved on parasite–macrophage interaction.  相似文献   

20.
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