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1.
Escherichia coli is able to utilize l-galactonate as a sole carbon source. A metabolic pathway for l-galactonate catabolism is described in E. coli, and it is known to be interconnected with d-galacturonate metabolism. The corresponding gene encoding the first enzyme in the l-galactonate pathway, l-galactonate-5-dehydrogenase, was suggested to be yjjN. However, l-galactonate dehydrogenase activity was never demonstrated with the yjjN gene product. Here, we show that YjjN is indeed an l-galactonate dehydrogenase having activity also for l-gulonate. The K m and k cat for l-galactonate were 19.5?±?0.6 mM and 0.51?±?0.03 s?1, respectively. In addition, YjjN was applied for a quantitative detection of the both of these substances in a coupled assay. The detection limits for l-galactonate and l-gulonate were 1.65 and 10 μM, respectively.  相似文献   

2.
A sensitive fluorescence liquid chromatographic analytical method was developed for the simultaneous determination of carnosine enantiomers in rat plasma. The method was applied to pharmacokinetic studies. Chiral separation of carnosine enantiomers was achieved by pre-column derivatization with o-phthaldialdehyde and the thiol N-acety-l-cysteine as derivating reagents. They were separated on an ODS column and detected by fluorescence detection (λex = 350 nm, λem = 450 nm). γ-Aminobutyric acid was used as internal standard. The method was linear up to 6,000 ng mL?1 for l-carnosine, 4,000 ng mL?1 for d-carnosine. Low limit of quantitation (LLOQ) was 40 ng mL?1 for each isomer. The relative standard deviations obtained for intra- and inter-day precision were lower than 12% and the recoveries were higher than 75% for both enantiomers. The method was applied to a stereoselective study on the pharmacokinetics of carnosine after oral administration with a single dose (carnosine, 75 mg kg?1 for each isomer) to a rat. The initial data indicated that l-carnosine had a larger value of the highest plasma concentration than d-carnosine (C max 5,344 vs. 1,914 ng mL?1), and that of l-carnosine had a lower value of AUC(0?∞) and t 1/2(h) (AUC(0?∞) 5,306 vs. 6,321 ng h mL?1, t 1/2 1.43 vs. 3.37 h). Our results indicated that the pharmacokinetic of l-carnosine and d-carnosine revealed enantioselective properties significantly.  相似文献   

3.
l-Glutaminase (E.C.3.5.2.1) extracellularly produced by Bacillus cereus MTCC 1305 was purified to apparent homogeneity with a fine band. The molecular weight of native enzyme and its subunit were found to be approximately 140 and 35 kDa, respectively, which indicates its homotetrameric nature. The substrate specificity test of this enzyme showed its specificity for l-glutamine. The purified enzyme showed maximum activity at optimum pH 7.5 and temperature 35 °C. The enzyme retained stability up to 50 and 20 % even after treatment at 50 and 55 °C, respectively, for 30 min. Monovalent cations (Na+, K+) and phosphate ion activated the enzyme activity, while divalent cations (Mg2+, Mn2+, Zn2+, Pb2+, Ca2+, Co2+, Hg2+, Cd2+, Cu2+) inhibited its activity. Reducing agents (cysteine, glutathione, dithiothreitol, l-ascorbic acid, and β-mercaptoethanol) stimulated its activity, whereas thiol-binding agents (iodoacetamide, p-chloromercuribenzoic acid) resulted in the inhibition of this enzyme. Kinetic parameters, K m, V max, K cat, of purified enzyme were found to be 6.25 mM, 100 μmol/min/mg protein and 2.22?×?102 M?1s?1, respectively. The gradual inhibition in growth of hepatocellular carcinoma (Hep-G2) cell lines was found with IC50 value of 82.27 μg/ml in the presence of different doses of l-glutaminase (10–100 μg/ml).  相似文献   

4.
l-DOPA (3,4-dihydroxyphenyl-l-alanine), the most widely used drug for the treatment of Parkinson??s disease, was produced in buffer using biomass of Brevundimonas sp. SGJ. The effects of enhancers, such as carrageenan, diatomaceous earth, and activated charcoal, on the l-DOPA production were evaluated to obtain the maximum yield. The optimal process conditions found were pH?8, 2?g?l?1 cell mass, 2?g?l?1 l-tyrosine, 0.04?g?l?1 CuSO4, 0.02?g?l?1 l-ascorbic acid, 0.5?g?l?1 carrageenan, and 40?°C temperature. In addition, repeated use of cells resulted in the highest yield of 3.81?g?l?1 (95.2%) of l-DOPA with utilization of 4?g?l?1 l-tyrosine, and the highest tyrosinase activity (9,201?U?mg?1) was observed at 18?h of incubation. Furthermore, the produced l-DOPA was confirmed by high-performance thin-layer chromatography, high-performance liquid chromatography, and gas chromatography?Cmass spectroscopy. Kinetic studies showed significant values of Y p/s, Q s, and q s after optimization of the process. Thus, Brevundimonas sp. SGJ could be an eventual new source for large-scale production of l-DOPA.  相似文献   

5.
A simple and accurate chiral liquid chromatographic method was developed for the enantiomeric purity determination of d-nateglinide and quantitative determination of l-nateglinide in bulk drug samples. Good resolution (R s  > 6.0) between d-enantiomer and l-enantiomer of nateglinide were achieved with Chiralpak AD-H (250 × 4.6 mm, 5 μm particle size) column using hexane and ethanol (90:10 v/v) as mobile phase at 25 °C temperature. Flow rate was kept as 1.0 mL min?1 and elution was monitored at 210 nm. The effects of the mobile phase composition, the flow rate and the temperature on the chromatographic separation were investigated. Developed method is capable to detect (LOD) and quantitate (LOQ) l-nateglinide to the levels of 0.3 and 1.0 μg mL?1 respectively, for 10 μL injection volume. The percentage RSD of the peak area of six replicate injections of l-nateglinide at LOQ concentration was 5.2. The percentage recoveries of l-nateglinide from d-nateglinide ranged from 97.9 to 99.7. The test solution and mobile phase was found to be stable up to 24 h after preparation. The developed method was validated with respect to LOD, LOQ, precision, linearity, accuracy, robustness and ruggedness.  相似文献   

6.
Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO4 which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu2+ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm?3 (KNO3) in aqueous solution. The ternary species [Cu(L)(l-His)H]+ and [Cu(L)(d-His)H]+ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO4.
Figure
Schematic view of the separation process  相似文献   

7.
l-asparaginase is a widely used cancer chemotherapy enzyme. The source for the enzyme with this property is mainly bacterial and its synthesis is strongly regulated by oxygen. In this study, we utilized two recombinant systems: one carried the gene (vgb) for the Vitreoscilla hemoglobin (VHb), a protein of prokaryotic origin which confers a highly efficient oxygen uptake to its host and the other carried the l-asparaginase gene (ansB). The host bacteria were Escherichia coli, Enterobacter aerogenes, and Pseudomonas aeruginosa. Of these three bacteria, all gram-negative, E. coli and its recombinant strain showed up to sevenfold higher l-asparaginase activity in lactose than in other carbon sources. Although, in this bacterium glycerol was the poorest source for l-asparaginase synthesis, it supported the highest biomass production. In glucose medium, l-asparaginase activity of E. aerogenes was about threefold higher than its vgb and ansB recombinants. ansB recombinant showed significantly higher enzyme levels than both host and vgb recombinants in glycerol and lactose media. In this bacterium, VHb/vgb clearly caused a decrease in the enzyme synthesis under all conditions. As seen for E. aerogens, glycerol was the most favorable carbon source for P. aeruginosa and its vgb strain in terms of both l-asparaginase synthesis and biomass production. The cultures grown in glycerol had more than two- and threefold biomass than in glucose and lactose, respectively, and up to elevenfold than in mannitol. Indeed, the highest biomass production for all bacteria and their recombinants was in glycerol. The VHb/vgb system is clearly advantageous for production of l-asparaginase in P. aeruginosa. The same, however, does not hold true for E. aerogenes.  相似文献   

8.
l-Ribose is an important precursor for antiviral agents, and thus its high-level production is urgently demanded. For this aim, immobilized recombinant Escherichia coli cells expressing the l-arabinose isomerase and variant mannose-6-phosphate isomerase genes from Geobacillus thermodenitrificans were developed. The immobilized cells produced 99 g/l l-ribose from 300 g/l l-arabinose in 3 h at pH 7.5 and 60 °C in the presence of 1 mM Co2+, with a conversion yield of 33 % (w/w) and a productivity of 33 g/l/h. The immobilized cells in the packed-bed bioreactor at a dilution rate of 0.2 h?1 produced an average of 100 g/l l-ribose with a conversion yield of 33 % and a productivity of 5.0 g/l/h for the first 12 days, and the operational half-life in the bioreactor was 28 days. Our study is first verification for l-ribose production by long-term operation and feasible for cost-effective commercialization. The immobilized cells in the present study also showed the highest conversion yield among processes from l-arabinose as the substrate.  相似文献   

9.
A sensitive, simple and rapid LC–MS–MS method has been developed and validated for the simultaneous determination of l-dopa and l-dopa n-pentyl ester hydrochloride in rat plasma in the present study. The analytes were separated on a C18 column (5 μm, 2.1 × 150 mm) with a security guard C18 column (5 μm, 4 × 20 mm) and a triple-quadrupole mass spectrometer was applied for detection. The method was linear over the concentration ranges of 25–5,000 ng mL?1 for l-dopa and 12.5–2,500 ng mL?1 for l-dopa n-pentyl ester hydrochloride. Finally, the method was successfully applied to support the pharmacokinetic study.  相似文献   

10.
Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was cloned and overexpressed in Lactococcus lactis using a phosphate-depletion-inducible expression system. The purified B. longum l-AI was characterized using d-galactose and l-arabinose as the substrates. The enzyme was active and stable at acidic pH with an optimum at pH 6.0?C6.5. The enzyme showed the highest activity at 55?°C during a 20-min incubation at pH 6.5. The K m value was 120?mM for l-arabinose and 590?mM for d-galactose. The V max was 42?U mg?1 with l-arabinose and 7.7?U mg?1 with d-galactose as the substrates. The enzyme had very low requirement for metal ions for catalytic activity, but it was stabilized by divalent metal ions (Mg2+, Mn2+). The enzyme bound the metal ions so tightly that they could not be fully removed from the active site by EDTA treatment. Using purified B. longum l-AI as the catalyst at 35?°C, equilibrium yields of 36?% d-tagatose and 11?% l-ribulose with 1.67?M d-galactose and l-arabinose, respectively, as the substrates were reached.  相似文献   

11.
Extracellular tyrosinase from Auricularia auricula RF201 was purified in a three-step procedure involving ammonium sulfate precipitation, Sephadex G-100, and DEAE-Sepharose column chromatography. The partially purified enzyme showed a single protein band of 12.6 kDa on SDS-PAGE. The optimum pH for tyrosinase activity was 7, and the enzyme was stable between pH 6 and 9. Tyrosinase has optimal activity at 40 °C and retained most of its activity between 4 and 50 °C. A. auricula tyrosinase could oxidize l-tyrosine, l-DOPA, catechol, and caffeic acid and displayed dark brown or peach color. However, the enzyme was unable to catalyze l-phenylalanine and ferulic acid. In comparison with other substrates, l-tyrosine displayed the highest affinity (K m of 0.11 mM) and the maximal reaction velocity (V max of 102.58 μmol/min). Tyrosinase activity was reduced in the presence of numerous tested compounds. Particularly SDS, it significantly inhibited enzyme activity. CuSO4 and NaCl showed an activation effect on enzyme activity, with the maximum activation found in the presence of CuSO4.  相似文献   

12.
The reactions of a racemic four-coordinate Ni(II) complex [Ni(rac-L)](ClO4)2 with l- and d-alanine in acetonitrile/water gave two six-coordinate enantiomers formulated as [Ni(RR-L)(l-Ala)](ClO4)·2CH3CN (1) and [Ni(SS-L)(d-Ala)](ClO4) (2) (L = 5,5,7,12,12,14-hexamethyl-1,4,8,11-tetraazacyclo-tetradecane, Ala? = alanine anion), respectively. Evaporation from the remaining solutions gave two four-coordinate enantiomers characterized as [Ni(SS-L)](ClO4)2 (S-3) and [Ni(RR-L)](ClO4)2 (R-3), respectively. Single-crystal X-ray diffraction analyses of complexes 1 and 2 revealed that the Ni(II) atom has a distorted octahedral coordination geometry, being coordinated by four nitrogen atoms of L in a folded configuration, plus one carboxylate oxygen atom and one nitrogen atom of l- or d-Ala? in mutually cis-positions. Complexes 1 and 2 are supramolecular stereoisomers, constructed via hydrogen bonding between [Ni(RR-L)(l-Ala)]+ or [Ni(SS-L)(d-Ala)]+ monomers to form 1D hydrogen-bonded zigzag chains. The homochiral natures of complexes 1 and 2 have been confirmed by CD spectroscopy.  相似文献   

13.
Xylose effective utilization is crucial for production of bulk chemicals from low-cost lignocellulosic substrates. In this study, an efficient l-lactate production process from xylose by a mutant Bacillus coagulans NL-CC-17 was demonstrated. The nutritional requirements for l-lactate production by B. coagulans NL-CC-17 were optimized statistically in shake flask fermentations. Corn steep liquor powder and yeast exact were identified as the most significant factors by the two-level Plackett–Burman design. Steepest ascent experiments were applied to approach the optimal region of the two factors, and a central composite design was employed to determine their optimal levels. The optimal medium was used to perform batch fermentation in a 3-l bioreactor. A maximum of 90.29 g l?1? l-lactic acid was obtained from 100 g l?1 xylose in 120 h. When using corn stove prehydrolysates as substrates, 23.49 g l?1? l-lactic acid was obtained in 36 h and the yield was 83.09 %.  相似文献   

14.
The exchange extraction constants corresponding to the general equilibrium C+(aq) + Cs+(nb) ? C+ (nb) + Cs+(aq) occurring in the two-phase water–nitrobenzene system (C+ = protonated α-amino acid methyl ester, 1 = hexaethyl p-tert-butylcalix[6]arene hexaacetate; aq = aqueous phase, nb = nitrobenzene phase) were evaluated on the basis of extraction experiments and γ-activity measurements. Further, the stability constants of the C+ cationic complex species in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: protonated l-tryptophan methyl ester < protonated l-phenylalanine methyl ester < protonated l-leucine methyl ester < protonated l-methionine methyl ester < protonated l-valine methyl ester.  相似文献   

15.
Amperometric detection of 3-(3,4-dihydroxyphenyl)-l-alanine (l-dopa) on a glassy carbon electrode at oxidation potential of +0.70 V in Mucuna pruriens after micro-high performance liquid chromatography separation is reported. Optimised eluent consisted of 0.87 mM 1-octane sulphonic acid sodium salt, 18.2 mM citric acid, and 82.8 mM sodium acetate with pH adjusted to 2.18 using 85% orthophosphoric acid. Detection of low concentrations of l-dopa up to 5.12 ng mL?1 was achieved. The method was employed to determine l-dopa in raw and cooked beans after water extraction through a 0.45 μm membrane with no further sample treatment.  相似文献   

16.
(R,S)-Atenolol was derivatized with Marfey’s reagent, (MR; 1-fluoro-2,4-dinitrophenyl-5-l-alanine amide or FDNP-l-Ala-NH2) and its four structural variants (FDNP-l-Phe-NH2, FDNP-l-Val-NH2, FDNP-l-Leu-NH2 and FDNP-l-Pro-NH2). MR reacts quantitatively with 1° and 2° amino groups and atenolol has a secondary amino group. The derivatization reactions were carried out under conventional and microwave heating and compared. The resulting diastereomers were separated on RP-TLC and on a C18 column with detection at 340 nm. (R)-Isomer eluted before (S). The conditions of derivatization and chromatographic separation were optimized. The method was validated for linearity, repeatability, limits of detection and limit of quantification.  相似文献   

17.
β-Aminopeptidases exhibit both hydrolytic and aminolytic (peptide bond formation) activities and have only been reported in bacteria. We identified a gene encoding the β-aminopeptidase homolog from a genome database of the filamentous fungus Aspergillus oryzae. The gene was overexpressed in A. oryzae, and the resulting recombinant enzyme was purified. Apart from bacterial homologs [β-Ala-para-nitroanilide (pNA)], the enzyme preferred d-Leu-pNA and d-Phe-pNA as substrates. Therefore, we designated this gene as d-stereoselective aminopeptidase A (damA). The purified recombinant DamA was estimated to be a hexamer and was composed of two subunits with molecular masses of 29.5 and 11.5 kDa, respectively. Optimal hydrolytic activity of DamA toward d-Leu-pNA was observed at 50 °C and pH 8.0. The enzyme was stable up to 60 °C and from pH 4.0–11.0. DamA also exhibited aminolytic activity, producing d-Leu-d-Leu-NH2 from d-Leu-NH2 as a substrate. In the presence of 3.0 M NaCl, the amount of pNA liberated from d-Leu-pNA by DamA was 3.1-fold higher than that in the absence of NaCl. Thus, DamA is a halophilic enzyme. The enzyme was utilized to synthesize several hetero-dipeptides containing a d-amino acid at the N-terminus as well as physiologically active peptides.  相似文献   

18.
The protonation constants of some α-amino acids (glycine (Gly), l-alanine (Ala), l-valine (Val), l-serine (Ser), l-leucine (Leu) and l-isoleucine (Ile)) were studied in water and DMSO–water solution mixtures containing 30, 50 and 70 vol-% DMSO; in addition the complex formation equilibria of their copper(II) complexes were studied by potentiometric technique using a combined pH electrode system calibrated in concentration units of the hydrogen ion at 25 ± 0.1 °C under a nitrogen atmosphere, and at an ionic strength of 0.10 mol·dm?3 NaNO3. The protonation constants and the overall stability constants of copper(II) complexes were influenced by changes in solvent composition, and their variations are discussed in terms of solvent and structural properties.  相似文献   

19.
The racemisation ofcyclo-(l-Pro?l-Pro) (2) with metal amides in liq. ammonia was examined. The K-kation causes more extensive racemisation than Na-kation, which in turn is more effective than Li+. This, the racemisation of2 int-butyl alcohol with K+C6H5O? and the data gained from corresponding deuterated medium show that the racemisation of2 proceeds in two steps: in the first, the less stabletrans-cyclo-(l-Pro?d-Pro) (3) is formed, followed by the rapid conversion of3 to a mixture ofcyclo-(l-Pro?l-Pro) andcyclo-(d-Pro?d-Pro) in the second step.  相似文献   

20.
Liquid chromatography with electrochemical detection (LC-ED), coupled with in vivo microdialysis sampling, has been used for analysis of thiols. An acetylene black–dihexadecyl hydrogen phosphate (AB–DHP) composite film-modified electrode was used as working electrode. The AB–DHP-modified electrode enabled efficient electrocatalytic oxidation of l-cysteine (l-Cys) and glutathione (GSH) with relatively high sensitivity, stability, and longevity. The peak currents of l-Cys and GSH were linear in the concentrations ranges 2.0 × 10?7–2.0 × 10?4 and 3.0 × 10?7–5.0 × 10?4 mol L?1, respectively, with calculated detection limits (S/N = 3) of 1.0 × 10?7 and 2.0 × 10?7 mol L?1, respectively. The method has been successfully used to measure the amounts of l-Cys and GSH in striatal microdialysate of freely moving rats.  相似文献   

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