首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 62 毫秒
1.
Sorbitol dehydrogenase (SDH) originating from recombinant Escherichia coli cells is immobilized on gold electrodes. First of all, (4-carboxy-2,5,7-trinitrofluorenyliden)malon-nitrile (CTFM) is adsorbed on the surface as mediator. In a second step, the cofactor β-nicotinamide adenine dinucleotide (NAD+) is immobilized on the gold electrode. Due to the formation of a complex between the mediator and the cofactor, the electron transfer rate can be enhanced by adding calcium ions to the buffer. The immobilization of NAD+ and SDH on the surface has been achieved by cross-linking with the glutaraldehyde/bovine serum albumin system. The successful biofunctionalization is monitored by cyclic voltammetry.Paper presented at the “Jahrestagung der Fachgruppe Angewandte Electrochemie der Gesellschaft Deutscher Chemiker, Düsseldorf, 11.-14.09.2005”.  相似文献   

2.
Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD+), β-nicotinamide adenine dinucleotide phosphate NADP+, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD+ or NADP+ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)+ and NAD(P)H cofactors in the concentration range of 1×10−6 to 1×10−3 M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD+ and lactate dehydrogenase using the NADH-imprinted polymer film.  相似文献   

3.
The reagentless and oxygen‐independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD+) on chitosan (CHIT) chains. The CHIT‐NAD+‐ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8–30 µM ethanol; dynamic range up to 20 mM). The CHIT‐NAD+‐dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases‐based electrochemical biosensors. Interestingly, the CHIT‐NAD+ solutions preserved their enzymatic activity even after five years of storage at 4 °C.  相似文献   

4.
Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD+ less effectively than NADP+. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD+ or NADP+. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD+ and NADP+ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD+ or NADP+. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.  相似文献   

5.
NADP+-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg2+ was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP+ and no NAD-dependent activity could be detected. The K m values displayed for NADP+ and isocitrate were 59 and 31 μM (Mg2+), 120 μM and 58 μM (Mn2+), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K m values for NADP+ of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD+-dependent activity was detected for R322D mutant and the K m and k cat values for NAD+ were 47,000 μM and 0.38 s?1, respectively. Although the R322D mutant showed low activity with NAD+, it revealed the feasibility of engineering an eukaryotic IDP to a NAD+-dependent one.  相似文献   

6.
Two different self‐contained ethanol amperometric biosensors incorporating layered [Ru(phend)2bpy]2+‐intercalated zirconium phosphate (ZrP) as the mediator as well as yeast‐alcohol dehydrogenase (y‐ADH) and its cofactor nicotinamide adenine dinucleotide (NAD+) were constructed to improve upon a design previously reported where only this mediator was immobilized in the surface of a modified electrode. In the first biosensor, a [Ru(phend)2bpy]2+‐intercalated ZrP modified carbon paste electrode (CPE) was improved by immobilizing in its surface both y‐ADH and NAD+ using quaternized Nafion membrane. In the second biosensor, a glassy carbon electrode was modified with [Ru(phend)2bpy]2+‐intercalated ZrP, y‐ADH, and NAD+ using Nafion as the holding matrix. Calibration plots for ethanol sensing were constructed in the presence and absence of ZrP. In the absence of ZrP in the surface of the modified glassy carbon electrode, leaching of ADH was observed as detected by UV‐vis spectrophotometry. Ethanol sensing was also tested in the presence and absence of ascorbate to measure the selectivity of the sensor for ethanol. These two ethanol biosensors were compared to a previously reported one where the y‐ADH and the NAD+ were in solution, not immobilized.  相似文献   

7.
L-lactate dehydrogenase (LDH) catalyzes the interconversion of an oxoacid (pyruvate) and hydroxy-acid (lactate) using the NADH/NAD+ pair as a redox cofactor. The enzyme has a commercial significance, as it can be used to produce chiral building blocks for the synthesis of key pharmaceuticals and agrochemicals. However, the substrate inhibition which is due to an abortive NAD+-pyruvate complex reducing the steady state concentration of functional LDH limits its use in industry. This substrate inhibition can be overcome by weaking the binding of NAD+. The conserved aspartic acid residue at position 38 was replaced by the longer basic arginine side chain (D38R) using PCR based overlap extension mutagenesis technique in the hope of weakening NAD+-binding. The mutant gene was overexpressed in theEscherichia coli high-expression vector pKK223-3 in JM105 cells; then, the mutant protein was produced. Comparing the effect of substrate inhibition in the arginine-38 mutant with wild-type, substrate inhibition is decreased threefold.  相似文献   

8.
It is believed that the biological effects of chelating agents such as crown ethers are largely related to their ability to form complexes with ions and/or to facilitate ion transport across membranes. Specific influences are rarely related. Here we present the evidence that even one of the simplest representatives of the crown ether super-family, 1,4,7,10,13,16-hexaoxacyclooctane (18-crown-6), is able to affect the activity of Na+, K+-ATPase directly. Using nonlinear regression fitting to kinetic data we have found that the crown ether diminishes the apparent Michaelis constant, K m , and the maximal rate of ATP hydrolysis, V m , acting as noncompetitive inhibitors. The apparent dissociation constants, K i , for the crown interaction with the free ATPase and with the enzyme-substrate complex were established to be of 77 ± 3 mM and 21 ± 2 mM, respectively. So 18-crown-6 possesses weak but “direct” pharmacological activity on Na+, K+-ATPase hinders the formation of enzyme–substrate complex and detains the enzyme in this state.  相似文献   

9.
A ferrocene-labeled high molecular weight cofactor derivative (PEI-Fc-NAD) was prepared by attaching both ferrocene and nicotinamide adenine dinucleotide (NAD+) to a water soluble polyelectrolyte, polyethylenimine (PEI). Approximately 9.8% and 2.9% of all the primary amino groups of PEI were coupled with ferrocene and the bioactive cofactor, respectively. The cyclic voltammograms of PEI-Fc-NAD exhibited a one-electron transfer process, and the difference between the anodic and cathodic peak potential was found to be 80 mV. The PEI-Fc-NAD was used together with NAD-dependent dehydrogenase to construct a reagentless biosensor. An NAD-dependent alcohol dehydrogenase (ADH) was selected as the model enzyme, and both ADH and PEI-Fc-NAD were retained onto the sensing area of gold electrodes by a dialysis membrane or immobilized by layer-by-layer adsorption method. In both cases, the modified electrodes showed current response to ethanol without the addition of native NAD+ to the system, which suggested that the electrical communication between ADH and the electrode was achieved through PEI-Fc-NAD. In summary, PEI-Fc-NAD provides a new way for immobilization of mediator and cofactor, and exhibits the potential as a platform for constructing reagentless NAD-dependent dehydrogenase biosensors.  相似文献   

10.
Different soluble NAD+-dependent alcohol dehydrogenase (ADH) isozymes were detected in cell-free homogenates from aerobically grown mycelia of YR-1 strain of Mucor circinelloides isolated from petroleumcontaminated soil samples. Depending on the carbon source present in the growth media, multiple NAD+-dependent ADHs were detected when hexadecane or decane was used as the sole carbon source in the culture media. ADH activities from aerobically or anaerobically grown mycelium or yeast cells, respectively, were detected when growth medium with glucose added was the sole carbon source; the enzyme activity exhibited optimum pH for the oxidation of different alcohols (methanol, ethanol, and hexadecanol) similar to that of the corresponding aldehyde (≈7.0). Zymogram analysis conducted with partially purified fractions of extracts from aerobic mycelium or anaerobic yeast cells of the YR-1 strain grown in glucose as the sole carbon source indicated the presence of a single NAD+-dependent ADH enzyme in each case, and the activity level was higher in the yeast cells. ADH enzyme from mycelium grown in different carbon sources showed high activity using ethanol as substrate, although higher activity was displayed when the cells were grown in hexadecane as the sole carbon source. Zymogram analysis with these extracts showed that this particular strain of M. circinelloides has four different isozymes with ADH activity and, interestingly, one of them, ADH4, was identified also as phenanthrene-diol-dehydrogenase, an enzyme that possibly participates in the aromatic hydrocarbon biodegradation pathway.  相似文献   

11.
Three novel dinucleotide analogues of nicotinamide adenine dinucleotide (NAD+) have been synthesised from d-ribonolactone. These compounds incorporate a thiophene moiety in place of nicotinamide and are hydrolytically stable. They have been evaluated as inhibitors of adenosine diphosphate ribosyl cyclase, glutamate dehydrogenase and Sir2 acyltransferase activities. Enzyme specificity and a high level of inhibition was observed for the dehydrogenase.  相似文献   

12.
An enzymatic method for determining L-malic acid in wine based on an L-malate sensing layer with nicotinamide adenine dinucleotide (NAD+), L-malate dehydrogenase (L-MDH) and diaphorase (DI), immobilized by sol-gel technology, was constructed and evaluated. The sol-gel glass was prepared with tetramethoxysilane (TMOS), water and HCl. L-MDH catalyzes the reaction between L-malate and NAD+, producing NADH, whose fluorescence (λ exc = 340 nm, λ em = 430 nm) could be directly related to the amount of L-malate. NADH is converted to NAD+ by applying hexacyanoferrate(III) as oxidant in the presence of DI. Some parameters affecting sol-gel encapsulation and the pH of the enzymatic reaction were studied. The sensing layer has a dynamic range of 0.1–1.0 g/L of L-malate and a long-term storage stability of 25 days. It exhibits acceptable reproducibility [s r(%)≈10] and allows six regenerations. The content of L-malic acid was determined for different types of wine, and polyvinylpolypyrrolidone (PVPP) was used as a bleaching agent with red wine. The results obtained for the wine samples using the sensing layer are comparable to those obtained from a reference method based on UV-vis molecular absorption spectrometry, if the matrix effect is corrected for.  相似文献   

13.
The effects of La3+ on inward K+ channels at plasma membrane in vicia guard cells are investigated using the whole-cell patch-clamp recording mode. It is shown that La3+ on both sides of plasma membrane blocks inward K+ currents in a concentration-dependent manner, indicating that La3+ binding sites may exist on both sides of plasma membrane in guard cells in vicia. The dose response is fitted by the Michaelis-Menten relation characterized by an inhibitor constant K i of 2.56±0.25 μmol · L−1 (outside membrane) and (1.18±0.11)×10−15 mol · L−1 (inside membrane). Intracellular La3+ has much stronger inhibitory effect on inward K+ currents than extracellular La3+ does, suggesting there may exist stronger binding sites inside membrane than outside membrane. Since ion channel activities of guard cells directly affect plant stomatal movement and water status, our results imply that rare earth elements might have potential practical values in regulating plant water status and strengthening plant drought endurance.  相似文献   

14.
The complexation reactions between dibenzo-24-crown-8 (DB24C8) and K+, Rb+, Cs+ and Tl+ ions were studied conductometrically in different acetonitrile–nitromethane mixtures at various temperatures. The formation constants of the resulting 1:1 complexes were calculated from the computer fitting of the molar conductance–mole ratio data at different temperatures. At 25 °C and in all solvent mixtures used, the stability of the resulting complexes varied in the order Tl+ > K+ > Rb+ > Cs+. The enthalpy and entropy changes of the complexation reactions were evaluated from the temperature dependence of formation constants. It was found that the stability of the resulting complexes increased with increasing nitromethane in the solvent mixture. The TΔS° vs. ΔH° plot of all thermodynamic data obtained shows a fairly good linear correlation indicating the existence of enthalpy–entropy compensation in the complexation reactions.  相似文献   

15.
The reactive adenosine derivative, adenosine 5′-O-[S-(4-hydroxy-2,3-dioxobutyl)]-thiophosphate (AMPS-HDB), contains a dicarbonyl group linked to the purine nucleotide at a position equivalent to the pyrophosphate region of NAD+. AMPS-HDB was used as a chemical label towards Candida boidinii formate dehydrogenase (CbFDH). AMPS-HDB reacts covalently with CbFDH, leading to complete inactivation of the enzyme activity. The inactivation kinetics of CbFDH fit the Kitz and Wilson model for time-dependent, irreversible inhibition (KD = 0.66 ± 0.15 mM, first order maximum rate constant k3 = 0.198 ± 0.06 min−1). NAD+ and NADH protects CbFDH from inactivation by AMPS-HDB, showing the specificity of the reaction. Molecular modelling studies revealed Arg174 as a candidate residue able to be modified by the dicarbonyl group of AMPS-HDB. Arg174 is a strictly conserved residue among FDHs and is located at the Rossmann fold, the common mononucleotide-binding motif of dehydrogenases. Arg174 was replaced by Asn, using site-directed mutagenesis. The mutant enzyme CbFDHArg174Asn was showed to be resistant to inactivation by AMPS-HDB, confirming that the guanidinium group of Arg174 is the target for AMPS-HDB. The CbFDHArg174Asn mutant enzyme exhibited substantial reduced affinity for NAD+ and lower thermostability. The results of the study underline the pivotal and multifunctional role of Arg174 in catalysis, coenzyme binding and structural stability of CbFDH.  相似文献   

16.
Water-soluble CuInS2 quantum dots (QDs) stabilized with 3-mercaptopropionic acid were synthesized in aqueous solution and then coated with bovine serum albumin. The resulting particles display fluorescence with a peak at 680 nm that is effectively quenched by 1, 4-dihydro-nicotinamide adenine dinucleotide (NADH), but not by 1, 4-nicotinamide adenine dinucleotide (NAD+). The enzyme lactate dehydrogenase catalyzes the reduction of pyruvate and dehydrogenation of lactic acid using NAD+ or NADH as a cosubstrate. The new QDs were applied to monitor the course of lactate dehydrogenase-catalyzed reaction of pyruvate by detecting NADH via its quenching effect. This resulted in a convenient and selective detection scheme for pyruvate. The detection limit is as low as 25 nM.
Figure
Bovine serum albumin coated CuInS2 quantum dots (QDs) are quenched by 1,4- dihydronicotinamide adenine dinucleotide (NADH) that could react with pyruvic acid and lactate dehydrogenase. Therefore, the CuInS2 QDs could be used to detect pyruvic acid.  相似文献   

17.
A sequential injection system to monitor glycerol in a Saccharomyces cerevisiae fermentation process was developed. The method relies on the rate of formation of nicotinamide adenine dinucleotide in its reduced form (NADH, measured spectrophotometrically at 340 nm) from the reaction of glycerol with NAD+ cofactor, catalysed by the enzyme glycerol dehydrogenase present in solution. This procedure enables the determination of glycerol between 0.046 and 0.46 g/l, (corresponding to yeast fermentation samples with concentrations up to 50 g/l) with good repeatability (relative standard deviation for n = 10 lower than 2.2% for three different samples) at a sampling frequency of 25/h. The detection and quantification limits using a miniaturised spectrophotometer were 0.13 and 0.44 mM, respectively. Reagent consumption was of 0.45 μmol NAD+ and 1.8 μg enzyme per assay, and the waste production was 2.8 ml per determination. Results obtained for samples were in agreement with those obtained with a high-performance liquid chromatography method.  相似文献   

18.
The dominant voltammetric response of a yeast suspension in neutral or slightly alkaline media can be assigned to the redox transformations of nicotinamide adenine dinucleotide (NAD+/NADH) and nicotinamide adenine dinucleotide phosphate (NADP+/NADPH). By immobilization of yeast on platinum, a stable electrode can be prepared which shows an electrocatalytic activity towards the reduction of NADP+ and the reoxidation of the product formed. Reversible cyclic voltammetric responses were obtained. The peak currents depend practically linearly on the NADP-Na2 concentration and on the square root of the scan rate. The surface mass changes accompanying the redox transformations were monitored by an electrochemical quartz crystal nanobalance.  相似文献   

19.
He X  Ni X  Wang Y  Wang K  Jian L 《Talanta》2011,83(3):937-942
An electrochemical method for nicotinamide adenine dinucleotide (NAD+) detection with high sensitivity and selectivity has been developed by using molecular beacon (MB)-like DNA and Escherichia coli DNA ligase. In this method, MB-like DNA labeled with 5′-SH and 3′-biotin was self-assembled onto a gold electrode in its duplex form by means of facile gold-thiol chemistry, which resulted in blockage of electronic transmission. It was eT OFF state. In the presence of NAD+, E. coli DNA ligase was activated, and the two nucleotide fragments which were complementary to the loop of the MB-like DNA could be ligated by the NAD+-dependent E. coli DNA ligase. Hybridization of the ligated DNA with the MB-like DNA induced a large conformational change in this surface-confined DNA structure, which in turn pushed the biotin away from the electrode surface and made the electrons exchange freely with the electrode. Then the generated electrochemical signals can be measured by differential pulse voltammetry (DPV). Under optimized conditions, a linear response to logarithmic concentration of NAD+ range from 3 nM to 5 μM and a detection limit of 1.8 nM were obtained. Furthermore, the proposed strategy had sufficient selectivity to discriminate NAD+ from its analogues.  相似文献   

20.
Recombination rate coefficients of protonated and deuterated ions KrH+, KrD+, XeH+ and XeD+ were measured using Flowing Afterglow with Langmuir Probe (FALP). Helium at 1600 Pa and at temperature 250 K was used as a buffer gas in the experiments. Kr, Xe, H2 and D2 were introduced to a flow tube to form the desired ions. Because of small differences in proton affinities of Kr, D2 and H2 mixtures of ions, KrD+/D3+ and KrH+/H3+ are formed in the afterglow plasma, influencing the plasma decay. To obtain a recombination rate coefficient for a particular ion, the dependencies on partial pressures of gases used in the ion formation were measured. The obtained rate coefficients, αKrD+(250 K) = (0.9 ± 0.3) × 10−8 cm3 s−1 and αXeD+(250 K) = (8 ± 2) × 10−8 cm3 s−1 are compared with αKrH+(250 K) = (2.0 ± 0.6) × 10−8 cm3 s−1 and αXeH+(250 K) = (8 ± 2) × 10−8 cm3 s−1.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号