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1.
An assay for the determination of NAD has been developed utilizing the coupled oxidoreductase activity of liver alcohol dehydrogenase. The coupled reaction between ethanol and lactaldehyde is driven by the removal of one of the products, acetaldehyde, into a semicarbazide solution. Under the stated conditions, a linear relationship exists between the absorbance of acetaldehyde semicarbazone and NAD concentration in the reaction mixture. The principal advantages of this method are speed and simplicity. NAD+ and NADH are assayed by the same procedure, which is also used to measure NADP+ and NADPH after these nucleotides have been converted to NAD+ and NADH, respectively. 相似文献
2.
A new, simple and very sensitive assay for NADH-ferredoxin or flavodoxin reductase activity is described. The assay is based on the nonenzymatic reduction of the metronidazole by ferredoxin or flavodoxin. In the presence of NADH, ferredoxin or flavodoxin and cell-free extract of clostridia, no metronidazole reduction is observed; the reaction occurs only if acetyl-CoA is added to the reaction mixture. Metronidazole reduction is quantitated by the spectrophotometric measurement at 320 nm. In this assay the change in absorbance is linearly related to the amount of clostridial extract for concentration of 0.1 to 0.8 mg/ml and to the flavodoxin or ferredoxin for concentrations of 0.5 to 8 nmol/ml. 相似文献
3.
J Hupert S Mobarhan T J Layden V M Papa D J Lucchesi 《Biochimie et biologie cellulaire》1991,69(8):509-514
Enzymatic conversion of retinal to retinoic acid in rat liver cytosol was detected using a rapid and sensitive assay based on high pressure liquid chromatography (HPLC). This retinal oxidase assay system did not require extraction steps or any other manipulation of the sample mixture once the sample vial was sealed for incubation. The product (retinoic acid) and the reactant (retinal) were separated by HPLC in 14.0 min with a sensitivity of 15 and 40 pmol per injection for retinoic acid and retinal, respectively. Enzymatic activity was observed to be linear with protein concentration (0-2.4 mg/mL) and time (0-30 min) and displayed a broad pH maximum of 7.7-9.7. The enzyme exhibited Michaelis-Menten single-substrate kinetics with an apparent Km of 0.25 mM. The average specific activity in nine normal rats was 35.6 +/- 3.3 nmol retinoic acid formed/h per mg protein. Incubation of the enzyme with zinc did not affect the rate of retinoic acid synthesis. Dithiothreitol inhibited the reaction. Both NAD and NADH stimulated retinoic acid formation. Formation of retinol was also observed when these pyridine nucleotides were added to the reaction mixture, indicating the presence of retinal reductase activity. The results of kinetic studies suggest that NADH may act indirectly to stimulate retinoic acid formation. 相似文献
4.
The purification and steady-state kinetic behaviour of rabbit heart mitochondrial NAD(P)+ malic enzyme. 总被引:1,自引:1,他引:0
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The mitochondrial NAD(P)+ malic enzyme [EC 1.1.1.39, L-malate:NAD+ oxidoreductase (decarboxylating)] was purified from rabbit heart to a specific activity of 7 units (mumol/min)/mg at 23 degrees C. A study of the reductive carboxylation reaction indicates that this enzymic reaction is reversible. The rate of the reductive carboxylation reaction appears to be completely inhibited at an NADH concentration of 0.92 mM. A substrate saturation curve of this reaction with NADH as the varied substrate describes this inhibition. The apparent kinetic parameters for this reaction are Ka(NADH) = 239 microM and Vr = 1.1 mumol/min per mg at 23 degrees C. The steady-state product-inhibition patterns for pyruvate and NADH indicate a sequential binding of the substrates: NAD+ followed by L-malate. These data also indicate that NADH is the last product released. A steady-state kinetic model is proposed that incorporates NADH-enzyme dead-end complexes. 相似文献
5.
Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate 总被引:4,自引:0,他引:4
A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Km of 74 mg polysaccharide/liter was determined, which corresponds to 2.1 X 10(-4) M, expressed as concentration of uronic acid; Vmax was 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3'-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mM), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mM. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry. 相似文献
6.
The sequential metabolism of substrates by heterogeneous bacterial populations has been previously reported from this laboratory in studies with high substrate concentrations. This phenomenon has now been shown to occur at very dilute substrate concentrations, i.e., 5 mg/liter of glucose plus 5 mg/liter of sorbitol, in studies conducted under the conditions of the standard biochemical oxygen demand (BOD) test. Sequential metabolism of these substrates resulted in a diphasic curve of accumulated oxygen uptake wherein the two phases were separated by a discernible plateau. These findings illustrate one possible explanation for the generation of discontinuity in the kinetic course of carbonaceous BOD exertion. 相似文献
7.
Metabolic engineering of Bacillus subtilis for ethanol production: lactate dehydrogenase plays a key role in fermentative metabolism 总被引:1,自引:0,他引:1
Romero S Merino E Bolívar F Gosset G Martinez A 《Applied and environmental microbiology》2007,73(16):5190-5198
Wild-type Bacillus subtilis ferments 20 g/liter glucose in 48 h, producing lactate and butanediol, but not ethanol or acetate. To construct an ethanologenic B. subtilis strain, homologous recombination was used to disrupt the native lactate dehydrogenase (LDH) gene (ldh) by chromosomal insertion of the Zymomonas mobilis pyruvate decarboxylase gene (pdc) and alcohol dehydrogenase II gene (adhB) under the control of the ldh native promoter. The values of the intracellular PDC and ADHII enzymatic activities of the engineered B. subtilis BS35 strain were similar to those found in an ethanologenic Escherichia coli strain. BS35 produced ethanol and butanediol; however, the cell growth and glucose consumption rates were reduced by 70 and 65%, respectively, in comparison to those in the progenitor strain. To eliminate butanediol production, the acetolactate synthase gene (alsS) was inactivated. In the BS36 strain (BS35 delta alsS), ethanol production was enhanced, with a high yield (89% of the theoretical); however, the cell growth and glucose consumption rates remained low. Interestingly, kinetic characterization of LDH from B. subtilis showed that it is able to oxidize NADH and NADPH. The expression of the transhydrogenase encoded by udhA from E. coli allowed a partial recovery of the cell growth rate and an early onset of ethanol production. Beyond pyruvate-to-lactate conversion and NADH oxidation, an additional key physiological role of LDH for glucose consumption under fermentative conditions is suggested. Long-term cultivation showed that 8.9 g/liter of ethanol can be obtained using strain BS37 (BS35 delta alsS udhA+). As far as we know, this is the highest ethanol titer and yield reported with a B. subtilis strain. 相似文献
8.
The online photoreaction of the rose bengal photosensitized luminol–copper (II) chemiluminescence (CL) system was used for the determination of β-nicotinamide adenine dinucleotide (NADH) and ethanol (EtOH) in pharmaceutical formulations combined with a flow injection technique. NADH can significantly enhance the CL emission of the reaction. For EtOH, alcohol dehydrogenase in soluble form was utilized in the presence of nicotinamide adenine dinucleotide resulting in NADH production. The limit of detection (3σ blank, 𝑛 = 3) of 4.0 × 10−8 and 2.17 × 10−5 M, and linear range 1.3 × 10−7 to 2.5 × 10−5 M (R2 = 0.9998, n = 6) and 0.11–2.17 × 10−3 M (R2 = 0.9996, n = 6) were obtained for NADH and EtOH respectively. The injection rate was 100 h−1 with a relative standard deviation (n = 3) of 1.5–4.8% in the range studied for both analytes. The procedure was satisfactorily applied to pharmaceutical formulations with recoveries in the range 91.6 ± 3.0% to 110 ± 2.0% for NADH and 88 ± 3.0% to 95.4 ± 4.0% for EtOH. The results obtained were very consistent and did not differ considerably from the reported approaches at a 95% confidence limit. The possible mechanism of the CL reaction is also explained briefly. 相似文献
9.
The inhibitory effect of ethanol is studied during alcoholic fermentation in strict anaerobiosis (initial dissolved oxygen stripped by gasing pure nitrogen). It is demonstrated that the ethanol produced during the batch fermentation is more inhibitory than the added ethanol (in the range of 0 to 72.6g/liter). By analogy with noncompetitive enzyme kinetic inhibition, the inhibition constant for added ethanol is 105.2 g/liter and 3.8 g/liter for produced ethanol, which exhibits the same inhibition effects in all experiments where ethanol was added. The measurement of the intracellular alcohol concentration can explain the dual inhibitory effects of ethanol. 相似文献
10.
Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: central role of mitochondrial NADH redox state 总被引:1,自引:0,他引:1
A K Orpana M M Orava R K Vihko M H?rk?nen C J Eriksson 《Journal of steroid biochemistry》1990,36(6):603-608
The mechanisms by which ethanol (EtOH) inhibits the human chorionic gonadotropin (hCG)-stimulated testosterone synthesis was studied in isolated rat Leydig cells in vitro. EtOH inhibited steroidogenesis, but this inhibition was reversed by L-glutamate (Glu) and an uncoupler of the oxidative phosphorylation, 2,4-dinitrophenol (DNP). The mechanism of EtOH-induced inhibition was studied by measuring steroidogenic precursors and comparing them with the cytosolic and mitochondrial NADH redox states during uncoupling or in the presence of Glu. DNP had a dual effect. Low concentrations abolished the EtOH-induced inhibition of progesterone to testosterone formation suggesting that the inhibitory step was at or before progesterone formation. A large concentration led to an overall decrease in steroidogenesis indicating toxic effects on steroidogenesis. The mitochondrial NADH/NAD+ ratio, measured as the 3-hydroxybutyrate/acetoacetate ratio, decreased simultaneously when steroidogenesis was stimulated, either during uncoupling or in the presence of Glu, whereas cytosolic NADH/NAD+ ratio, measured as lactate/pyruvate ratio showed no response. These results demonstrate that the rise in the mitochondrial NADH/NAD+ ratio rather than in the cytosolic one is connected with the inhibition of testosterone synthesis by EtOH in isolated Leydig cells. The EtOH-induced high mitochondrial NADH/NAD+ ratio may deplete mitochondrial oxalacetate concentrations. This can decrease the activity of several transport shuttles and interrupt the flow of mitochondrial citrate into the smooth endoplasmic reticulum, which then reflects to decreased rate of steroidogenesis in the presence of ethanol. 相似文献
11.
Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system. 相似文献
12.
Continuous enzymatic production of xylitol with simultaneous coenzyme regeneration in a charged membrane reactor 总被引:5,自引:0,他引:5
We have developed a new process for the production of xylitol from D-xylose by enzyme technology. An NADH-dependent xylose reductase (XR) from Candida tenuis catalyzes the reduction of xylose, which is coupled to enzymatic oxidations of D-glucose or D-xylose by glucose dehydrogenase from Bacillus cereus to make achievable an up to 10,000-fold regeneration of NADH per cycle of discontinuous conversion. Using a simple kinetic model as a tool for process optimization, suitable conditions with regard to performance and stability of the multi-component reaction system were established, and 300 g/L of substrate could be converted in yields above 96% in one single batch reaction. Due to selective and over 98% complete retention of the native coenzyme by negatively charged nanofiltration membranes used in a continuously operated enzyme reactor, a specific productivity of 80 g xylitol per liter, day, and kilounit of XR was maintained over the 150-h reaction time with only a single dosage of NADH. (c) 1996 John Wiley & Sons, Inc. 相似文献
13.
Monitoring of phenylketonuria: a colorimetric method for the determination of plasma phenylalanine using L-phenylalanine dehydrogenase 总被引:1,自引:0,他引:1
A simple, rapid, accurate, and precise colorimetric assay for the determination of L-phenylalanine in plasma samples using L-phenylalanine dehydrogenase [L-phenylalanine:NAD+-oxidoreductase (deaminating)] from Rhodococcus sp. M 4 is described. The enzyme catalyzes the NAD-dependent oxidative deamination of L-phenylalanine. However, the equilibrium of reaction favors L-phenylalanine formation. By stoichiometric coupling of this reaction with diaphorase/iodonitro tetrazolium chloride (INT) the formed NADH converts INT to a formazan whereby the reaction is displaced in favor of phenylpyruvate. Using a kinetic approach the increase in absorbance at 492 nm shows linearity over more than 30 min. Deproteinized standard solutions of L-phenylalanine in the range from 30 to 1200 mumol/liter show a linearity between the dAformazan/30 min and the substrate concentration. In phenylketonuria (PKU) plasma samples no interferences caused by L-tyrosine or phenylpyruvic acid are seen. Applicability is demonstrated by comparative determination of plasma L-phenylalanine of treated PKU patients by the colorimetric method and automated amino acid analysis. 相似文献
14.
O V Lebedeva N N Ugarova T V De?ko E I Ra?nina T A Makhlis 《Prikladnaia biokhimiia i mikrobiologiia》1985,21(1):114-121
The luminous bacteria Beneckea Harveyi were immobilized on BrCN-sepharose and cellulose films activated with cyanuric chloride. Preparations with high luciferase and FMN-reductase activities were obtained, which showed no background luminescence without NADH being added. The storage conditions for the preparations obtained were optimized, and their kinetic parameters and thermostability were studied. Standard curves for NADH determining within the concentration range 1 nM-1 microM were plotted with the detection level of 1 picomol NADH. The preparations are very promising for bioluminescent assay due to their high activity, simple production, high stability during storage and a possibility for the repeated use. 相似文献
15.
C. M. Watteeuw W. B. Armiger D. L. Ristroph A. E. Humphrey 《Biotechnology and bioengineering》1979,21(7):1221-1237
The production of single cell protein (SCP) form ethanol is an interesting process to study from a biochemical engineering viewpoint. The cellular yield mainly depends upon the metabolic activity of the cells and the amount of substrate available. Fedbatch fermentations Were run in a 70 liter highly instrumented computer-coupled fermentor using Candida utilis. Respiratory quotient and culture fluorescence, measuring NADH, indicate by which pathway ethanol is utilized. By monitoring these parameters it is possible to control the ethanol concentration so that accumulation of acetate is minimized and the conversion of ethanol to cell mass is maximized. 相似文献
16.
N W Heinsman A M Valente H G Smienk A van der Padt M C Franssen A de Groot K van 't Riet 《Biotechnology and bioengineering》2001,76(3):193-199
The Novozym 435(R) catalyzed esterification and hydrolysis reactions of 4-methyloctanoic acid (ethyl ester) were investigated. In both the hydrolysis and esterification reactions, the increase of ethanol concentration led to an increase in enantiomeric ratio (E). For hydrolysis of the ethyl ester, the E-value increased from 5.5 [0% (v/v) EtOH] up to 12 [20% (v/v) EtOH]. In case of esterification, the E-value was already 16 [14% (v/v) EtOH] and rose to 57 [73% (v/v) EtOH]. When combining these results of esterification and hydrolysis, an enantiomeric ratio of 350 can be estimated for the sequential kinetic resolution of 4-methyloctanoic acid. In this way, enantiopure 4-methyloctanoic acid could be obtained after two consecutive reaction steps. 相似文献
17.
18.
A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure. 相似文献
19.
P Gauduchon J J Baumann E Bar J Y Le Tala?r 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1985,300(13):475-478
A kinetic spectrophotometric method in which galactose transfer is coupled to the production of NADH, has been adapted to the assay of galactosyltransferase activity in human serum. Under the described conditions, the rate of NADH production is linear with regard to enzyme concentration, and directly depends upon the various biochemical factors which control galactosyltransferase activity. 相似文献
20.
The overall goal of this work was to develop a saccharification method for the production of third generation biofuel (i.e. bioethanol) using feedstock of the invasive marine macroalga Gracilaria salicornia. Under optimum conditions (120 °C and 2% sulfuric acid for 30 min), dilute acid hydrolysis of the homogenized invasive plants yielded a low concentration of glucose (4.1 mM or 4.3 g glucose/kg fresh algal biomass). However, two-stage hydrolysis of the homogenates (combination of dilute acid hydrolysis with enzymatic hydrolysis) produced 13.8 g of glucose from one kilogram of fresh algal feedstock. Batch fermentation analysis produced 79.1 g EtOH from one kilogram of dried invasive algal feedstock using the ethanologenic strain Escherichia coli KO11. Furthermore, ethanol production kinetics indicated that the invasive algal feedstock contained different types of sugar, including C(5) -sugar. This study represents the first report on third generation biofuel production from invasive macroalgae, suggesting that there is great potential for the production of renewable energy using marine invasive biomass. 相似文献