Specific detection of nicotinamide coenzymes by liquid chromatography and amperometric detection with immobilized glucose-6-phosphate dehydrogenase

A liquid chromatographic system for the specific and simultaneous detection of nicotinamide coenzymes is constructed by combining an immobilized glucose-6-phosphate dehydrogenase reactor with an amperometric system based on a phenazine methosulphate-mediated reaction, after separation on a reversed-phase column. The calibration graphs are linear from 0.05 to 20 nmol for all four coenzymes. The detection limits are 3.2, 5.2, 7.9 and 9.4 pmol for NADP⁺, NADPH, NAD⁺ and NADH, respectively. The enzyme reactor retains most of its original activity after repeated use for 2 months.     

Determination of dehydrogenase substrates by Clark-type oxygen electrodes and photosensitized coenzyme oxidation

The photosensitized oxidation of NADPH by oxygen can be used for the determination of the reduced coenzymes by means of a Clark oxygen electrode. This method is suitable for coupling to enzyme-catalysed dehydrogenation reactions and thus for the determination of glucose-6-phosphate with glucose-6-phosphate dehydrogenase and of glucose with the combined ATP/hexokinase/glucose-6-phosphate dehydrogenase system, even with the use of immobilized mediators.     

Nanomolar Level Amperometric Determination of ATP Through Substrate Recycling in an Enzyme Reactor in a FIA System

Abstract

ATP, adenosine-5′-triphosphate, was determined by recycling in an enzyme reactor with co-immobilized pyruvate kinase and hexokinase in the presence of glucose, NAD⁺, and phosphoenolpyruvate, PEP. Recycling produces glucose-6-phosphate which is converted to an equivalent amount of NADH by glucose-6-phosphate dehydrogenase. The NADH is detected at a graphite flow-through electrode modified with an adsorbed 3,3′-bis(benzo[a]phenoxazin-7-ium, 5-amino-9-(diethylamino))1,4,N,N′-diamidobenzene, BPT. Oxidation of NADH takes place at 0 mM vs Ag/AgCl due to the adsorbed phenoxazine. The amplification factor is directly proportional to the residence time in the reactor and it is increased as the flow rate decreases; it becomes350 at a flow rate of 0.07 ml/min. The amplification factor can be increased further by a controlled stop-time recycling; it became 1200 at a stop-time of 12 min. A theoretical expression for the amplification factor was derived and it shows that the amplification depends o n the residence time and the pseudo-first order rate coefficients of the recycling enzyme systems. The response was linear over more than three decades, from 1 nM to 5 μM ATP. The detection limit, 1 nM ATP was set by cofactor impurities in the reagent rather than by system sensitivity or noise.     

Determination of glucose in alcoholic beverages by flow injection with two internally coupled injection valves and an enzyme reactor

Two new flow configurations are described for the determination of a component in samples for which the matrix provides varying blank values. They are applied to the determination of glucose in alcoholic beverages, by using immobilized hexokinase and glucose-6-phosphate dehydrogenase, and spectrophotometric determination of the NADPH formed.     

Sequential determination of glucose and fructose in foods by flow-injection analysis with immobilized enzymes

A sequential method for determination of glucose and fructose involving the use of enzymes (hexokinase and glucose-6-phosphate dehydrogenase) immobilized on controlled-pore glass is proposed. The flow is selected so as to determine glucose or both sugars by fluorimetric determination of the NADH formed. The method is applied to the determination of these compounds in fruit juices, yoghourt and dessert powder with good results.     

Optimization of flow-injection systems for determination of substrates by means of enzyme amplification reactions and chemiluminescence detection

A flow-injection system is described that incorporates a small column reactor containing two co-immobilized, synergistically operating oxidoreductases, allowing determination of minute amounts of substrates by means of enzyme amplification and subsequent chemiluminescence detection of the hydrogen peroxide generated in the repeated redox cycling. With lactate oxidase and lactate dehydrogenase, and taking advantage of the fact that the enzymatic degradation step and the ensuing detection step can be individually optimized, the FIA-system has been optimized by factorial experiments to yield an amplification factor of over 140 for each of the two substrates lactate and pyruvate. With a linear calibration range of 0-6muM, the limits of detection for the two species were 48 and 103nM, respectively, and the sampling rate was 50-60/hr. The optimized system has also been employed for assay of glucose by utilizing a column reactor with immobilized glucose oxidase and glucose dehydrogenase, but yielded amplification factors of only 3-4. The large discrepancy in the performance of the two enzyme systems is discussed.     

Fructose determination using immobilized enzymes in a flow system with special emphasis on the effect of isomerism

A flow injection method for the determination of fructose has been developed using immobilized hexokinase, glucose phosphate isomerase and glucose-6-phosphate dehydrogenase. The produced NADH was monitored amperometrically with a graphite electrode modified by an adsorbed Nile-blue derivative. The response was linear from the detection limit, 1M to 3 mMD-fructose for a sample volume of 25l. Glucose, the only interfering substrate, was eliminated with a reactor containing immobilized glucose oxidase, mutarotase and catalase. The experiments indicate that hexokinase is selective for the-fructo-furanose isomer. The other isomers will spontaneously isomerize to-fructo-furanose with time, if the latter is consumed. The fructose response factor of an enzyme reactor or biosensor will therefore depend on the isomer distribution in the original sample solution. The effect of pH and temperature can be controlled, but complexing ligands like borate interfere in a complex way and should be absent.     

Use of various types of column reactors for flow-injection analysis.

Two or three different kinds of immobilized enzymes can be aligned in a minireactor so that sequential enzymatic reactions are carried out from upstream to downstream during flow-injection analysis. A lactate oxidase-catalase reactor, used as precolumn for removing pre-existing lactate in serum before the lactose dehydrogenase (LDH) reactions, was useful for the determination of serum LDH activity, which did not require any blank correction. A sequential glutamate dehydrogenase-glutamate oxidase reactor was also useful for a novel chemiluminometric determination of ammonia. On the other hand, a co-immobilized creatininase-creatinase-sarcosine oxidase reactor, in spite of containing creatininase which catalyses the reversible reaction, was the most efficient for the determination of serum creatinine.     

Highly selective and sensitive detection of NADP coenzymes using co-immobilized glucose-6-phosphate dehydrogenase/diaphorase reactors as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system

Two enzyme reactors prepared by the co-immobilization of two different glucose-6-phosphate dehydrogenases (G6PDH; from Leuconstoc mescenteroides (LM) and yeast (Y) and diaphorase are employed to enhance the sensitivity of NAD(P) coenzymes as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system. The NAD(P) coenzymes are recycled enzymatically during passage through the reactor in the presence of sufficient glucose-6-phosphate and oxygen in the carrier solution to produce a large amount of hydrogen peroxide, which is detected chemiluminometrically in the subsequent flow line. The G6PDH(LM)/diaphorase co-immobilized reactor is not specific between the NAD and NADP coenzymes, but shows a six fold selectivity towards NADP coenzymes compared to NAD coenzymes; the amplification factors for NAD and NADP coenzymes are 60 and 380, respectively, at a flow rate of 0.3 ml min(-1). In contrast, the G6PDH(Y)/diaphorase co-immobilized reactor is specific for NADP coenzymes with an amplification factor of about 600 (at a flow rate of 0.3 ml min(-1)). The detection limit is 6 fmol for both NADP(+) and NADPH.     

Chemiluminometric flow-injection method for determination of free l-malate in wine with co-immobilized malate dehydrogenase/NADH oxidase

A flow-injection system with a co-immobilized malate dehydrogenase/reduced nicotineamide adenine dinucleotide (NADH) oxidase reactor and a chemiluminometer is described for the determination of free l-malate in wine. Malate dehydrogenase and NADH oxidase were co-immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm x 4 mm i.d.). The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear from 3 x 10(-7) M to 2.5 x 10(-4) M (the linear correlation coefficient was 0.9998); the detection limit (signal-to-noise ratio, 3) was 8 x 10(-8) M. The sample throughput was 30 h(-1) without carryover. The ractor was renewed every 2 weeks.     

A bypass trapped-flow analysis system evaluation of enzyme kinetic parameters with a coupled enzyme assay and fluorescence detection

The newly developed flow instrument, bypass trapped-flow analysis system (ByT-FAS), was used to perform enzyme assays and generate data for evaluation of an enzyme's kinetic parameters. The enzyme assay was yeast hexokinase coupled to glucose-6-phosphate dehydrogenase and the production of reduced NADPH was monitored fluorometrically. The kinetic parameters of hexokinase that were evaluated with the system were the apparent K(m)'s of the substrates glucose and ATP, and the K(i) of the competitive inhibitor for ATP, ADP. The ByT-FAS generated data were comparable to other manually derived published values of the kinetic constants for hexokinase and validates the utility of the ByT-FAS system for making absolute enzyme measurements. The semi-automated ByT-FAS system can perform analyses more rapidly than manual assay techniques with less manual manipulation and smaller sample and/or reagent volumes.     

Micro-flow in vivo analysis of L-glutamate with an on-line enzyme amplifier based on substrate recycling.

A micro-flow enzyme system with a microdialysis probe is proposed for the amperometric detection of trace amounts of neurotransmitter L-glutamate released from rat brain cells. The L-glutamate oxidase (EC 1.4.3.11)/glutamate dehydrogenase (EC 1.4.1.4) coimmobilized reactor was used to enhance the sensitivity of L-glutamate as an on-line amplifier based on substrate recycling. A poly(1,2-diaminobenzene) film-coated platinum electrode was also used to selectively detect only the hydrogen peroxide generated into a upstream enzyme reactor, without interference from oxidizable species, such as L-ascorbate, the adsorption of low molecular-weight proteins in a dialysate, and NADPH added to the carrier solution to initiate substrate recycling. By the present in vivo system, L-glutamate was selectively assayed with about a 600-fold increase in sensitivity compared with the unamplified responses. The detection limit was 0.08 mumol dm-3. This method was applied to an in vivo assay of L-glutamate in the extracellular space of rat brain; also, monitoring of the L-glutamate level changed after a continuous stimulation of KCl to demonstrate the reliability of the system.     

Indirect Flow-Injection Assays for Glucose-6-Phosphate Dehydrogenase/Glucose-6-Phosphate and Malate Dehydrogenase/L-Malate Using Immobilized Bacterial Luciferase

Abstract

Flow-injection procedures for the indirect determination of glucose-6-phosphate dehydrogenase, glucose-6-phosphate, malate dehydrogenase and L-malate are described. They are based on a coupled reaction with bacterial luciferase and oxidoreductase co-immobilized on cyanogen bromide activated Sepharose 4B. the limits of detection are 0.015 pmol for glucose-6-phosphate dehydrogenase, 0.03 pmol for malate dehydrogenase, 10^?8 mol^?1 for glucose-6-phosphate and 10^?6 mol l^?1 for L-malate. the reproducibility is less than 5% relative standard deviation (n=5) for all assays and the sample throughput is 60 h^?1.     

Enzymatic production and analysis of d-xylulose with a recirculating flow system and post-column liquid chromatographic detection using a co-immobilized enzyme reaction and a chemically modified electrode

A pure d-xylulose and standard was produced by isomerization of d-xylose in a recirculating flow system incorporating an enzyme reactor containing immobilized xylose isomerase. The d-xylulose formed was purified chromatographically. A selective chromatographic detection system was used in the post-column mode. It consisted of a co-immobilized enzyme reactor (CIMER) with xylose isomerase, mutarotase and glucose dehydrogenase on-line with a chemically modified electrode for selective elctrochemical oxidation of NADH. The pure standard was compared with commercially available d-xylulose, which was confirmed to contain impurities of d-glucose and d-xylose.     

Highly sensitive detection of l-glutamate by on-line amperometric micro-flow analysis based on enzymatic substrate recycling

A highly selective and sensitive on-line monitoring system is proposed for amperometric assay of trace amounts of l-glutamate. The system includes a microdialysis probe, immobilized enzyme reactor, and poly(1,2-diaminobenzene)-coated platinum electrode. The enzyme reactor prepared by the co-immobilization of l-glutamate oxidase and glutamate dehydrogenase are here employed to enhance the sensitivity of l-glutamate as an on-line amplifier based on the substrate recycling. The l-glutamate in the dialysate from the probe are recycled enzymatically during passage through the reactor in the presence of sufficient amounts of NADH and oxygen to produce a large amount of hydrogen peroxide, which is detected if selectively at a downstream poly(1,2-diaminobenzene)-coated platinum electrode without interference from oxidizable species such as l-ascorbate in the sample and NADH added to the carrier buffer. The cycle is also initiated with 2-oxoglutarate, and so saccharopine dehydrogenase reactor is positioned in series before the amplifier reactor to remove 2-oxoglutarate in the dialysate. By the present method, l-glutamate is selectively assayed with a 160-fold increase in sensitivity compared with the unamplified responses. The detection limit is 0.5x10(-7) M of l-glutamate.     

Determination of aliphatic amino acids in serum by HPLC with fluorimetric detection using co-immobilized enzyme reactor

A simple and selective method is presented for the determination of aliphatic amino acids such as L-alanine, L-valine, L-isoleucine and L-leucine in serum using HPLC with detection by co-immobilized alanine dehydrogenase/leucine dehydrogenase post-column reactor and fluorimeter. The enzymes were simultaneously immobilized on chitosan beads. The separation was achieved by means of an ods column with elution with phosphate buffer (pH 7.0). The system gave linear responses over two orders of magnitude and detection limits at 1-2muM levels.     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.     

Spectrophotometric and fluorimetric enzymatic determination of serum creatine kinase mb isoenzyme by using immuno-inhibition

Two fully enzymatic methods, colorimetric and fluorimetric, are reported for the determination of creatine kinase (EC 2.7.3.2) MB isoenzyme after immune-inhibition with the use of goat anti-human CK-M IgG antibodies. The residual creatine kinase activity is assayed by using hexokinase and glucose-6-phosphate dehydrogenase systems; the resulting NADPH is determined spectrophotometrically by reaction with p-iodonitro-tetrazolium violet in the presence of diaphorase as an intermediate electron carrier, or fluorimetrically by coupling the NADPH with resazurin/diaphorase to form resorufin. Both assays take only 12 min and require only 100 or 25μl of serum. The calibration plots of enzyme activities are linear up to 580 and 435 U l^-1 of CK-MB in serum for the spectrophotometric and fluorimetric assays, respectively, the coefficients of variation being 2.3% and 4.6%, and the recovery values 103% and 100% respectively. The results correlate very well with those obtained by the Helena electrophoresis—fluoridensitometric methods. As little as 4 U l^-1 and 2 U l^-1 CK-MB could be measured reproducibly.     

Expression of NAD(H) Kinase and Glucose-6-Phosphate Dehydrogenase Improve NADPH Supply and l-isoleucine Biosynthesis in Corynebacterium glutamicum ssp. lactofermentum

Corynebacterium glutamicum is the workhorse for the production of amino acids, including l-isoleucine (Ile). During Ile biosynthesis, NADPH is required as a crucial cofactor. In this study, four NADPH-supplying strategies based on NAD kinase, NADH kinase, glucose-6-phosphate dehydrogenase, and NAD kinase coupling with glucose-6-phosphate dehydrogenase were compared, and their influences on Ile biosynthesis were examined. PpnK is a NAD kinase of C. glutamicum ssp. lactofermentum JHI3-156 that predominantly phosphorylates NAD⁺ to produce NADP⁺. Pos5 is a NADH kinase of Saccharomyces cerevisiae that predominantly phosphorylates NADH to produce NADPH. Zwf is a glucose-6-phosphate dehydrogenase of JHI3-156. The ppnK, POS5, zwf, and zwf-ppnK genes were overexpressed in the Ile-producing strain JHI3-156. The expression of all four genes increased intracellular NADPH concentration and Ile production. The increase of NADPH concentration and Ile production in a POS5-expressing strain (229 and 75.6 %, respectively) was higher than that in a ppnK-expression strain. The expression of zwf also increased NADPH supply and Ile biosynthesis, but the constitutive expression of zwf was not as effective as the inducible expression of zwf. Coexpression of zwf and ppnK genes greatly enhanced NADPH supply and thus improved Ile production by up to 85.9 %, indicating that this strategy was the most effective one. These results are helpful for improving Ile biosynthesis and other biosynthetic processes.     

Immobilization of L-glutamate oxidase and peroxidase for glutamate determination in flow injection analysis system

Streptomyces SP.N 14, isolated from soil samples, produced extracellular L-glutamate oxidase (GOD) in liquid culture. After a two-step ammonium sulfate purification and dextran G-150 chromatography, the specific activity was reached at 28.2 U/mg. The partial purified enzyme and horseradish peroxidase (HRP) were covalently coupled to alkylamine controlled pore glass (CPG) by means of glutaraldehyde. About 200–300 U/g of immobilized GOD and 300–400 U/g of immobilized HRP were obtained. The immobilized enzymes were packed into a teflon tube and used in flow injection analysis (FIA) for glutamate in broth. A good linear range was observed for this immobilized enzyme system at 0.1–2.0 mM, and the precision was 2.8% (n = 25). More than 80 samples were measured within an hour. One enzyme column with about 4 U of immobilized GOD and 5 U of immobilized HRP, applied for 50 assays/d, has been used for more than 50 d. The concentration of L-glutamate remaining lower than 2.0 mM, the determination of glutamate in this system was not affected by pH and temperature within the range of 6.0–7.0 and 25–35‡C, respectively. The system was applied to determine L-glutamate in broth samples during L-glutamate fermentation, and good correlation was achieved between results obtained with the system and with the Warburg’s method.