A Highly Sensitive Bioluminescent Assay of β-D-Galactosidase from Escherichia coli Using 2-Nitrophenyl-β-D-galactopyranoside as a Substrate

Abstract

A highly sensitive bioluminescent assay of β-D-galactosidase from Escherichia coli is described. D-Galactose was released from 2-nitrophenyl-β-D-galactopyranoside as a substrate by the catalytic action of β-D-galactosidase, and subsequently NADH was formed using galactose dehydrogenase. NADH was measured by a bioluminescent assay using NAD(P)H:FMN oxidoreductase and luciferase from Photobacterium fischeri. The detection limits of β-D-galactosidase for 100 and 1,000 min assays were 2 × 10^?21 mol and 2 × 10^?22 mol, respectively. When the volume of the reaction mixture for β-D-galactosidase assay was reduced from 2 μ to 0.5 μ1, the detection limits were reduced to half.     

Highly Sensitive Bioluminescent Assay of Dehydrogenases using Nad (P) H: Fmn Oxidoreductase and Luciferase from Photoeacterium Fischeri

Abstract

A highly sensitive bioluminescent assay of dehydrogenases was performed. NADH was produced by the catalytic action of alcohol and glucose-6-phosphate dehydrogenases and subsequently measured with high sensitivity by a bioluminescent assay using NAD (P) H : FMN oxidoreductase and luciferase from Photobacterium fischeri. The minimal amount of dehydrogenases that could be measured was 0.0055 amol (5.5 × 10^?-²¹ mol).     

Multi-function fibre-optic sensor for the bioluminescent flow determination of ATP or NADH

A multi-function biosensor for the determination of either ATP or NADH using a single bioluminescence-based fibre-optic probe is described. This was made possible by co-immobilizing the firefly luciferase from Photinus pyralis for ATP analysis with the bacterial/oxidoreductase system from Vibrio harveyi for NADH analysis, on the same preactivated polyamide membrane. Compatible analytical conditions with regard to the activity and stability of each bioluminescent system were selected, enabling them to attain their highest performances. It was possible to perform continuous-flow measurements of ATP and NADH over a wide (log-log) linear calibration range with a relative standard deviation of 4.0–4.5% and detection limits of 0.25 pmol ATP and 5 pmol NADH.     

FLUORESCENT POLYENE ALIPHATICS AS SPECTROSCOPIC AND MECHANISTIC PROBES FOR BACTERIAL LUCIFERASE: EVIDENCE AGAINST CARBONYL PRODUCT FROM ALDEHYDE AS THE PRIMARY EXCITED SPECIES

The fluorescent α-parinaric acid (α-PAC) and β-parinaric acid (β-PAC) were converted to the corresponding aldehydes and alcohols all of which exhibited absorption and fluorescence properties closely resembling those of the parent acids. α-PAC and β-PAC each binds to luciferase in competition with aldehyde. The hydrophobic nature of the aldehyde site was indicated by the enhanced fluorescence quantum yields of the bound α-PAC and β-PAC. These two polyene acids and the β-parinaryl alcohol were shown to stabilize the luciferase flavin-peroxide intermediate. α-Parinaraldehyde (α-PAD) and bt-parinaraldehyde (β-PAD) were active substrates for Vibrio harveyi and Vibrio fischeri luciferases and, for the former enzyme, exhibited K_m, values similar to and quantum yields about20–30% as those for decanal and dodecanal. For the V. harveyi luciferase with reduced FMN as a co-substrate, the α-PAD- or β-PAD-initiated luminescence was indistinguishable from the normal emission obtained with octanal (γ_max 495 nm) showing no additional 430-nm component correlatable with emission from excited α-PAC or β-PAC. In reactions using reduced 2-thioFMN for V. harveyi luciferase or reduced FMN for V. fischeri luciferase plus yellow fluorescent protein, the replacement of octanal by β-PAD again resulted in no additional 430-nm emission. The lack of any emission correlatable with excited α-PAC, β-PAC, or equivalent carbonyl product was not due to the quenching of the polyene moiety by chemical transformation, binding to luciferase, or a 100% energy transfer to the flavin 4a-hydroxide emitter. These results strongly favor singlet state of flavin 4a-hydroxide rather than singlet or triplet carbonyl product from aldehyde as the primary excited species in the normal luciferase reaction in the absence of any additional fluorescent protein.     

Bioluminescent Flow Sensors: L-Alanine Determination in Serum and Urine

Abstract

A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).     

Bioluminescence-based fibre-optic sensor with entrapped co-reactant: an approach for designing a self-contained biosensor

The possibility of designing a self-contained fibre-optic biosensor, i.e., that can be used without renewing reagents in the probe, was investigated. A probe specific for NADH involves immobilized bioluminescent enzymes which require the presence of two co-reactants [a flavin substrate (FMN) and an aldehyde] to catalyse light emission. The FMN was non-covalently immobilized in a synthetic film and was internally released in the vicinity of the bound enzymes, at the sensing tip of the bioprobe. Release of FMN was achieved from the two different matrices tested: a collagen film and a poly(vinyl alcohol) (PVA) network. Continuous-flow assays of NADH could be performed over a linear dynamic range from 10 pmol to 1 nmol. A PVA matrix appears to be a promising support for designing a self-contained biosensor, as 30–35 reliable measurements (R.S.D.=5%) could be achieved without a decrease in the sensor signal, compared with only 10–15 assays with a collagen film.     

Consistency of the Mediated Electrochemical Method and the Fluorescence Method in Monitoring the Catabolic Activities of Yeasts

Abstract

The mediated electrochemical method and the intrinsic NADH fluorescence method were employed in evaluating the catabolic activities of three yeasts. The responses from the menadione/ferricyanide system were 70.00 ± 2.25 nA, 61.39 ± 1.76 nA, and 57.18 ± 1.51 nA, respectively, for Saccharomyces cerevisiae, Pachysolen tannophilus, and Pichia stipitis. The NADH fluorescence intensities were 1638 ± 25.46 FI, 1039 ± 18.67 FI, and 963.4 ± 15.78 FI, respectively,for S. cerevisiae, P. tannophilus, and P. stipitis. It was evident that there is a positive relationship between the mediated electrochemical method and the intrinsic NADH fluorescence method in cellular metabolic activity assays.     

Hexavalent Chromium Reduction by Free and Immobilized Cell-free Extract of <Emphasis Type="Italic">Arthrobacter rhombi-RE</Emphasis>

In the present study, hexavalent chromium (Cr(VI)) reduction potential of chromium reductase associated with the cell-free extracts (CFE) of Arthrobacter rhombi-RE species was evaluated. Arthrobacter rhombi-RE, an efficient Cr(VI) reducing bacterium, was enriched and isolated from a chromium-contaminated site. Chromium reductase activity of Arthrobacter rhombi-RE strain was associated with the cell-free extract and the contribution of extracellular enzymes to Cr(VI) reduction was negligible. NADH enhanced the chromium reductase activity. The enzyme activity was optimal at a pH of 5.5 and a temperature of 30 °C. Among the ten electron donors screened, sodium pyruvate was the most effective one followed by NADH and propionic acid. Michaelis–Menten constant, K _m, and maximum reaction rate, V _max, obtained from the Lineweaver–Burk plot were 48 μM and 4.09 nM/mg protein/min, respectively, in presence of NADH as electron donor and 170.5 μM and 4.29 nM/mg protein/min, respectively, in presence of sodium pyruvate as electron donor. Ca²⁺ enhanced the enzyme activity while Hg²⁺, Cd²⁺, Ba²⁺, and Zn²⁺ inhibited the enzyme activity. Among the various immobilization matrices screened, calcium alginate beads seemed to be the most effective one. Though immobilized enzyme system was able to reduce Cr(VI), the performance was not very encouraging in continuous mode of operation.     

Study of the Electrochemical Behavior of Disperse Blue 1‐Modified Graphite Electrodes. Application to the Flow Determination of NADH

The preparation and the electrochemical study of Disperse Blue 1‐chemically modified electrodes (DB1‐CME), as well as their efficiency for the electrocatalytic oxidation of NADH is described. The proposed mediator was immobilized by physical adsorption onto graphite electrodes. The electrochemical behavior of DB1‐CME was studied with cyclic voltammetry. The electrochemical redox reaction of DB1 was found to be reversible, revealing two well‐shaped pair of peaks with formal potentials 152 and ?42 mV, respectively, (vs. Ag/AgCl/3M KCl) at pH 6.5. The current I_p has a linear relationship with the scan rate up to 800 mV s^?1, which is indicative for a fast electron transfer kinetics. The dissociation constants of the immobilized DB1 redox couple were calculated pK₁=4 and pK₂=5. The electrochemical rate constants of the immobilized DB1 were calculated k₁°=18 s^?1 and k₂°=23 s^?1 (Γ=2.36 nmol cm^?2). The modified electrodes were mounted in a flow injection manifold, poised at +150 mV (vs. Ag/AgCl/3M KCl) and a catalytic current due to the oxidation of NADH was measured. The reproducibility was 1.4% RSD (n=11 for 30 μM NADH) The behavior of the sensor towards different reducing compounds was investigated. The sensor exhibited good operational and storage stability.     

Structure-Toxicity Relationships for Aliphatic Compounds Encompassing a Variety of Mechanisms of Toxic Action to Vibrio fischeri

Abstract

QSARs based upon the logarithm of the octanol-water partition coefficient, logP, and energy of the lowest unoccupied molecular orbital, E_LUMO were developed to model the toxicity of aliphatic compounds to the marine bacterium Vibrio fischeri. Statistically robust, hydrophobic-dependent QSARs were found for chloroalcohols and haloacetonitriles. Modelling of the toxicity of the haloesters and the diones required the use of terms to describe both hydrophobicity and electrophilicity. The differences in intercepts, slopes, and fit of these models suggest different electrophilic mechanisms occur between classes, as well as within the diones and haloesters. In order to model globally the toxicity of aliphatic compounds to V. fischeri, all the data determined in this study were combined with those determined previously for alkanones, alkanals, and alkenals. A highly predictive two-parameter QSAR [pT₁₅ = 0.760(log P) ?0.625(E _LUMO) ?0.466; n = 63, s = 0.462, r ² = 0.846, F = 171, Pr > F = 0.0001] was developed for the combined data that models across classes and is independent of mechanisms of action. The toxicity of these compounds to V. fischeri compares well to the toxicity (50% population growth inhibition) to the ciliate Tetrahymena pyriformis (r ² = 0.850).     

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose

NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10^-5 M and 5.8% at 2 × 10^-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10^-4 M and 3.2% at 1 × 10^-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.     

Amplified Detection of NAD+ and NADH by the Enzymatic Cycling with the Use of Immobilized Enzymes in a Flow System

Abstract

The enzymatic cycling for the detection of NAD⁺ and NADH in low level was carried out in the flow system with immobilized enzymes. Alcohol dehydrogenase and glutamate dehydrogenase immobilized on Sepharose 4B were used for the cycling reaction. The compound produced in the enzymatic cycling reaction was subjected to an enzymatic reaction to yield NADH, which was detected fluorometrically.     

Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane

Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters K_m and V_max for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors.     

Whole cell immobilised biosensors for toxicity assessment of a wastewater treatment plant treating phenolics-containing waste

Wastewater treatment plants dealing with industrial wastes are often susceptible to overload of toxic influent that can partially or completely destroy treatment for extended periods. An obvious candidate for monitoring toxicity in such wastewater systems is bioluminescent bacteria. However, the natural bioluminescent bacteria can be particularly sensitive to some industrial wastes and therefore their response to normal operational conditions does not reflect the status of the microbial community responsible for treatment. Moreover, the salt dependence of the marine bioluminescent bacteria, and the temperature sensitivity of some strains, further complicate their use. Here we describe the construction of whole cell genetically modified bioluminescent biosensors and their immobilisation for use in monitoring the toxicity of a complex industrial wastewater containing phenolic materials. A hand-held luminometer was designed for laboratory or field use, and the immobilisation system designed with several things in mind: the geometry of the instrument; the need for containment of GM bacteria; the maximisation of the bioavailability of the wastewater to the biosensor. The performance of a candidate GM sensor was compared with Vibrio fischeri in liquid culture and after immobilisation in thin films of poly(vinyl alcohol) (PVA) cryogels. The biosensors were tested against pure phenol and 3-chlorophenol as a reference toxic chemical known to be much more toxic to bacteria than phenol. The biosensors were then tested with the phenolics-containing industrial wastewater. The immobilisation system proved to operate predictably with pure toxicants, and was able to discriminate toxicity of various zones within the wastewater treatment plant.     

Synthetic Routes to Coelenterazine and Other Imidazo[1,2‐a]pyrazin‐3‐one Luciferins: Essential Tools for Bioluminescence‐Based Investigations

In the last few decades, bioluminescent systems based on the expression of a luciferase and the addition of a luciferin to monitor the emission of light have become very important tools for biological investigations. A growing proportion of these systems use coelenterazine or analogues of imidazo[1,2‐a]pyrazine luciferins along with photoproteins or luciferases from sea creatures such as Aequorea, Renilla, Gaussia or Oplophorus. Central to the success of these tools are the synthetic pathways developed not only to prepare the naturally occurring luciferins, but also to design altered compounds that exhibit improved bioluminescence. Current work is indeed focused on the design of systems exhibiting extended luminescence (“glow” systems) or redshifted wavelengths, as well as constructions better adapted to conditions in cells or in vivo. This review describes the synthetic pathways used to prepare imidazo[1,2‐a]pyrazine luciferins along with the research efforts aimed at preparing analogues even better suited to the design of assays.     

Enzyme/semiconductor nanoclusters combined systems for novel amperometric biosensors

In this work quantum-sized CdS nanocrystals were synthesized using a quaternary water-in-oil microemulsion and immobilized onto gold working electrode by self-assembled monolayers techniques. Formaldehyde dehydrogenase was covalently immobilized onto a protecting membrane, which was stratified on part of the semiconductor nanoparticles modified electrode. The covalent enzyme immobilization has been required to improve the stability of the catalytic oxidation of formaldehyde, which occurs after light stimulation of the semiconductor through the electron/hole recombination. A study about the best electrochemical oxidation potentials under different flow conditions was performed. Preliminary sensor stability and interferences tests were also carried out, for a sensitive and selective detection of formaldehyde. A detection limit of 41 ppb of formaldehyde was calculated and an operational stability of 6 h was achieved under flow conditions by means of this novel amperometric biosensor based on FDH-semiconductor hybrid systems, not requiring NAD⁺/NADH as charge transfer in the enzymatic reaction.     

Determination of Glycerol and Serum Triglycerides with Collagen-Immobilized Glycerol Dehydrogenase and Amperometric Detection

Abstract

An amperometric procedure is described for the determination of glycerol and triglycerides in aqueous samples and in serum using glycerol dehydrogenase immobilized on a collagen membrane. Glycerol is determined by measurement of the steady-state oxidation currents generated at a platinum electrode by NADH produced in the enzyme-catalyzed reaction. The triglycerides were first hydrolyzed by the enzyme lipase in solution and the resulting glycerol determined similarly. Olive oil, determined to contain 78 % triolein, was used as the source of triglycerides in this study. For both glycerol and triglycerides the calibration plots are linear in the range from 0 to 12 μM, with detection limits of 0.2 and 0.7 μM, respectively. The immobilized glycerol dehydrogenase retained high operational activity for a period longer than 30 days.     

Electrochemical Sensing of NADH and Glutamate Based on Meldola Blue in 1,2‐Diaminobenzene and 3,4‐Ethylenedioxythiophene Polymer Films

The redox mediator Meldola blue (MB) was entrapped into two polymers, poly‐1,2‐diaminobenzene (p‐DAB) and poly‐3,4‐ethylenedioxythiophene (p‐EDOT) by potential cycling and films were applied to NADH oxidation with subsequent glutamate detection using immobilized glutamate dehydrogenase. Both polymer films were tested for electrocatalysis of NADH using amperometry at E_app=0.1 V vs. Ag/AgCl and similar response characteristics were obtained with sensitivity values of 6.1 nA μM^?1, linear range up to 0.5 mM (R²=0.9972) and LOD of 50 μM. Subsequent amperometric determination of glutamate resulted in sensitivity 0.7 nA μM^?1, linearity 0–100 μM and detection limit of 2 μM glutamate.     

Properties of pectinesterase and endo-d-polygalacturonase coimmobilized in a porous glass support

Derivatives of pectinesterase and polygalacturonase, both individually immobilized and coimmobilized, were obtained and characterized. Homologous soluble systems were also studied to establish differences between the effect of the immobilization process and the presence of the other enzyme. Immobilization or coimmobilization did not change the optima pH or temperature for the enzymes. However, optimum ionic strength was displaced toward higher values for immobilized pectinesterase, while for polygalacturonase immobilization resulted in a wider range for activity.K _m value remained nearly unchanged for pectinesterase, and decreased for polygalacturonase. TheV _m value decreased with the immobilization process for the two enzymes, except for polygalacturonase immobilization in presence of pectinesterase. Soluble pectinesterase activity showed a competitive inhibition by polygalacturonic acid (K_i = 0.44 mg/mL). Either immobilization or presence of polygalacturonase rendered the enzyme insensitive to the inhibitory effect. Thermal stability of pectinesterase was not improved after immobilization. On the contrary, the thermal stability of endo-D-polygalacturonase was improved slightly by presence of pectinesterase, and in a greater extent by immobilization. Individually immobilized and coimmobilized pectinesterase activities kept 90 and 60%, respectively, of their initial values after more than one year stored at 3-5 °C. The two endo-D-polygalacturonase derivatives showed the same activity decay pattern along 10 mo storage at 3-5 °C. The two immobilized pectinesterase derivatives showed similar operational stabilities during continuous operation. The presence of pectinesterase remarkably increased the operational stability of the immobilized endo-D-poly galacturonase.     

Immobilization of pig liver microsomes

Microsomes from pig liver were covalently coupled to Sepharose activated by CNBr and to Sephadex activated by 1,1’-carbonyldiimidazole. Microsomes were also entrapped inside Ca-alginate andk-carrageenan gels. The concentration of immobilized cytochrome P-450 was determined by CO-difference spectra. The activity of the monooxygenase system was demonstrated by theN-demethylation of aminopyrine, theO-demethylation ofp-nitroanisole, and the hydroxylation of perhexiline maleate. Upon immobilization, a 30–40% and a 60–70% decrease in V _max ^app for theOandN-demethylations were respectively observed. The V _max ^app values for the hydroxylation of perhexiline maleate were essentially the same for the different immobilized forms and for the freely suspended microsomal cytochrome P-450. Under storage at 4°C, microsomes entrapped insidek-carrageenan and Ca-alginate were less stable than the free microsomes, whereas immobilization on CNBr-activated Sepharose improved the stability of the hepatic microsomal monooxygenase system at the same temperature. These types of immobilized microsomes have the advantage of being easily recovered and reused for other assays. Finally, microsomes entrapped insidek-carrageenan or Ca-alginate can be used to follow up the continuous metabolization ofp-nitroanisole for several hours in a stirred-batch reactor.