Enzyme activity fingerprinting with substrate cocktails

In the postgenomic era, emphasis is shifting from protein identification to protein functional analysis. Enzyme function can be characterized by measuring activity across series of substrates, which generates an activity profile or fingerprint. Activity fingerprinting is particularly useful to differentiate closely related enzymes. Previously reported fingerprinting methods use series of parallel measurements, which are complex and difficult to reproduce. Here we report a new method for fingerprinting enzyme activities based on using mixtures of substrates, or substrate cocktails, in a single reaction that is then analyzed by HPLC. The fingerprints produced are highly reproducible and allow functional differentiation and classification of closely related enzymes, as demonstrated for a series of lipases and esterases. The method is practical, general, and flexible in terms of reaction conditions and can be adapted to any reaction type.     

Enzyme monolayer- and bilayer-modified electrodes with diaphorase and dehydrogenases

Diaphorase was immobilized covalently as a monolayer on a tin(IV) oxide electrode, and the diaphorase electrode thus obtained responded to NADH amperometrically in the presence of ferricyanide or 2,6-dichloroindophenol as the electron mediator. The response was one to two orders of magnitude larger than that of a bare electrode. Further derivatization of the diaphorase electrode with a dehydrogenase (glucose, lactate or alcohol dehydrogenase), which reduces NAD to NADH by reaction with the substrate, yielded dehydrogenase/diaphorase heterobilayer-modified electrodes. These electrodes functioned as sensors for the respective substrate with NAD and ferricyanide as the mediators. Each bilayer electrode responded to the substrate only in the presence of added NAD; this provides evidence for the essential contribution of diaphorase to the sensor performance. As much as 60 to 80% of the electron mediator reduced by the enzymatic reaction was utilized in the amperometric response.     

Signal amplification by substrate recycling on polyaniline/lactate oxidase/lactate dehydrogenase bienzyme electrodes

The bienzyme electrodes were fabricated by coimmobilization of lactate oxidase (LOD) and lactate dehydrogenase (LDH) onto electrochemically prepared polyaniline (PANI) films. These PANI/LOD/LDH bienzyme electrodes were shown to provide signal amplification by substrate recycling, making it possible to detect l-lactate at lower concentrations (0.1-1 mM). The PANI/LOD/LDH bienzyme electrodes were found to be stable for about 21 d at 4–10°C.     

Enzyme electrodes for continuous in-vivo monitoring

Blood metabolite levels can alter rapidly in the acutely ill patient and may need to be monitored continuously. Enzyme electrodes show promise as selective transducers for organic species and could provide the basis for practical monitoring systems.     

Enzyme electrodes for the sugar substitute aspartame

A sensor of aspartame (l-aspartyl-l-phenylalanine methyl ester) is prepared by chemical immobilization of l-aspartase on an ammonia-selective electrode. Semi logarithmic response (E vs. log C) was observed in the 1 × 10^?3?1 × 10^?2 M range with a slope of ?30 mV/decade. The sensor is stable for more than eight days. The probe is successfully used for the assay of aspartame in commercially available sweeteners.     

Electrodes with extremely high hydrogen overvoltages as substrate electrodes for stripping analysis based on bismuth-coated electrodes

Hydrogen evolution bothers stripping analysis significantly. Dioctyl phthalate-based carbon paste electrode exhibits extremely wide cathodic potential window. It is explored as a powerful substrate electrode to solve the problem of hydrogen evolution and further improve reproducibility for stripping analysis using bismuth-coated electrodes for the first time. It was successfully applied to the simultaneous determination of Zn²⁺, Cd²⁺, and Pb²⁺. Linear responses are obtained for Zn²⁺ in the range of 10–100 μg L⁻¹ and for Pb²⁺ and Cd²⁺ in the range of 5–100 μg L⁻¹. The detection limits for Zn²⁺, Cd²⁺, and Pb²⁺ are 0.1 μg L⁻¹, 0.22 μg L⁻¹ and 0.44 μg L⁻¹, respectively. The method has been successfully applied to the determination of Zn²⁺, Cd²⁺, and Pb²⁺ in waste water samples. The detection strategy based on the combination of dioctyl phthalate-based carbon paste electrode and bismuth-coated electrodes holds great promise for stripping analysis.     

Enzyme electrodes using ultrafiltration membranes as enzyme carriers

Two kinds of commercially available ultrafiltration membranes were used for binding enzymes. These enzyme-bound membranes were connected with oxygen electrodes to build up enzyme electrodes. Quantitative assays of glucose, sucrose, and hydrogen peroxide were carried out using these enzyme electrodes in concentration ranges for glucose from 1 to 20 mg/dl, sucrose from 0.5 to 5 mg/ml, and hydrogen peroxide from 5 to 200 ppm. Similarly, D-alanine was measured in the range 0.005 to 0.05 mg/ml, but the calibration curve was not a straight line.     

Enzyme modified microband electrodes: cross-talk effects and their elimination

One microband of an array of four microband electrodes (1 mm long and 25 microm wide with a 25 microm gap) was modified with glucose oxidase by direct electrochemically assisted immobilisation, giving a stable microbiosensor with an apparent Michaelis-Menten constant of 12 mM and an i(max) of 80 nA. Cross-talk effects on the adjacent microbands were studied and three different methods for their elimination were tested: the most efficient one involved catalase deposition on the adjacent microband. Under these conditions, the maximum response at the unmodified microbands was in the worst case about 3% compared with the response of the modified microband. This approach has the potential to fabricate a multianalyte microbiosensor.     

l-Glutamate enzyme electrode involving amplification by substrate recycling

An enzyme electrode with an amplified response for l-glutamate is constructed by co-immobilizing l-glutamate oxidase and glutamate-pyruvate transaminase on a platinum disk electrode. With l-alanine (1 mM) in the solution, the sensitivity of the electrode to l-glutamate is greatly increased by the substrate recycling reaction between the two enzymes; the detection limit is 0.2 nM in the presence of l-alanine compared with 0.2 μM in its absence. The amplification factor for 1 μM l-glutamate is ca. 53.     

Enzyme electrodes with improved mechanical and analytical characteristics obtained by binding enzymes to nylon nets

Enzyme electrodes obtained by binding enzymes on nylon net show improved mechanical and analytical characteristics. The chemical binding process is mild so that the final membrane retains a high enzyme activity. The resulting membrane also has high mechanical strength and can be assembled on the electrode surface several times without damage. Response times without nylon net are excellent. Examples with single and dual enzymes coupled with an oxygen or ammonia electrode are discussed.     

The kinetic mechanism of horse liver alcohol dehydrogenase with thio-nad as co-enzyme

The kinetic mechanism of horse liver alcohol dehydrogenase with thio-NAD as a co-enzyme and ethanol as a substrate has been investigated by steady-state analysis at a pH of 7.0. The enzyme obeys an ordered Bi-Bi mechanism, with the co-enzyme binding first and dissociating last. Central ternary complexes are shown, to be kinetically important for the turnover number: in fact, non-competitive inhibition by acetaldehyde is observed when ethanol is the variable substrate. The position of the rate-limiting step for enzyme turnover has been located by using stopped-flow and isotope-replacement techniques. The absence of a “burst” reaction and the presence of a primary isotope effect on the steady-state rate shows that the turnover number contains a large contribution from hydride transfer.The rate acceleration observed with thio-NAD relative to NAD can thus be explained by assuming a shift of the rate-limiting step from co-enzyme dissociation to interconversion of the central ternary complexes.The influence of substrate concentration on the isotope effect has also been studied in detail for the system thio-NAD/benzyl alcohol.In the system thio-NAD/ethanol, inhibition by ethanol was observed at concentrations above 80 mmol l⁻¹, which could indicate the formation of a ternary enzyme-thio-NADH-ethanol dead-end complex. In addition, the product inhibition patterns of acetaldehyde in the thio-NAD/ethanol reaction are consistent with the existence of a ternary enzyme-thio-NAD-acetaldehyde complex. All these data have been compared with those mentioned in the literature for the NAD/ethanol system, and the differences between the kinetic mechanisms in these two cases are discussed.     

Oxidase-cofactor electrodes aimed at the quantitative measure of substrate concentrations

Two approaches are described briefly for utilizing oxidase enzymes in electrochemical sensors. In one approach an oxidase enzyme flavin cofactor was covalently coupled to the electrode surface with the anticipated readout being a current proportional to the concentration of enzyme substrate. In the second approach the enzyme glucose oxidase was immobilized on platinum such that the potential of the platinum varied with the concentration of glucose in a solution buffered at pH 7.4. The status of the two projects is described.     

Enzyme production and profile by Aspergillus niger during solid substrate fermentation using palm kernel cake as substrate

The oil palm sector is one of the major plantation industries in Malaysia. Palm kernel cake is a byproduct of extracted palm kernel oil. Mostly palm kernel cake is wasted or is mixed with other nutrients and used as animal feed, especially for ruminant animals. Recently, palm kernel cake has been identified as an important ingredient for the formulation of animal feed, and it is also exported especially to Europe, South Korea, and Japan. It can barely be consumed by nonruminant (monogastric) animals owing to the high percentages of hemicellulose and cellulose contents. Palm kernel cake must undergo suitable pretreatment in order to decrease the percentage of hemicellulose and cellulose. One of the methods employed in this study is fermentation with microorganisms, particularly fungi, to partially degrade the hemicellulose and cellulose content. This work focused on the production of enzymes by Aspergillus niger and profiling using palm kernel cake as carbon source.     

Enzyme sequence and competition electrodes based on immobilized glucose oxidase,peroxidase and catalase

The toxicologically important peroxidase substrates bilirubin and aminopyrine can be determined by combination of immobilized glucose oxidase, horseradish peroxidase and catalase, forming so-called enzyme sequence and enzyme competition electrodes. Bilirubin and aminopyrine are determined in the concentration range 5–50 μM.     

Deposition of polyaniline on RVC electrodes: effect of substrate thickness

Polyaniline films, obtained by either chemical or electrochemical deposition on reticulated vitreous carbon (RVC), were investigated as a function of the substrate thickness. The electrochemical properties of these RVC/Pani electrodes were assessed by cyclic voltammetry and electrochemical impedance spectroscopy (EIS), whereas the morphology of the Pani films on RVC was analyzed by scanning electron microscopy (SEM). The cyclic voltammetric results revealed that the oxidation/reduction charges for electrodeposited polyaniline decrease as the RVC thickness is increased. Conversely, the charge densities for the chemically deposited films do not present a significant dependence on the substrate thickness. Two time constants, appearing in all the EIS spectra, indicate that an ohmic drop effect within the RVC substrate affects the polymer electrodeposition and the electrochemical behavior of the obtained electrodes. Therefore, an electric equivalent circuit considering the different electrochemical environments at the outer and inner RVC surfaces was proposed to analyze the EIS data.     

514— Oxidase-cofactor electrodes aimed at the quantitative measure of substrate concentrations

Two approaches are described briefly for utilizing oxidase enzymes in electrochemical sensors. In one approach an oxidase enzyme flavin cofactor was covalently coupled to the electrode surface with the anticipated readout being a current proportional to the concentration of enzyme substrate. In the second approach enzyme glocose oxidase was immobilized on platinum such that the potential of the platinum varied with the concentration of glucose in a solution buffered at pH 7.4. The status of the two projects is described.     

UPD metal electrocatalysis of formic acid oxidation at teflon-bonded carbon substrate electrodes

Teflon-bonded porous electrodes have been prepared for the study of the upd metal effect on the electrocatalysis of formic acid oxidation. The influence of upd lead on finely divided platinum with porous carbon as substrate was tested in fuel cell anodes for the oxidation of formic acid in 1 M HClO₄ electrolyte.In order to investigate the increase in activity through the use of upd lead, the co-adsorption of lead and a strongly adsorbed intermediate was followed by a flux cell technique. From this study it follows that the third-body effect is of minor importance. Obviously upd lead itself catalyses the direct oxidation to CO₂.     

Real-time monitoring of strand-displacement DNA amplification by a contactless electrochemical microsystem using interdigitated electrodes

This paper reports the design and implementation of a contactless conductivity detection system which combines a thermal control cell, a data processing system and an electrochemical (EC) cell for label-free isothermal nucleic acid amplification and real-time monitoring. The EC cell consists of a microchamber and interdigitated electrodes as the contactless conductivity biosensor with a cover slip as insulation. In our work, contactless EC measurements, the effects of trehalose on amplification, and chip surface treatment are investigated. With the superior performance of the biosensor, the device can detect the amount of pure DNA at concentrations less than 0.1 pg μl(-1). The EC cell, integrated with a heater and a temperature sensor, has successfully implemented nicking-based strand-displacement amplification at an initial concentration of 2.5 μM and the yields are monitored directly (dismissing the use of probes or labels) on-line. This contactless detector carries important advantages: high anti-interference capability, long detector life, high reusability and low cost. In addition, the small size, low power consumption and portability of the detection cell give the system the potential to be highly integrated for use in field service and point of care applications.     

Enzyme fingerprints of activity,and stereo- and enantioselectivity from fluorogenic and chromogenic substrate arrays

A series of stereochemically and structurally diverse fluorogenic and chromogenic substrates for hydrolytic enzymes has been synthesized and used to characterize enzyme activity profiles of esterases, lipases, proteases, peptidases, phosphatases, and epoxide hydrolases. The substrates used are particularly resilient to nonspecific reactions due to their mechanism of activation. The activities recorded with the individual substrates are therefore remarkably reproducible, and enable us to use the overall pattern of activity as a specific fingerprint for the enzyme sample. Fingerprints of activity, and enantio- and stereoselectivity are displayed as arrays of color-scale squares that are easily analyzed visually. Such fingerprints might be useful for quality control, enzyme discovery, and possibly for addressing the issue of functional convergence in enzymes.