Extraction of free phenols from blood by a liquid membrane enzyme reactor

The development of a liquid membrane enzyme reactor for the extraction of phenols from blood and plasma is described. Phenol permeation across the liquid membrane is studied, the transport of the toxins is linked with an enzymatic reaction. The encapsulation of the enzyme is described in detail. The stability of the multi-emulsion as well as the loss of enzyme activity during encapsulation and treatment axe examined thoroughly. The resulting liquid surfactant membrane emulsion is applied in in-vitro experiments to remove phenols from blood and plasma.     

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.     

Photolithographically patterned enzyme membranes for the detection of pesticides and copper(II) based on enzyme inhibition

Summary A non-aqueous and an aqueous photopolymer system with an enzyme are used to prepare photolithographically patterned enzyme membranes for amperometric (thinfilm platinum electrode) and potentiometric (ISFET) sensors based on enzyme inhibition. Flow methods for enzyme inhibition tests are described. The decrease in enzyme (AChE) activity after incubation in a solution of dichlorvos as inhibitor is detected amperometrically. The enzyme urease is immobilized onto the pH-sensitive gate area of an ISFET. Such a biosensor is able to detect copper-(II) in water in the ppm-range without preconcentration.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

Enzyme peptide synthesis by an iterative procedure in a nucleophile pool

An effective method for enzyme solubility-controlled synthesis of peptides, consisting in an iterative addition of equivalent amounts of acyl and amine components to a solution (nucleophile pool) containing the enzyme and a large excess of the amine component, is described.     

A potentiometric enzyme electrode for monitoring in organic solvents

A potentiometric enzyme electrode is described for monitoring reactions in organic solvents. By use of an enzyme deposited on magnetic particles which are attracted to the tip of the electrode by means of a magnetic field, it is possible to produce an electrode in which the enzyme can easily be exchanged. As an example, studies of the chymotrypsin-catalyzed ester synthesis in diisopropyl ether and in toluene at varying water contents are reported. The results are consistent with those obtained from batch experiments. Operational behaviour and signal stability of the system makes this kind of potentiometric enzyme electrode attractive for monitoring bioorganic processes.     

Amperometric Enzyme Based Biosensor For the Detection of Xanthine Via Superoxide

Abstract

An amperometric enzyme modified carbon paste electrode for the detection of xanthinc by the xanthine oxidase catalyzed reaction is described. the product of the enzyme reaction monitored is the highly unstable radical superoxide, detected at O mV (vs SCE) poised potential. A dual working electrode configuration consisting of an enzyme modified carboa paste electrode and an unmodified glassy carbon electrode are utilized for this purpose.     

Development of an organo- and enzyme-catalysed one-pot,sequential three-component reaction

A one-pot, three-component process is described which involves both organo- and enzyme-catalysed carbon–carbon bond-forming steps. In the first step, an organocatalyst catalyses the aldol reaction between acetaldehyde and a glyoxylamide. After dilution with additional aqueous buffer, and addition of pyruvate and an aldolase enzyme variant, a second aldol reaction occurs to yield a final product. Crucially, it was possible to develop a reaction in which both the organo- and enzyme-catalysed reactions could be performed in the same aqueous buffer system. The reaction described is the first example of a one-pot, three-component reaction in which the two carbon–carbon bond-forming processes are catalysed using the combination of an organocatalyst and an enzyme.     

Amperometric Enzyme Sensor-Based Enzyme Immunoassay for Factor VIII-Related Antigen

Abstract

The coupling of an enzyme immunoassay for factor VIII-related antigen with a commercial glucose oxidase based amperometric sensor permits the determination of 1.6 to 16 ng of factor XIII-related antigen in human plasma. Further pure amperometric sensors or amperometric enzyme sensors for determination of the main marker-enzymes of enzyme immunoassays are described.     

Xanthine and hypoxanthine sensors based on xanthine oxidase immobilized in poly(mercapto-p-benzoquinone) film

The construction and performance of an enzyme electrode as an amperometric sensor of xanthine and hypoxanthine is described. Xanthine oxidase has been immobilized in a conductive redox polymer, poly(mercapto-p-benzoquinone), by means of an electropolymerization of mercaptohydroquinone in the presence of the enzyme. An Au-electroplated glassy carbon electrode coated with the resulting polymer film functioned well as a direct response type of sensor, where the polymer chain served as a conductive molecular chain between the active sites in the enzyme and the substrate electrode. Response characteristics as well as kinetic parameters have been evaluated.     

Homogeneous enzyme-linked assays mediated by enzyme antibodies; a new approach to electrode-based immunoassays

A new homogeneous enzyme—immunoassay system is described. The assay employs an ammonia-liberating enzyme covalenty coupled to protein antigens along with two antibodies. An anti-enzyme antibody inhibits the enzyme. However, an antibody selective for the antigen reverses the inhibition process. When samples containing free antigen are present in the assay mixture, there is competition for anti-antigen antibody sites and protection against the anti-enzyme antibodies is diminished. The extent of the enzymatic reaction is monitored with an ammonium ion-selective electrode. Preliminary data demonstrating the feasibility of this approach for human serum albumin are presented.     

Fluorimetric assay of phospholipase A acting on biomembrane phospholipids

A sensitive phospholipase A assay suitable for organelle activities is described. Activation of the enzyme produces an increase in membrane lysophospholipids. The amino groups of extracted lipids were derivatized with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and the amounts of phosphatidylethanolamine and its lysoform were quantitated using HPLC equipped with a SiO2 column and a fluorescence detector. The procedure was tested with porcine enzyme acting on liposomes and mitochondria, and with endogenous mitochondrial enzyme.     

Inhibition of the enzyme phosphoenolpyruvate-carboxylase (PEPC) by different pollutants

The inhibitory effect of different pollutants on the enzyme phosphoenolpyruvate-carboxylase was investigated. For this enzyme, different procedures are described for determining the activity of the enzyme. Determination of the presence of the enzymatically-formed oxaloacetate was found to be more suitable for this purpose because of the higher sensitivity compared with that of the enzymatically-formed phosphate. In order to detect possible inhibitors of this enzyme, inhibition tests were carried out with different pesticides and metal ions. The enzyme activity was decreased by ziram, ferbam and pentachlorophenol. Of the tested metal ions, the enzyme reacts very sensitively to mercury ions, however cobalt, lead, zinc and copper also showed an inhibition effect.     

Enzyme nanoparticles-based electronic biosensor

A simple and effective method to prepare an enzyme electronic biosensor by immobilizing enzyme nanoparticles directly onto the gold electrode surface is described; prepared horseradish peroxidase nanoparticles have been successfully used to develop reagentless electronic biosensors for H2O2 detection without promoters and mediators and offer great potential to develop enzyme-based electronic biosensors.     

Structure-based design of potent CDK1 inhibitors derived from olomoucine

Cyclin-dependent kinase 1 (CDK1), an enzyme participating in the regulation of the cell cycle, constitutes a possible target in the search for new antitumor agents. Starting from the purine derivative olomoucine and following a structure-based approach, potent inhibitors of this enzyme were rapidly identified. The molecular modeling aspects of this work are described.     

Dealkoxycarbonylations of Gem Diesters and β-Ketoesters via an Enzyme Catalyzed Hydrolysis

A new method for the dealkoxycarbonylation of malonate esters and β-ketoesters, involving an enzyme catalyzed hydrolysis followed by a high temperature Kugelroher distillation, is described.     

Pseudobond ab initio QM/MM approach and its applications to enzyme reactions

This perspective article mainly focuses on the development and applications of a pseudobond ab initio QM/MM approach to study enzyme reactions. The following aspects of methodology development are discussed: the approaches for the QM/MM covalent boundary problem, an efficient iterative optimization procedure, the methods to determine enzyme reaction paths, and the approaches to calculate free energy change in enzyme reactions. Several applications are described to illustrate the capability of the methods. Finally, future directions are discussed.     

Trace analysis and speciation of copper by application of an urease reactor

Summary A method for metal speciation using an enzyme reactor is described. The enzyme urease is immobilized on a polymer support. The parameters of the inhibition procedure are investigated for the determination of copper in drinking and surface water samples. The results are compared with those obtained by atomic absorption spectrometry. It has been found that the enzyme urease was inhibited only by free copper ions.Dedicated to Professor Dr. Wilhelm Fresenius on the occasion of his 80th birthday     

An activated enzyme electrode for creatinine

A highly selective enzyme electrode for creatinine, based on tripolyphosphate-activated creatininase enzyme, is described and evaluated. Kinetic studies comparing purified creatininase enzyme in the activated and non-activated forms show that the activation mechanism involves an increase in V_max but no change in K_m. The analytical effect of enzyme activation is to extend the sensitivity of the electrode to lower limits and to improve the response slope of calibration curves. As a result, this activated creatininase enzyme electrode shows promise as a sensor for urine and serum samples.     

Separation-free amperometric enzyme immunoassay

A new technique for conducting a separation-free amperometric enzyme immunoassay is described using DNP-aminocaproic acid as the analyte. The technique is based on the combined use of a recently described separation-free enzyme immunoassay (19) and an electrode system that senses H₂O₂. Oxidation of glucose to gluconate and H₂O₂ by the enzyme reconstituted from DNP-conjugate apoglucose oxidase (DPN-CAGO) and FAD was continuously measured amperometrically. The reconstitution was inhibited by preincubation with anti-DNP antibody before adding FAD. This antibody-induced inhibition of the reconstituting of the holoenzyme was reversed by adding DNP-amino caproic acid to DNP-CAGO before adding the antibody to DNP-CAGO. Based on (a) the antibody-induced inhibition of holoenzyme reconstitution, (b) a specific ligand-induced reversal of the inhibition, and (c) an electrochemical system that measures H₂O₂, we developed a separation-free (homogeneous) amperometric enzyme immunoassay.     

Biochemical studies of soluble and immobilized aldehyde dehydrogenase from yeast

Aldehyde dehydrogenase from baker’s yeast was purified to homogeneity. The soluble and immobilized forms of this enzyme were characterized and compared biochemically. These included steady state kinetics, stability study, fluorescence, and NMR spectroscopy. Evidence of the coenzyme binding to this enzyme in the absence of aldehyde substrates was obtained by fluorescence and NMR studies. A significant quenching of protein fluorescence was observed upon the addition of coenzymes to the aldehyde-free enzyme solution. Significant shifts and broadening of coenzyme proton resonances in the presence of enzyme also indicate the enzyme-coenzyme interactions in the aldehyde-free solution. the enzyme was immobilized on glass beads by three different methods. The immobilized enzyme was found to exhibit physical and biochemical properties similar to those of the soluble enzyme. A system in which the immobilized alcohol, aldehyde, and steriod dehydrogenases are included in an enzyme reactor is described.