Showing papers on "Immobilized enzyme published in 1995"

Aldehyde Biosensor Based on the Determination of NADH Enzymically Generated by Aldehyde Dehydrogenase

Abstract: We describe the preparation, characterization, and performance of an aldehyde biosensor based on the determination of NADH generated by the enzymatic activity of immobilized (on a nylon mesh membrane) aldehyde dehydrogenase. The enzymatically generated NADH is, in turn, electrocatalytically oxidized at a glassy carbon electrode modified with an electropolymerized film of 3,4-dihydroxybenzaldehyde (3,4-DHB). We have characterized the response of the biosensor in terms of the effects of the immobilization procedure, enzyme loading, pH of the solution, and the presence of anionic species with particular emphasis on the role of phosphate anions. In addition, we have carried out studies of the kinetics of the catalytic reaction, as well as permeability studies. The sensor exhibits high sensitivity and a limit of detection in the micromolar regime (5.0 μM), as well as rapid response (60 s to reach 90% of its steady state value). We have also carried out analytical determinations of aliphatic and aromatic aldehydes and consistently find that aromatic aldehydes give superior results.

Conducting polymer based amperometric enzyme electrodes

Abstract: The construction and the properties of conducting-polymer based amperometric enzyme electrodes are reviewed. The main aim is to focus on the properties of conducting polymer films which are important for the construction of amperometric enzyme electrodes. Additionally, the review is focused on electron-transfer pathways between conducting-polymer integrated immobilized enzyme molecules and the modified electrode using free-diffusing redox mediators as well as direct electron transfer via the conducting-polymer wires. Possible future applications using microstructured conducting-polymer films will be discussed.

Methylphenazonium-modified enzyme sensor based on polymer thick films for subnanomolar detection of phenols

Abstract: A methylphenazonium-zeolite-modified enzyme sensor based on a planar, screen-printed, two-electrode arrangement is described for the subnanomolar detection of phenols. Using tyrosinase (EC 1.14.18.1), a novel polyurethane hydrogel was applied for immobilization and stabilization of the enzyme, which forms a self-adhering layer on the active surface of the strip sensor. Performance and characteristics of the sensor were evaluated with regard to response time, detection limit, selectivity, and dependences on temperature and pH as well as operating and storage stabilities. The sensor shows marked sensitivity to eight phenolic compounds usually present in industrial waste waters. The detection limit for phenol was obtained with 0.25 nM

Improved fiber-optic chemical sensor for penicillin.

Abstract: An optical penicillin biosensor is described, based on the enzyme penicillinase. The sensor is fabricated by selective photodeposition of analyte-sensitive polymer matrices on optical imaging fibers. The penicillin-sensitive matrices are fabricated by immobilizing the enzyme as micrometer-sized particles in a polymer hydrogel with a covalently bound pH indicator. An array of penicillin-sensitive and pH-sensitive matrices are fabricated on the same fiber. This array allows for the simultaneous, independent measurement of pH and penicillin. Independent measurement of the two analytes allows penicillin to be quantitated in the presence of a concurrent pH change. An analysis was conducted of enzyme kinetic parameters in order to model the penicillin response of the sensor at all pH values. This analysis accounts for the varying activity of the immobilized penicillinase at different pH values. The sensor detects penicillin in the range 0.25-10.0 mM in the pH range 6.2-7.5. The sensor was used to quantify penicillin concentration produced during a Penicillium chrysogenum fermentation.

Biosensors based on oxidases immobilized in various conducting polymers

Abstract: The electrodeposited organic polymers polypyrrole, poly(N-methylpyrrole), poly(o-phenylenediamine) and polyaniline are compared as matrices for the immobilization of glucose oxidase in the preparation of amperometric glucose biosensors. Enzyme entrapment in the polymer layer is obtained by electrodeposition of polymers from solutions of monomers containing dissolved enzyme. For all examined sensors a useful and almost linear range of response to glucose is observed up to at least 20 mM of glucose. The best sensitivity of response is obtained for a glucose sensor made of poly(o-phenylenediamine) and polypyrrole. A linear response up to 20 mM glucose is also obtained in flow-injection measurements for a glucose/polypyrrole sensor. Poly(o-phenylenediamine) is also used for satisfactory immobilization of choline oxidase in the preparation of a choline sensor, whereas a lactate biosensor has been prepared by immobilization of lactate oxidase in polypyrrole.

Do organic solvents affect the catalytic properties of lipase? Intrinsic kinetic parameters of lipases in ester hydrolysis and formation in various organic solvents

Abstract: When it is assumed that organic solvents do not interfere with the binding process nor with the catalytic mechanism, the contribution of substrate-solvent interactions to enzyme kinetics can be accounted for by just replacing substrate concentrations in the equations by thermodynamic activities. It appears from the transformation that only the affinity parameters (K(m), K(sp)) are affected by this. Thus, in theory, the values of these corrected, intrinsic parameters (K(m) (int), k(sp) (int)) and the maximal rate (V(1)) should be equal for all media. This was tested for hydrolysis, transesterification, and esterification reactions catalyzed by pig pancreas lipase and Pseudomonas cepacia lipase in various organic solvents. Correction was carried out via experimentally determined activity coefficients for the substrates in these solvents or, if not feasible, from values in data bases. However, although the kinetic performances of each enzyme in the solvents became much more similar after correction, differences still remained. Analysis of the enzyme suspensions revealed massive particles, which explains the low activity of enzymes in organic solvents. However, no correlation was found between estimates of the amount of catalytically available enzyme (present at the surface of suspended particles or immobilized on beads) and the maximal rates observed. Moreover, the solvents had similar effects on the intrinsic parameters of suspended and immobilized enzyme. The possible causes for the effects of the solvents on the catalytic performance of the enzymes, remaining after correction for solvent-substrate interactions and the amount of participating enzyme, are discussed with respect to the premises on which the correction method is based. (c) 1995 John Wiley & Sons, Inc.

Applications of “Wired” Peroxidase Electrodes for Peroxide Determination in Liquid Chromatography Coupled to Oxidase Immobilized Enzyme Reactors

Abstract: An osmium poly(vinylpyridine) redox polymer «wired» horseradish peroxidase electrode has been used to detect H 2 O 2 for the determination of glucose, lactate, and acetylcholine/choline with liquid chromatography (LC) and postcolumn immobilized enzyme reactors (IMERs). The redox polymer film containing peroxidase is coated on the surface of a glassy carbon electrode and operated at +100 mV vs Ag/AgCl for the reduction of H 2 O 2 . Compared with a conventional platinum electrode oxidizing H 2 O 2 at +500 mV vs Ag/AgCl, the peroxidase cathode exhibits a 2-10-fold improvement in sensitivity and detection limit. The enzyme electrode also shows better operational stability than the Pt electrode. When the enzyme electrode is coupled to LC oxidase IMER systems, the initial stabilization of the background current is significantly faster than that for the conventional Pt electrode. The enzyme electrode has been used to determine glucose and lactate concentrations in rat subcutaneous microdialysates. After 8 days of continuous injection of the dialysate samples, the enzyme electrode sensitivity dropped by only 5%

Imaging of immobilized antibody layers with scanning electrochemical microscopy.

Abstract: Visualization of immobilized antibodies can be achieved with scanning electrochemical microscopy (SECM) by saturation of the antigen binding sites with an alkaline phosphatase-antigen conjugate, which catalyzes hydrolysis of the redox-inactive 4-aminophenyl phosphate to the redox-active 4-aminophenol (PAP). PAP was detected in the collection mode at an amperometric SECM tip. The tip current reflects the density of active binding sites in the immobilized antibody layer. The application of this approach for immunosensing research has been demonstrated with the optimization of a covalent immobilization procedure of antibodies on glass. The special advantages and present limitations of the procedures are discussed.

Online Microreactors/Capillary Electrophoresis/Mass Spectrometry for the Analysis of Proteins and Peptides

Abstract: We report the use of trypsin and carboxypeptidase Y-modified capillary microreactors (50 μm i.d. and 20 nL volume per centimeter length) in combination with mass spectrometry for peptide molecular mass mapping of various peptides and proteins. Advantages of immobilized enzyme capillary microreactors include picoliter to nanoliter volume requirements, longer enzyme lifetimes, higher stability, and the ability to reuse enzymes conveniently. Additionally, extremely efficient sample handling modes are used, and the reaction products are easily separated from the enzyme reagents. Plasma desorption and matrix-assisted laser desorption/ionization were used for off-line analyses of digestion products. The use of MassMap for the identification of proteins is also discussed. Finally, a trypsin microreactor was integrated on-line with capillary electrophoresis/ion spray mass spectrometry for fast peptide mapping. Digestion of the oxidized insulin B-chain in an on-line trypsin microreactor and electromigration of aliquots from the capillary microreactor into the CE separation capillary allowed the entire peptide mapping procedure to be completed in <1 h. The on-line technique is especially well-suited for the characterization of minute quantities of proteins, because it transfers picoliter to nanoliter volumes of digestion products with minimum sample handling which could lead to losses or contamination.

Polyphenoloxidases immobilized in organic gels: Properties and applications in the detoxification of aromatic compounds.

Abstract: Gelatine gels originate from water in oil microemulsions in which the ternary system consists of isooctane/ sulfosuccinic acid bis [2-ethyl hexyl] ester/water; the solubilization of gelatin in the water pool of these microemulsions transforms them into viscous gels in which it is possible to cosolubilize various reactive molecules. These gels were used to immobilize two phenoloxidases, a laccase from Trametes versicolor and a tyrosinase from mushroom. The best balance between gel retention and catalytic activity was reached at a gelatine concentration of 2.5% (w/v) in the case of tyrosinase, while laccase immobilization was independent of gelatine concentration. Both enzymes kept the same optimum pH as the corresponding soluble controls, while a partial loss of activity was observed when they were immobilized. Immobilized enzymes showed an increased stability when incubated for several days at 4 degrees C with a very low release from the gels in the incubation solutions. The immobilization of tyrosinase and of laccase enhanced stability to thermal inactivation. Furthermore, gel-entrapped tyrosinase was almost completely preserved from proteolysis: more than 80% of the activity was maintained, while only 25% of the soluble control activity was detected after the same proteolytic treatments. A column packed with gel-immobilized tyrosinase was used to demonstrate that enzymes immobilized with this technique may be reused several times in the same reaction without loosing their efficiency. Finally, gel-entrapped tyrosinase and laccase were capable of removing naturally occurring and xeno-biotic aromatic compounds from aqueous suspensions with different degrees of efficiency. (c) 1995 John Wiley & Sons, Inc.

Four‐factor response surface optimization of the enzymatic modification of triolein to structured lipids

Abstract: The ability of an immobilized lipase to modify the fatty acid composition of (88.8% C18:1, 4.3% C16:0, 3.1% C18:0, and 3.8% C18:2 as determined by gas chromatography, and approximately 90% triolein) in hexane by incorporation of a medium-chain fatty acid, capric acid (C10), to form structured triacylglycerol was studied. Response surface methodology was used to evaluate the effect of synthesis variables, such as reaction time (12–36 h), temperature (25–65°C), molar substrate ratio of capric acid to triolein (2:1–6:1), and enzyme amount (10–30% wt% of triacylglycerol), on the yield of structured lipid. Optimization of the transesterification was attempted to obtain maximum yield of structured lipid while using the minimum molar substrate ratio and enzyme amount as much as possible. Computer-generated contour plot interpretation revealed that a relatively high molar substrate ratio (6:1) combined with low enzyme amount (10%) after 30 h of reaction at 25°C gave optimum incorporation of capric acid. A total yield for combined monoand dicaproolein of up to 100% was obtained.

Working at controlled water activity in a continuous process: the gas/solid system as a solution.

Abstract: Fusarium solani cutinase and Candida cylindracea lipase were used to catalyze a transesterification reaction in a continuous gas/solid bioreactor. In this system, a solid phase composed of a packed enzymatic preparation was continuously percolated with carrier gas which fed substrate and removed reaction products simultaneously. Different conditions of immobilization were used and compared to the results obtained with a nonsupported enzyme. The enzymatic activity was found to be highly dependent of a key parameter: water activity (a(w)). Biocatalyst stability was greatly influenced by water activity and the choice of immobilization technique for the enzymatic material. For free and adsorbed enzymes, water requirements exhibited optima which corresponded to the complete hydration coverage of the protein. These optima presented a good correlation with the isotherm sorption curves obtained for the different preparations. In this work are reported the results concerning the possibility of using a continuous system able to operate at controlled water activity in a heterogeneous medium. Lipolytic enzyme in such a system appears to be a new process for the biotransformation of volatile esters.

Electrochemical Sensors Based on Impedance Measurement of Enzyme-Catalyzed Polymer Dissolution: Theory and Applications

Abstract: A novel sensor approach based on ac impedance measurement of capacitance changes produced during enzyme-catalyzed dissolution of polymer coatings on electrodes, leading to a 4 orders of magnitude change in capacitance, is described. Electrodes were coated with an enteric polymer material, Eudragit S 100, which is based on methyl methacrylate, and dissolution was exemplified by utilizing the catalytic action of the enzyme urease. The resulting alkaline pH change caused dissolution of the polymer film with a consequent large increase in capacitance. A mechanism for polymer breakdown is proposed which has been validated experimentally using both ac impedance measurements and electron microscopy. The large changes in capacitance that are apparent using this technique allow much greater sensitivity of measurement than, for example, potentiometric electrodes. The potential broad clinical analytical application of this technique is demonstrated in this report by application to urea measurement and to enzyme immunoassay. Urea measurement between 2 and 100 mM has been achieved with a change in response over this concentration range by over 4 orders of magnitude. We have taken account of both the effect of protein adsorption on the surface of the polymer-coated and bare electrodes and the effect of buffer capacity when carrying out these measurements in buffered solutions containing 8% (w/v) protein and have demonstrated that the method should allow simple, interference-free measurement of urea in serum and whole blood. In addition, both competitive and noncompetitive enzyme immunoassays for human IgG based on the use of urease-antibody conjugates are reported. Human IgG, or goat anti-human IgG (Fab specific), were immobilized covalently onto cellulosic membranes via a diamine spacer group and the membranes placed over enteric polymer-coated electrodes. Specific measurement of IgG in both formats was achieved over the concentration range 0.0001-100 μg mL -1 . The performances of the impedance-based enzyme immunoassays were compared directly with identical assays employing spectrophotometric detection.

On-line glucose monitoring by using microdialysis sampling and amperometric detection based on ‘wired’ glucose oxidase in carbon paste

Abstract: In-vitro on-line glucose monitoring is described, based on microdialysis sampling and amperometric detection operated in a flow-injection system. Samples were injected into a two-electrode microcell containing an Ag/AgCl quasi-reference electrode and a glucose enzyme electrode as the working electrode, operated at + 0.15 Vvs. Ag/AgCl. The enzyme electrode is constructed by mixing the ‘wired’ glucose oxidase into carbon paste. {Poly[1-vinylimidazole osmium(4,4′-dimethylbipyridine)2Cl)]}+/2+ was used to ‘wire’ the enzyme. The non-coated electrodes, cross-linked with poly(ethylene glycol) diglycidyl ether, responded linearly to glucose concentrations up to 60 mM, and were characterized by a sensitivity of 0.23 μA mM−1 cm−2, when operated in flow injection mode and of 5.4 μAmM –1 cm–2 in steady-state conditions. This sensitivity of the resulting enzyme electrode was 50% lower than that of similarly prepared but non-cross-linked electrodes. However, the cross-linked electrodes showed superior operational and storage stabilities, which were further improved by coating the electrodes with a negatively charged Eastman AQ film. An in-house designed microdialysis probe, equipped with a polysulphone cylindrical dialysis membrane, yielded a relative recovery of 50–60% at a perfusion rate of 2.5 μl/min–1 in a well stirred glucose solution. The on-line set up effectively rejected common interferences such as ascorbic acid and 4-acetaminophen when present at their physiological concentrations.

Studies on the lipozyme-catalyzed synthesis of butyl laurate.

Abstract: The effects of temperature, speed of agitation, enzyme concentration, etc., on butyl laurate synthessis using Mucor miehei lipase (Lipozyme™) have been studied. Although the soluble enzyme was quite thermcstable in aqeous solution, it deactivated rapidly at and above 40°C in the presence of butanol. This enzyme immobilized on an anion-exchange resin (Lipozyme™) showed enhanced stability (as compared to the soluble form) to denaturation by butanol under the same conditions. The denaturation of M. miehei lipase was found to be a function of the butanol concentration in the aqueous phase, and rapid denaturation takes place at the concentration corresponding to its saturation at that temperature. © 1995 John Wiley & Sons, Inc.

Amperometric Detection of Peroxides with Poly(anilinomethylferrocene)-Modified Enzyme Electrodes

Abstract: An amperometric enzyme electrode employing covalently attached horseradish peroxidase to glassy carbon electrode and a ferrocene-modified polyaniline film deposited by the electrochemical polymerization of N-(ferrocenylmethyl)aniline monomer is reported. The electrode responded rapidly to micromolar concentrations of peroxides, in accordance with the following trend: hydrogen peroxide>cumene hydroperoxide>tert-butyl hydroperoxide. The effect of operational parameters, such as methods of enzyme immobilization, operating potential of the working electrode, pH, and effect of molecular oxygen, were explored for optimum analytical performance