Immobilized Enzyme Column Research Articles

19] Flow enthalpimetric determination of glucose

The most important thermal condition for the flow enthalpimetric determination is not high precision thermostatting of the detection unit, but the thermal equilibration of the sample solution. In principle, flow enthalpimetry requires a relatively large sample volume, because the sample pulse broadening caused by the thermal equilibration coil influences the signal change with concomitant decrease in the analytical sensitivity. Therefore, the sample volume giving the most sensitive analytical signal is mainly set by the volume of the heat exchanger. An optimum flow rate is determined mainly by a combination of column adiabaticity or column length and the activity of the immobilized enzyme provided a sufficient sample volume is supplied. A limiting factor of this and other flow enthalpimetry methods is the nonspecific heat effect seen as peak height changes caused by slight differences in sample composition. Thus, the reproducibility (coefficient of variation) of this method is slightly poorer than those of other immobilized enzyme columns with end-point detection. By considering the analytical sensitivity based on the heat of reaction and the magnitude of nonspecific heat, the lower limit of determination is estimated to be 0.1 m M glucose. Therefore, on application of the method to various practical samples satisfactory results are obtained with samples above 1 m M (18 mg/dl) in glucose. The important feature of the method is its practicability and its ease of adaptability for routine analysis.

Flow-injection determination of glutathione with amperometric monitoring of the enzymatic reaction

Glutathione sulfhydryl oxidase is immobilized on oxirane-acrylic beads (Eupegit-C) and packed in a small column. The simple system for glutathione comprises the immobilized enzyme column and a flow-through membrane-covered platinum/silver/silver chloride electrode pair for detection of hydrogen peroxide. The calibration graph for glutathione was linear from 0.05 to 1.0 mM for 200-μl samples. The assay took 3 min. The relative standard deviation for 0.5 mM glutathione was 2% ( n=10).

23] Use of immobilized enzyme column reactors in clinical analysis

Publisher Summary A column reactor is essentially a minicolumn, usually made of a plastic tubing, which is packed with uniformly sized solid particles. An enzyme or a given number of enzymes are immobilized on the surface of the solid particles which are usually porous glass beads. The enzyme proteins are covalently bound to the alkylamino groups of derivatized glass. An immobilized enzyme reactor thus produced can be inserted into the flow system of an analyzer such as the Technicon type. This chapter describes the methods for making a minicolumn with enzyme-immobilized glass beads. Several examples of applications of such column reactors are presented, which include conventional flow analysis as well as flow-injection analysis. These methods are specific for serum samples, but the methods may also be used, with appropriate modifications, for the analysis of urine and other body fluids, as well as to industrial and environmental systems.

15] Flow-injection analysis with immobilized chemiluminescent and bioluminescent columns

Publisher Summary Flow analysis using immobilized chemi- and bioluminescent packed-bed enzyme reactors as detectors is rapid, sensitive, and precise. The reusability and stability of the immobilized enzyme columns make them potential and low-cost tools for automated determinations of different analytes in both research and routine analysis. There are many matrices and many different procedures used for enzyme immobilization. High coupling efficiency, high remaining activity, good stability of the final product during storage and continuous use, and good mechanical stability of the support are the main criteria when the immobilization procedure is optimized. For chemi- and bioluminescent systems there is also a special demand for the matrix and the coupling reaction: the enzyme–matrix conjugate should not absorb any light at 400–600 nm. This chapter describes the immobilization of peroxidase, choline oxidase, firefly luciferase, and the bacterial bioluminescence enzymes with different dehydrogenases, including the use of resulting enzyme preparations in flow-injection analysis of some metabolites.

Assay of adenosine deaminase in serum by flow-injection analysis with fluorescence detection.

A sensitive assay method for adenosine deaminase activity in human serum is described based on the flow-injection determination of inosine formed enzymatically from substrate adenosine. Serum (10μl) is incubated with adenosine in the presence of urate oxidase and catalase, and the resulting mixture, after deproteinization with perchloric acid, is introduced into a flow-injection system in which immobilized enzyme columns of purine nucleoside phosphorylase, xanthine oxidase, urate oxidase and horseradish peroxidase are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of inosine is measured fluorimetrically by reaction with 3-(p-hydroxyphenyl) propionic acid in the system. The lower determinable limit of adenosine deaminase activity is 0.17 U/1 serum.

“One-Shot” Flow Injection Analysis with Immobilized Enzyme Columns: Clinical Applications

“One-Shot” Flow Injection Analysis with Immobilized Enzyme Columns: Clinical Applications

Sensitive flow-injection determination of l-lactate in human blood with immobilized enzyme columns and fluorimetric detection

Enzyme columns prepared by packing l-lactate oxidase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series. Hydrogen peroxide formed in the enzymatic conversion of l-lactate in the first column is mixed with 3-( p-hydroxyphenyl)propionic acid before passage through the peroxidase column and fluorimetric measurement. Linear calibration was obtained for 0.5–500 pmol of L-lactate in 20 μl of 1000-fold diluted, deproteinated whole blood. A rapid sampling rate (60 h −1) was possible.

Assay of purine nucleoside phosphorylase in erythrocytes by flow-injection analysis with fluorescence detection.

A sensitive assay for purine nucleoside phosphorylase (PNP) in human erythrocytes is described. Erythrocyte lysate is incubated with the substrate inosine in the presence of phosphate for the enzyme reaction, and the resulting mixture is deproteinized with perchloric acid and neutralized. The supernatant containing the reaction product, hypoxanthine, is applied to a flow-injection system in which immobilized enzyme columns of xanthine oxidase, urate oxidase and horseradish peroxidase are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of hypoxanthine is measured fluorimetrically by reaction with 3- (p-hydroxyphenyl) propionic acid in the system. The lower limit of determination (signal-to-noise ratio=5) for hypoxanthine is 0.3 pmol in a 20-μl injection volume, which corresponds to a PNP activity of 0.117 μmol/min/ml erythrocytes. This method permits the assay of PNP in only 10 nl of human erythrocytes in 50 μl of erythrocyte lysate.

Determination of Acetylcholine and Choline in Rat Brain Tissue by Liquid Chromatography/Electrochemistry Using an Immobilized Enzyme Post Column Reactor

Abstract Following separation by conventional LC, acetylcholine and choline are converted to hydrogen peroxide in a packed bed reactor consisting of covalently bound acetylcholinesterase and choline oxidase. Hydrogen peroxide, the ultimate enzymatic product, is detected amperometrically at a platinum electrode thin-layer cell. This method is simple and highly sensitive to the detection of acetylcholine and choline, with detection limits of about 100 femtomoles. The immobilized enzyme columns were stable for at least 60 days and conserved precious and expensive supplies of enzyme relative to continuous addition schemes. The apparatus is generally applicable to other enzymatic reactions which yield electroactive products.

Continuous production of homopolynucleotides by immobilized polynucleotide phosphorylase

A polynucleotide phosphorylase was immobilized with glutaraldehyde, via an aminopropyl spacer, on porous glass. The specific activity of the immobilized enzyme was effectively increased by the addition of an appropriate ribonucleoside diphosphate on immobilization. A homopolynucleotide could be synthesized continuously by passing a nucleoside diphosphate solution through the immobilized enzyme column. The chain length of the product depended upon the temperature and the flow rate. Polyinosinic acid, poly(I), was continuously synthesized with the immobilized enzyme for about one month without appreciable loss of activity. Polyinosinic acid-polycytidylic acid, poly(I)·poly(C), prepared from poly(I) and poly(C) synthesized with the immobilized polynucleotide phosphorylase, induced interferon-β (IFN-β) in human cultured cells as effectively as that prepared from homopolynucleotides synthesized with the free enzyme.

Simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue by high-performance liquid chromatography with electrochemical detection

A simple method for the simultaneous determination of acetylcholine, choline, noradrenaline, dopamine and serotonin in brain tissue was developed by using high- performance liquid chromatography with electrochemical detection. These compounds are analysed in a single chromatographic run within 30 min with a simple sample clean-up procedure. The detection system consists of two electrochemical detector cells aligned in series: a glassy-carbon electrode for catecholamines and serotonin, and a platinum electrode for acetylcholine and choline. For the detection of the latter compounds, they were converted enzymatically into hydrogen peroxide through a column reactor with immobilized acetylcholinesterase and choline oxidase. A column of boronic acid gel was placed just ahead of the immobilized enzyme column to remove catecholamines, which caused interfering responses on the platinum electrode. Two equivalent analytical columns and a column switching were employed to speed up the serotonin assay. Simultaneous determination of these major neurotransmitters in rat brain regions was successfully carried out with the system described.

Flow-injection determination of adenosine and inosine in blood plasma with immobilized enzyme columns connected in series and fluorimetric detection

A rapid and sensitive flow-injection method is described for the enzymatic determination of adenosine and inosine in human blood plama. Teflon columns prepared by packing adenosine deaminase. purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase immobilized chemically on controlled-pore glass beads are connected in series in that order in the flow line. Hydrogen peroxide formed in the enzymatic conversion of adenosine and inosine is measured fluorimetrically after reaction with 3-( p-hydroxyphenyl)propionic acid. Linear calibrations were obtained for 0.5–500 pmol of adenosine or inosine in hte 20 μl sample injected. Necessary deproteination routines are outlined.

Flow Injection Analysis of Hydrogen Peroxide Using Immobilized Horseradish Peroxidase and Its Fluorogenic Substrate 3-(p-Hydroxyphenyl)propionic Acid

A highly sensitive, rapid and precise method for the determination of hydrogen peroxide is described which employs flow injection analysis with fluorescence detection. Hydrogen peroxide solution is injected into a water stream, mixed with a solution of 3-(p-hydroxyphenyl)propionic acid, a fluorogenic substrate for horseradish peroxidase. The mixture is then passed through an immobilized horseradish peroxidase column prepared by coupling of the periodate-treated enzyme with aminopropyl glass beads. The fluorescent product in the stream is monitored with a conventional fluorimeter. A sampling rate of 120/h can be attained. The detection limit is 0.5pmol in a 20-μl injection volume. The immobilized enzyme column can be used for a year or longer without loss of the activity.

Application of an immobilized enzyme column reactor to measurement of creatine kinase isoenzyme activity by high performance liquid chromatography

Application of an immobilized enzyme column reactor to measurement of creatine kinase isoenzyme activity by high performance liquid chromatography

A highly sensitive determination of individual serum bile acids using high-performance liquid chromatography with immobilized enzyme

We have developed a highly sensitive method for the simultaneous determination of individual bile acids in serum using high-performance liquid chromatography (HPLC) combined with immobilized 3α-hydroxysteroid dehydrogenase. Both the HPLC column and the immobilized-enzyme column are suitable for use with alkaline solutions necessary in working with this enzyme system. With this method we were able to determine simultaneously fifteen different serum bile acids.

Biospecific thermal analyzer coupled with a flowthrough immobilized enzyme column

A thermal analyzer consisting of a pair of glass thermistors was coupled with a flowthrough immobilized enzyme reactor for the determination of ATP concentration. Hexokinase was covalently bound to poly(chloromethyl-styrene-divinyl benzene) beads and packed in the reactor. The thermal analyzer was designed so as to respond to heat generation in the reactor. A maximum temperature change was exhibited within 1.5 min after the injection of a sample solution containing ATP together with glucose. Thermal decay occurred within 1 min. The thermal analyzer responded reproducibly to ATP at a constant concentration. Assays could be repeated at 3-min intervals. The effects of sample volume and flow rate on the response were examined with regard to an empirical equation. Concentration of ATP was determined with the thermal analyzer in the range of 0-10 mM. The thermal analyzer may be a simple device for the rapid determination of ATP concentration.

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.

Immobilization of Glucose Isomerase in Microbial Cells. Part 2. Properties of Immobilized Glucose Isomerase in Cell Granule Preparations

AbstractSome properties of immobilized glucose isomerase in whole cell preparations were investigated. Immobilized glucose isomerase showed a similar velocity pattern to the native free enzyme. But the reaction system of immobilized enzyme was thought to be influenced by diffusion. The immobilized enzyme was much superior to the free enzyme in heat and pH stability and resistance to the inhibitory effect of several metal ions. In continuous process immobilized glucose isomerase showed a considerable durability. Decrease of activity of the immobilized enzyme column was approximately expressed by an exponential function of time.

Preparation and properties of immobilized naringinase using tannin-aminohexyl cellulose.

Naringinase from Aspergillus niger was immobilized with 58 to 100% yield by adsorbing the enzyme to tannin-aminohexyl cellulose prepared by reaction of aminohexyl cellulose and cyanogen bromide activated chinese gallotannin. Some enzymatic properties of the immobilized naringinase were investigated and compared with those of the native enzyme. The optimal pH (4_??_5) and optimal temperature (50°C) for enzyme reaction by the immobilized enzyme were the same as those of the native enzyme. The heat stability of the immobilized enzyme was better than that of the native naringinase. No significant difference of the Michaelis constant was detected. The operational stability of the immobilized naringinase column was investigated at 25°C and 37°C by passing a naringin solution and Natsudaidai juice continuously through it. In the case of a naringin solution, the activity of the immobilized enzyme column was very stable and its half-life was estimated to be 198 days at 25°C and 88 days at 37°C. However, in the case of Natsudaidai juice, the half-life of immobilized column was estimated to be 45 days at 25°C and 15 days at 37°C.

Analytical aspects of immobilized enzyme columns.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAnalytical aspects of immobilized enzyme columnsRichard S. Schifreen, D. Alan. Hanna, Larry D. Bowers, and Peter W. CarrCite this: Anal. Chem. 1977, 49, 13, 1929–1939Publication Date (Print):November 1, 1977Publication History Published online1 May 2002Published inissue 1 November 1977https://doi.org/10.1021/ac50021a014RIGHTS & PERMISSIONSArticle Views79Altmetric-Citations44LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts