Sensitive ultraviolet monitoring of aldoses in automated borate complex anion-exchange chromatography with 2-cyanoacetamide

A convenient method for postcolumn carbohydrate labeling has been developed. Eluates of borate complex anion-exchange columns are mixed with a reagent solution prepared from an aqueous solution of 2-cyanoacetamide and a borate buffer (pH 10.5), and the mixture is heated in a 10-m reaction coil at 100°C. Measurement of the absorbance of the product at 276 nm permits high reproducibility determination of 5 to 500 nmol of aldoses. Some carbonyl compounds are positive to this reaction, but most do not interfere with the analysis because their peaks do not appear in the aldose region. Ascorbate gives a small peak between those of mannose and fucose, but interference is negligible for equimolar amounts of ascorbate and these aldoses. This method is applied to and gives satisfactory results in the analysis of monosaccharides from various types of glycoconjugates.     

Production of penicillins and cephalosporins in an immobilized enzyme reactor

Summary Four enzymes required for the biosynthesis of pencillins and cephalosporins by Streptomyces clavuligerus have been immobilized on an anion exchange resin. The capabilities of the system have been studied by circulation of reaction mixtures through the immobilized enzyme reactor. Within 30 min, all of the substrate -(l--aminoadipyl)-l-cysteinyl-d-valine is consumed and converted to a mixture of penicillins and cephalosporins. After 60 min the major antibiotic products are (iso)penicillin N and desacetylcephalosporin C. The activity of the immobilized enzyme reactor activity is stable to storage at temperatures below 4°C but activity is lost on repeated use.     

Identification of N-acetylhexosamines produced by enzymes of the N-acetylneuraminic acid metabolic pathway by borate complex anion-exchange chromatography of the corresponding N-acetylhexosaminitols

A mixture of hexosaminitols obtained by reducing N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine with sodium borohydride was resolved by borate complex anion-exchange chromatography. This procedure yielded a complete separation of N-acetylglucosaminitol, N-acetylgalactosaminitol, and N-acetylmannosaminitol and provided a rapid and accurate means for identifying and measuring N-acetylhexosamines in biological samples. This method was applied to studies on N-acetylneuraminic acid metabolism in human skin fibroblasts. It was used to identify reaction products in two enzymatic reactions: the conversion of UDP-N-acetylglucosamine to N-acetylmannosamine and UDP by UDP-N-acetylglucosamine 2-epimerase and the conversion of N-acetylneuraminic acid to N-acetylmannosamine and pyruvate by N-acetylneuraminate pyruvate-lyase. It was also used to identify the free 3H-labeled N-acetylhexosamines found in fibroblasts cultured in the presence of N-[3H]acetylmannosamine.     

Dynamic affinity between dissociable coenzyme and immobilized enzyme in an affinity chromatographic reactor with single enzyme

The affinity chromatographic reactor (ACR) is a bioreactor which utilizes the dynamic interaction or the dynamic affinity between a free coenzyme and immobilized enzymes for the highly efficient regeneration of dissociable coenzymes. Dynamic affinity between free NAD and immobilized alcohol dehydrogenase (ADH) in ACR was investigated by three different methods. ADH catalyzed both oxidation and reduction of NAD, consuming propionaldehyde and ethanol. The theoretical model under consideration elucidated a criterion for the expression of the dynamic affinity as a relationship among the affinity constants and the concentrations of a coenzyme and immobilized enzyme. This criterion was confirmed experimentally by the measurements of the retention time of NAD and the half-life period of the reactor activity after one-shot pulse injection of NAD to ACR. In the stability measurement of the immobilized enzyme, it became clear that ADH was more stable at the higher concentration in immobilization. Although the present case of coenzyme cycling by a single enzyme is very special, with limited chance for the direct application, the results obtained here provide a theoretical basis for ACR with multienzymes-which is of more general use.     

Application of immobilized enzyme reactor in on-line high performance liquid chromatography: a review

This review summarizes all the research efforts in the last decade (1994-2003) that have been spent to the various application of immobilized enzyme reactor (IMER) in on-line high performance liquid chromatography (HPLC). All immobilization procedures including supports, kind of assembly into chromatographic system and methods are described. The effect of immobilization on enzymatic properties and stability of biocatalysts is considered. A brief survey of the main applications of IMER both as pre-column, post-column or column in the chemical, pharmaceutical, clinical and commodities fields is also reported.     

Quantification of ephedrines in urine by column-switching high-performance liquid chromatography

A method for the quantification of five congener ephedrines in urine samples without sample preparation was developed. The analytes were trapped on a C18 precolumn and separated on a C18 BDS analytical column. Baseline separation was achieved for all analytes. The method meets the requirements of the International Olympic Committee (IOC) medical commission regarding cut-off limits for positive doping cases with ephedrines.     

Study of an enzyme membrane reactor with immobilized fumarase for production of L-malic acid

The conversion of fumaric acid into L-malic acid by fumarase immobilized in a membrane reactor was analyzed experimentally. The enzyme was entrapped in asymmetric capillary membranes made of polysulfone. The performance of the reactor was evaluated in terms of conversion degree, reaction rate, and stability. The influence of operating conditions, such as amount of immobilized enzyme, substrate concentration, residence time, and axial flow rate, were investigated. The kinetic parameters K(m), V(max), and k(+2) were also measured. The stability of the immobilized enzyme was very good, showing no activity decay during more than 2 weeks of continuous operation.     

Macrokinetics of enzyme sorption on porous beads accompanied by complex formation with an immobilized ligand

A mathematical model has been proposed for enzyme sorption on porous beads accompanied by formation of a stable complex with an immobilized ligand. It has been experimentally verified by using the system trypsin (EC 3.2.21.4) - immobilized bovine basic polyvalent trypsin inhibitor on porous silica gel. The experimental results for kinetics of the non-specific/specific trypsin sorption on a carrier agree with the model. The value of the coefficient of trypsin diffusion in macroporous silica gel was calculated.     

Cofactor recycling in an enzyme reactor. A comparison Using free and immobilized dehydrogenases with free and immobilized NAD

The requirements for enzymic cofactor recycling have been investigated in a system employing alcohol and lactate dehydrogenases. The interactions of various combinations of free dehydrogenases or dehydrogenases immobilized either to the same or separate supports, with free NAD, a soluble highmolecular weight derivative of NAD or an insoluble derivative of NAD have been examined.     

An automated assay of γ-carboxyglutamate with an anion-exchange column

A method is presented for the assay of γ-carboxyglutamate with an amino acid analyzer equipped with an anion-exchange column. The polyacidic nature of γ-carboxyglutamate results in very good separation from other ninhydrin-reactive material.     

Determination of uric acid by an anion-exchange column chromatography

A column chromatography using a conventional anion-exchange resin for the separation of uric acid from other purine metabolites is described. It uses a HCl gradient, and the amount of uric acid is quantified directly by monitoring the absorbance of the effluent at 285 nm. The linear range of response is 0.5 to 100 nmol. The method was applied to the analysis of uric acid in urine and serum. Urine was injected directly into the system, while serum required removal of an interfering substance which absorbs the light and coelutes with uric acid. However, this substance was simply removed by heat coagulation of serum by heating in a boiling water bath for 2 min.     

Inversion of sucrose by immobilized beta-fructooxidase in an integral reactor

Experimental runs on the inversion of sucrose by means of immobilized beta-fructooxidase are reported. External mass-transfer and axial dispersion phenomena have been analyzed. It has been observed that external mass-transfer plays a significative role in the overall kinetics, while axial dispersion phenomena are negligible.     

Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated ¹⁴C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of ¹⁴C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).     

Post-column enzyme reactors for chemiluminometric detection of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in an anion-exchange chromatographic system

A liquid chromatographic system consisting of a co-immobilized 3-hydroxybutyrate dehydrogenase-NADH oxidase reactor and an immobilized pyranose oxidase reactor in series and a chemiluminometer was developed for the simultaneous determination of glucose, 1,5-anhydroglucitol and 3-hydroxybutyrate in plasma. The enzymes were immobilized on toresylated poly(vinyl alcohol) beads. Separation was achieved on a TSK gel SAX column (40×4 mm I.D.) with an eluent of 50 mM NaOH containing 30 mM sodium butyrate. The hydrogen peroxide produced was detected by measuring the chemiluminescence emitted on admixing with luminol and potassium hexacyanoferrate(III). The calibration curves were linear from 0.8 to 500 μM (7 ng−4 μg) for glucose, from 0.8 to 400 μM (7 ng−3 μg) for 1,5-anhydroglucitol and from 1 to 700 μM (5 ng−4 μg in a 50-μl injection) for 3-hydroxybutyrate. The sample throughput was four per hour. The reactors were stable for at least ten days.     

Purification of pharmaceutical-grade plasmid DNA by anion-exchange chromatography in an RNase-free process

Anion-exchange is the most popular chromatography technique in plasmid DNA purification. However, poor resolution of plasmid DNA from RNA often results in the addition of bovine-derived ribonuclease (RNase) A to degrade RNA impurities which raises regulatory concerns for the production of pharmaceutical-grade plasmid DNA. Low capacity for plasmid of most commercial media is another issue affecting the suitability of anion-exchange chromatography for large-scale processing. This study reports the use of anion-exchange chromatography to remove RNA in an RNase-free plasmid purification process. Resolution was achieved through careful selection of adsorbent and operating conditions as well as RNA reduction steps before chromatography. Dynamic capacity for plasmid was significantly increased (to 3.0mg/ml) so that it is now possible to envisage the large-scale manufacturing of therapeutic-grade plasmid DNA in the absence of added RNase using anion-exchange chromatography as a polishing step.     

Three-dimensional numerical approach to investigate the substrate transport and conversion in an immobilized enzyme reactor

This numerical study evaluates the momentum and mass transfer in an immobilized enzyme reactor. The simulation is based on the solution of the three-dimensional Navier-Stokes equation and a scalar transport equation with a sink term for the transport and the conversion of substrate to product. The reactor consists of a container filled with 20 spherical enzyme carriers. Each of these carriers is covered with an active enzyme layer where the conversion takes place. To account for the biochemical activity, the sink term in the scalar transport equation is represented by a standard Michaelis-Menten approach. The simulation gives detailed information of the local substrate and product concentrations with respect to external and internal transport limitations. A major focus is set on the influence of the substrate transport velocity on the catalytic process. For reactor performance analysis the overall and the local transport processes are described by a complete set of dimensionless variables. The interaction between substrate concentration, velocity, and efficiency of the process can be studied with the help of these variables. The effect of different substrate inflow concentrations on the process can be seen in relation to velocity variations. The flow field characterization of the system makes it possible to understand fluid mechanical properties and its importance to transport processes. The distribution of fluid motion through the void volume has different properties in different parts of the reactor. This phenomenon has strong effects on the arrangement of significantly different mass transport areas as well as on process effectiveness. With the given data it is also possible to detect zones of high, low, and latent enzymatic activity and to determine whether the conversion is limited due to mass transfer or reaction resistances.     

Oxidation of 1,5-anhydro-D-glucitol to 1,5-anhydro-D-fructose catalyzed by an enzyme from bacterial membranes

Bacteria which grow on 1,5-anhydro-D-glucitol (AG) were isolated from soil. One such strain showing the highest AG-assimilating activity was further characterized and identified as a new strain of the Pseudomonas family (named Pseudomonas sp. NK-85001). A subcellular membranous fraction obtained from this strain catalyzed the oxidation of AG to 1,5-anhydro-D-fructose. This oxidation reaction consumed molecular oxygen as the terminal electron acceptor. The AG-oxidizing activity was further purified after solubilization. The AG oxidation catalyzed by this solubilized enzyme utilized molecular oxygen only in the presence of an electron mediator such as 2,6-dichlorophenolindophenol or phenazine methosulfate. Thus, the enzyme was suggested to be a dehydrogenase rather than an oxidase. The solubilized enzyme preparation also showed a strict substrate specificity. The observed specificity indicated that application of the enzyme for AG assay in clinical samples might be possible.     

Simulation of a reactor for glucose isomerization to fructose by immobilized glucose isomerase with continuous enzyme renewal

Two different dispositions of laboratory-scaled columns have been tested to simulate the isomerization of glucose to fructose in a mobile bed reactor where exhausted immobilized glucose isomerase is continuously renewed. If the simulation columns working at 65°C are arranged in parallel and connected to a section for final enzyme exploitation at 75°C, a syrup with constant composition can be produced, at relatively constant total throughput, by feeding the individual columns at flow rate decreasing according to the enzyme decay profile and following a programmed disphased mode of operation.This revised version was published online in October 2005 with corrections to the Cover Date.     

Determination of gliclazide in human serum by high-performance liquid chromatography using an anion-exchange resin

A method for the routine clinical examination of serum gliclazide by high-performance liquid chromatography (HPLC) on a column packed with a macroporous anion-exchange resin, Diaion CDR-10, was developed. The elution was performed with acetonitrile—methyl alcohol—1.2 M ammonium perchlorate (4:3:7, v/v/v) at a flow-rate of 0.4 ml/min. The retention time of gliclazide was 15 min. It seems that the retention mechanism of gliclazide under the HPLC conditions described is not only ion-exchange mode but reversed-phase mode between the anion-exchange resin and the mobile phase. The detection limit of gliclazide was 0.2 μg/ml in plasma. The coefficient of variation for the within-day assay was 5.0% (0.2 μg/ml, n=8). The decay curve of serum gliclazide in diabetic patients was determined.