Publications
2003
M. Vodopivec, A. Podgornik, M. Berovič, A. Štrancar
Journal of Chromatography B, 795 (2003) 105-113
The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis–Menten constant Km and the turnover number k3 of the immobilized enzymes were found to be flow-unaffected. Furthermore, the Km values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.
2002
A. Podgornik, M. Barut, S. Jakša, J. Jančar, A. Štrancar
Journal of Liquid Chromatography & Related Technologies Vol. 25, No. 20, pp. 3097–3114, 2002
Convective Interaction Media® (CIM) disk monolithic columns are specific among the chromatographic columns because of their monolithic structure and extremely short column length. In this work, HETP values and Z factors for different groups of molecules—proteins, DNA, oligonucleotides, peptides, and organic acids on strong anion exchange CIM disk monolithic columns were determined. Results are discussed in terms of the molecule structures and applied to develop different approaches for successful separation of abovementioned group of molecules on these types of columns.
R. Hahn, M. Panzer, E. Hansen, J. Mollerup, A. Jungbauer
Separation Science and Technology, 37(7), 1545–1565 (2002)
The mass transfer properties of polyglycidylmethacrylate–ethylenedimethacrylate monolithic ion-exchangers (convective interaction media disks) were evaluated. As a reference material, the particulate ion-exchanger Source 30 was selected. The model proteins lysozyme, bovine serum albumin, and IgG were loaded at different concentrations and velocities. The mass transfer zones obtained with the monoliths were affected by neither the linear flow velocity nor the protein concentration in the mobile phase. The reduced height equivalent to one theoretical plate (HETP) of monoliths were independent of the reduced velocity. This was not the case for the particulate material.
A. Štrancar, A. Podgornik, M. Barut, D. Glover
BIOforum International 3/2002
In adsorptive chromatographic modes, the slope of the capacity factor k' (defined as the molar ratio of the separated compound in the stationary phase and the mobile phase) plot versus composition of the mobile phase is very steep. Up to a certain composition of the mobile phase, k' is so high that the protein is bound to the stationary phase and does not move along the column. Reaching a defined point, a small change of the mobile phase composition causes a rapid decrease in k' to a value near zero. At this point, the protein dissolves in the mobile phase and passes through the column practically without any retention. In other words, the protein remains adsorbed at the top of the column until the eluting power of the mobile phase reaches the point at which a small change in the composition of the mobile phase causes the movement of the protein without any retention. One can also speak about selective elution of the compound. As a result of this process, even very short columns can provide very good separations and recovery, while longer columns might cause problems due to unspecific binding, product degradation and minor changes in the structure of the protein which increase with the length of the column. On the other hand, short-beds are very difficult to pack with particles and form channels which eliminate the resolution power of the column. Monolithic supports offer an ideal solution to avoid most of these problems.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
J. Comb. Chem. 2002, 4, 33-37
Solid-phase peptide synthesis was performed on glycidyle methacrylate-co-ethylene dimethacrylate monoliths using Fmoc chemistry. The native epoxy groups were amino-functionalized by reaction with ethylenediamine or ammonia ions. A peptide directed against human blood coagulation factor VIII was synthesized as a model peptide. Amino acid analysis revealed the correct amino acid ratio as present in the sequence. The ligand density of 5 μmol/mL was equal to that achieved with conventional peptide immobilization via epoxy groups. These supports were directly used as peptide affinity chromatography matrixes. The functionality of the CIM monolithic supports was proven by affinity chromatography of factor VIII. The ammonia-functionalized support performed with low hydrophobicity and did not show unspecific adsorption of proteins.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
Biotechnology and Bioengineering 79 (2002) 733-740
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 733–740, 2002.
T. Tennikova, A. Štrancar
LabPlus international February - March 2002, Volume 16
Monolithic supports are a novel generation of stationary phases that can be used for liquid and gas chromatography, capillary electrochromatography, bioconversions, as well as supports for solid phase synthesis. In contrast to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They provide high rates of mass transfer at lower pressure drops and enable much faster separations. In addition to the speed, the nature of the pores allows easy permeability for large molecules. Monolithic supports are thus the method of choice for the separation of proteins, oligonucleotides, and nanoparticles such as pDNA and viruses. In this article we review the application of the monlithic columns to bioaffinity chromatography.
H. Podgornik, A. Podgornik
Enzyme and Microbial Technology 31 (2002) 855–861
The possibility of covalent attachment of LiP H2 and LiP H8 to CIM monoliths was studied. Due to negligible diffusional resistance, they can be useful tools to study characteristics of the immobilized lignin peroxidase (LiP). Immobilization to epoxy groups was performed using alkaline conditions (borate-phosphate buffer; pH 7.5). Characteristics of immobilized LiP were compared and factors that influence their biologic activity were evaluated using flow through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol (VA) into veratraldehyde (Vald). While VA oxidation rate increased by increasing flow rate (up to 1.5 ml/min) for LiP H2, it was almost constant in a wide flow rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison to free enzyme. The catalytic constant was found to be approximately 0.5 s-1 for both isoforms. Immobilized LiP H8 was used for decolorization of azo dye Mahogany.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
Biotechnology and Bioengineering 79 (2002) 733-740
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.
A. Podgornik, T. B. Tennikova
Advances in Biochemical Engineering/ Biotechnology, 2002, Vol. 76, 167-206
In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context.
H. Podgornik, A. Podgornik
Enzyme and Microbial Technology 31 (2002) 855–861
The possibility of covalent attachment of LiP H2 and LiP H8 to CIM monoliths was studied. Due to negligible diffusional resistance, they can be useful tools to study characteristics of the immobilized lignin peroxidase (LiP). Immobilization to epoxy groups was performed using alkaline conditions (borate-phosphate buffer; pH 7.5). Characteristics of immobilized LiP were compared and factors that influence their biologic activity were evaluated using flow through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol (VA) into veratraldehyde (Vald). While VA oxidation rate increased by increasing flow rate (up to 1.5 ml/min) for LiP H2, it was almost constant in a wide flow rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison to free enzyme. The catalytic constant was found to be approximately 0.5 s−1 for both isoforms. Immobilized LiP H8 was used for decolorization of azo dye Mahogany.
2001
I. Mihelič, M. Krajnc, T. Koloini, A. Podgornik
Ind. Eng. Chem. Res. 2001, 40, 3495-3501
Monolithic stationary phases are becoming more and more important in the field of liquid chromatography, because they enable extremely fast separations. Methacrylate-based monoliths are produced via a free-radical bulk polymerization of glycidyl methacrylate and ethylene dimethacrylate using a benzoyl peroxide as an initiator. Preparation of large monoliths represents a big problem because of the heat release during the polymerization, which consequently leads to the distortion of the structure. A closer investigation of the polymerization, using differential scanning calorimetry, was performed in order to determine global kinetic parameters. A multiple heating rate method, based on the work of Ozawa, Flynn, and Wall, was applied for estimation of the values of the apparent activation energy, preexponential factor, and reaction order. Global polymerization kinetics is of first order with A = 1.681 × 109 s-1 and Ea,app = 81.5 kJ/mol, where the heat of polymerization is approximately 190 J/g. In addition, the influence of air and nitrogen atmosphere on polymerization is presented.
M. Merhar, A. Podgornik, M. Barut, S. Jakša, M. Zigon, A. Štrancar
J. Liq. Chrom. & Rel. Technol., 24(16), 2429-2443 (2001)
Monoliths have already proven to be efficient chromatographic supports for the separation of various types of molecules. In this paper, the characterization of the novel reversed-phase support, CIM® RP-SDVB disk monolithic column is presented.
Using a 3 mm long RP-SDVB disk monolithic column, excellent separation of proteins within a very short time was achieved. The pressure drop observed on the column was considerably low (few bars), even at flow rates of the mobile phase up to 30 mL/min. Due to the low pressure drop, the use of high flow rates was preferred since they did not influence the quality of the gradient separation. The separation of test proteins was performed within only 14 seconds; faster separations were limited by the configuration of the HPLC system.
Furthermore, RP-SDVB disk monolithic columns were applied for fast separation of peptides. Five peptides of different lengths and composition were successfully separated in a very short time.
Finally, the preparative purification on the laboratory scale of the complex sample of oligodeoxynucleotide within a range of 1 minute demonstrates practical applicability of these columns.
D. Josić, A. Buchacher
J. Biochem. Biophys. Methods 49 (2001) 153–174
Monoliths are useful chromatographic supports, as their structure allows improved mass transport. This results in fast separation. Once the ligand of interest has been immobilized, chromatographic separation can also be accomplished in affinity mode. Ligands with low molecular mass have been shown to be the easiest to immobilize. Nowadays, ligands with low molecular mass are often designed by combinatorial chemical techniques. In addition, many applications have been described where ligands with high molecular mass, such as Proteins A and G, antibodies, lectins and receptors are used.
The immobilization of an enzyme on the monolithic support creates a flow-through reactor. Small proteins, such as carbonic anhydrase, can be directly immobilized on the support. However, in the case of large molecules, the active center of the enzyme is no longer accessible at all or only to a limited degree. An improvement can be achieved by introducing a spacer, which allows maximum enzymatic conversion. Fast conversion of substrates with high molecular mass has been investigated with immobilized trypsin. It was shown that in case of high-molecular-mass substrates, the conversion rate depends very much on the flow-rate. Most applications described have been performed on an analytical or semi-preparative scale. However, the technical problems of up-scaling are close to being definitely solved, enabling enzymatic conversion on a preparative scale in the future.
I. Mihelič, T. Koloini, A. Podgornik, M. Barut, A. Štrancar
Acta Chim. Slov. 2001, 48, 551-564
Monolithic stationary phases are becoming very important field of liquid chromatography. Methacrylate based CIM Convective Interaction Media® monolithic columns and are produced via radical polymerization, which results in a rigid and chemically very stable porous monolithic structure. Some characteristics of small-scale monolithic columns and an example of extremely fast separation of biomolecules are presented in the paper. However, the preparation of large and homogeneous monolithic columns represents a big problem, because the heat released during the polymerization causes distortion of the monolithic structure. A mathematical model employing the polymerization kinetics for the prediction of the temperature profiles and a comparison with the experimental results is presented with the emphasis on the conversion and the rate od the heat release profiles. Finally, the characteristics of a large-scale monolithic column are presented.
D. Josić, A. Buchacher
J. Biochem. Biophys. Methods 49 (2001) 153–174
Monoliths are useful chromatographic supports, as their structure allows improved mass transport. This results in fast separation. Once the ligand of interest has been immobilized, chromatographic separation can also be accomplished in affinity mode. Ligands with low molecular mass have been shown to be the easiest to immobilize. Nowadays, ligands with low molecular mass are often designed by combinatorial chemical techniques. In addition, many applications have been described where ligands with high molecular mass, such as Proteins A and G, antibodies, lectins and receptors are used.
The immobilization of an enzyme on the monolithic support creates a flow-through reactor. Small proteins, such as carbonic anhydrase, can be directly immobilized on the support. However, in the case of large molecules, the active center of the enzyme is no longer accessible at all or only to a limited degree. An improvement can be achieved by introducing a spacer, which allows maximum enzymatic conversion. Fast conversion of substrates with high molecular mass has been investigated with immobilized trypsin. It was shown that in case of high-molecular-mass substrates, the conversion rate depends very much on the flow-rate. Most applications described have been performed on an analytical or semi-preparative scale. However, the technical problems of up-scaling are close to being definitely solved, enabling enzymatic conversion on a preparative scale in the future.
H. Podgornik, M. Stegu, A. Podgornik, A. Perdih
FEMS Microbiology Letters 201 (2001) 265-269
High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.
2000
R. Hahn, A. Jungbauer
Analytical Chemistry, 7.2. 2000, (4853-4858)
Monoliths are stationary phases cast as a continuous medium which are interlaced by flow channels ramified with micropores. Pulse response experiments with bovine serum albumin as a model protein were applied for testing polymethacrylate-based monoliths, resulting in peak broadening that practically was not influenced by the chromatographic velocity. An empirical model was developed to describe peak broadening, allowing a term to account for the pore convection and a term for the pore diffusion. A diffusional distance lower than 10 nm was estimated. This corresponds to values observed with monodisperse 1-μm particles. Systematic investigations by changing the response time of the detector showed that the full potential of the monoliths could not be exploited, since the currently available chromatography systems are the limiting factor regarding the speed of data acquisition and virtual peak broadening by the infinite length of the detector. Inertia of the liquid and synchronization between liquid handling and electronic control introduced an additional disturbance. At the lowest possible response time, reliable peak data could be obtained up to a velocity of 35 cm/min. The pressure drop along the continuous bed was much smaller compared to a conventionally packed bed. Different flow patterns and significantly reduced eddy vortexes may be responsible for the high specific permeability.
H. LeThanh, B. Lendl
Analytica Chimica Acta 422 (2000) 63–69
A fully automated method for the rapid determination of organic acids (citric-, malic- and tartaric acid) and sugars (glucose, fructose, and sucrose) in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy is presented. A convective interaction media (CIM) disc carrying quaternary amino moieties was added as a solid phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM disc whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution and recorded in their fully deprotonated form as a second flow injection peak. In both cases, the FTIR spectra corresponding to the peak maxima were selected for data evaluation. Two partial least squares models, one for sugars and the other for organic acids, were constructed based on the analysis of standards containing all six analytes. The developed method was applied to natural samples yielding results which were in good agreement with those obtained by an external reference method (enzymatic test kits). Deviations in the results were 3.4. and 4.1% for citric and malic acid and ranged from 4.7–5.1% for the sugars. The developed method is characterized by its short analysis time, experimental simplicity and its potential applications in routine analysis and process control.
M. Vodopivec, M. Berovič, J. Jančar, A. Podgornik, A. Štrancar
Analytica Chimica Acta 407 (2000) 105-110
A new polymeric macroporous material, Convective Interaction Media (CIM) was applied as a support for glucose oxidase (GOD) immobilisation. CIM epoxy disks with the immobilised enzyme were integrated as an enzyme reactor in a flow injection analysis (FIA) system and applied to on-line monitoring of glucose during cultivation of Saccharomyces cerevisiae and citric acid production by Aspergillus niger. The developed CIM GOD disk–FIA system exhibited good signal reproducibility and satisfactory long-term stability with a linear response in the range 10–200 mg l-1. The CIM disk applied as an enzyme reactor proved to be a successful replacement for conventionally used packed-bed columns and as such it was well suited for on-line monitoring of bioprocesses.