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
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.
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.
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
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.
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
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.
1999
H. Podgornik, A. Podgornik, A. Perdih
Analytical Biochemistry 272, 43–47 (1999)
The HPLC separation of lignin peroxidase isoenzymes using Convective Interaction Media disks containing quaternary amine and diethylaminoethyl ion-exchange active groups is proposed. In contrast to standard HPLC procedures the separation can be performed within a few minutes without considerably affecting the separation resolution. The method is reproducible and gives a linear response of integrated peak area to protein concentration for all measured isoenzymes. The separation resolution is retained unchanged by applying crude culture filtrate instead of a sample previously frozen and dialyzed. The optimized method might therefore be used for on-line monitoring of lignin peroxidase isoenzyme composition during fermentation. On the other hand, the proposed method is comparable in time to the original method of lignin peroxidase activity measurement (proposed by Tien and Kirk), providing additionally the isoenzyme composition.
1998
D. Josić, H. Schwinn, A. Štrancar, A. Podgornik, M. Barut, Y. P. Lim, M. Vodopivec
Journal of Chromatography A, 803 (1998) 61–71
Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase IV, which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate–polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.
D. Josić, H. Schwinn, A. Štrancar, A. Podgornik, M. Barut, Y. P. Lim, M. Vodopivec
Journal of Chromatography A, 803 (1998) 61–71
Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase IV, which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate–polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.
R. Giovannini, R. Freitag, T. B. Tennikova
Anal. Chem. 1998, 70, 3348-3354
Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the threealbeit isolated bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 × 52 mm, 10-μm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.