Jiří Lenfeld

Oriented immobilization of galactose oxidase to bead and magnetic bead cellulose and poly(HEMA-co-EDMA) and magnetic poly(HEMA-co-EDMA) microspheres

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.

Enzymes immobilized on magnetic carriers: efficient and selective system for protein modification

In order to obtain an economical, efficient and selective system for glycoprotein modification we prepared reactors with immobilized neuraminidase or (and) galactose oxidase. High storage and operational stability of the enzyme reactors was obtained by their immobilization through the carbohydrate parts of the enzyme molecules to hydrazide-modified supports. Magnetic and non-magnetic forms of bead cellulose and poly(HEMA-co-EDMA) microspheres were used for immobilization. These reactors can be used almost universally for the activation of ligands and for labelling of substances having a carbohydrate moiety.

Immunomagnetic separation and detection of Salmonella cells using newly designed carriers.

Magnetic nonporous poly(HEMA-co-EDMA) and poly(HEMA-co-GMA) microspheres were prepared by dispersion copolymerisation of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) or glycidyl methacrylate (GMA) in the presence of magnetite. They were functionalized by polyclonal Salmonella antibodies via the trichlorotriazine method. Salmonella cells were then successfully identified using cultural and polymerase chain reaction (PCR) methods after their immunomagnetic separation. The PCR sensitivity of target cell detection was negatively influenced by the presence of some compounds used in the process of particle preparation. In some cases, magnetic poly(HEMA-co-EDMA) microspheres with immobilized proteinase K were used for degradation of intracellular inhibitors present in Salmonella cells.

Comparison of oriented and random antibody immobilization in immunoaffinity chromatography of cytokinins

Immunosorbents for the plant hormones cytokinins prepared by random antibody immobilization (to Affi-Gel 10) and by oriented approach via oxidized carbohydrate moieties on the Fc region (to Affi-Gel Hz or hydrazide derivative of Perloza MT 200) have been compared. Both approaches yielded immunosorbents with high dynamic capacity (ca. 5-10 nmol ml gel-1). Oriented antibody immobilization did not exhibit crucial effects in the case of low-molecular-mass cytokinins. Antibodies immobilized via a spacer to Affi-Gel 10 have probably enough conformational freedom to enable good accessibility to cytokinins. The sorbents were used in analysis of endogenous cytokinins in maize seeds. In phosphatase treated samples trans-zeatin and its riboside were predominant.

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles.

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.

Semisynthesis of C17:0 isoforms of sulphatide and glucosylceramide using immobilised sphingolipid ceramide N-deacylase for application in analytical mass spectrometry

Sphingolipid ceramide N-deacylase (SCDase, EC 3.5.1.69) is a hydrolytic enzyme isolated from Pseudomonas sp. TK 4. In addition to its primary deacylation function, this enzyme is able to reacylate lyso-sphingolipids under specific conditions. We immobilised this enzyme on magnetic macroporous cellulose and used it to semisynthesise C17:0 glucosylceramide and C17:0 sulphatide, which are required internal standards for quantification of the corresponding glycosphingolipids (GSL) by tandem mass spectrometry. A high rate of conversion was achieved for both lipids (80% for C17:0 sulphatide and 90% for C17:0 glucosylceramide). In contrast to synthesis with a soluble form of the enzyme, use of immobilised SCDase significantly reduced the contamination of the sphingolipid products with other isoforms, so further purification was not necessary. Our method can be effectively used for the simple preparation of specifically labelled sphingolipids of high isoform purity for application in mass spectrometry.

A tosyl-activated magnetic bead cellulose as solid support for sensitive protein detection.

Magnetic bead cellulose (MBC) was prepared using sol-gel transition of viscose in the presence of maghemite (γ-Fe₂O₃) nanoparticles. The MBC particles were then activated with p-toluenesulfonyl chloride to yield tosyl-activated magnetic bead cellulose (MBC-Ts). The microspheres were characterized by light and electron microscopy, elemental analysis and atomic absorption spectroscopy to determine morphology, size, polydispersity and content of iron and tosyl groups. The functionality of the MBC-Ts microspheres was demonstrated using proximity ligation assay (PLA) to detect vascular endothelial growth factor in femtomolar concentration range. The MBC-Ts microspheres performed equally well as commercially available microparticles that are routinely used as solid support in solid phase PLA.

3,5-Diiodo-l-tyrosine immobilized on bead cellulose

Abstract 3,5-Diiodo- l -tyrosine (DIT) was immobilized on bead cellulose activated by the reaction with 2,4,6-trichloro-1,3,5triazine (TCT). The amount of immobilized DIT depends on the TCT/DIT ratio in the reaction mixture up to the value 8 mol/mol, at higher values it becomes TCT/DIT-independent. Various sorbents containing 5–31 μtmol DIT/ml cellulose were prepared by varying the amount of DIT in the reaction mixture. The immobilization is also affected by pH. More DIT was bound in acid (pH 6.0) or neutral medium in comparison with the alkaline reaction conditions. Most of the experiments were performed at pH 9.1 equal to the ionization constant of NH 2 group of L-tyrosine. The DIT/bead cellulose materials were prepared for use as sorbents in affinity chromatography of proteases.

Functionalization of bead cellulose: azo coupling of diazotized 2-(4-aminophenylsulfonyl)ethyl derivative with 2-naphthol

Abstract The azo coupling reaction of 2-naphthol with diazotized 2-(4-aminophenylsulfonyl)ethyl groups bound to macroporous bead cellulose was studied. The reaction was followed spectrophotometrically by determining 2-naphthol in the reaction mixture. The amount of 2-naphthol bound to the sorbent decreases with increasing pH in the pH range 5–9 as a result of simultaneous influence of the sulfonyl groups on the stability of the diazonium salt and limited mobility of the diazonium groups bound to the sorbent. Approximately a 1.3 molar excess of 2-naphthol and a 1-h reaction time at 25°C are sufficient for a maximum reaction yield at a given pH. The method makes possible the semiquantitative analysis of the amount of amino groups in the sorbent which are accessible to coupling.

Preparation of gelatin spherical microparticles in aqueous polymer suspension

Abstract Gelatin spherical microparticles were prepared by suspending a 20 wt% aqueous solution of gelatin in a 10 wt% aqueous solution of polyvinylpyrrolidone. Gelatin was used either without modification, or it was extracted with water or ammonium hydroxide. Suspending was carried out at 60°C. The particle size decreased considerably when the extracted gelatin was used, or when ammonium hydroxide was added to the suspension medium. Increasing the stirring intensity decreased the particle size and also the stability of the particles. The particles were stabilized by cooling and crosslinking with formaldehyde and separated from the suspension medium by washing with methanol. The resulting particles were dried of ethyl acetate. Five samples of the gelatin microparticles were prepared, the diameters of which varied from 0.5 to 15 μm in dry state.