Milan Polakovič

Modelling of potato starch saccharification by an Aspergillus niger glucoamylase

The kinetics of hydrolysis of a soluble potato starch catalysed by free glucoamylase was investigated in a batch reactor. Seventeen progress curves of product concentration, obtained at varying initial substrate and enzyme concentrations, were fitted simultaneously using six different models of saccharification process. A good description of the whole course of the progress curves was obtained using a six-parameter model based on a kinetic equation, which contained product and substrate inhibition terms. An essential feature of the model was that starch was structured into susceptible and resistant fractions that differed in the rate constants of hydrolysis. The model parameters were estimated with a good accuracy and were in a good agreement with the available literature information.

Analysis of mechanism and kinetics of thermal inactivation of enzymes: Evaluation of multitemperature data applied to inactivation of yeast invertase

Abstract A method is presented that substantially improves the analysis of the inactivation mechanism when compared to the conventional evaluation of isothermal data. The method utilizes a simultaneous fit of inactivation data obtained at several temperatures with the kinetic models containing the temperature dependence of rate constants in the form of the Arrhenius equation. In a case study of thermal inactivation of yeast invertase in the temperature range of 40–60°C, it was demonstrated that the multitemperature evaluation method significantly diminished the lumped character of inactivation kinetics. While the isothermal evaluation could provide only a formal two-step kinetic equation without verifying a particular mechanism, the presented method efficiently discriminated among different two- and three-reaction mechanisms. The general assessment of the method was made by comparing the modeling results in relation to the degree of complexity and structure of the examined mechanisms. Based on the modeling results, the inactivation of invertase was assumed to proceed in at least three steps which were discussed in relation to the available structural data on the mechanism of yeast invertase inactivation.

Immobilization in biotechnology and biorecognition: from macro- to nanoscale systems

Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.

Characterization of pore structure of a strong anion-exchange membrane adsorbent under different buffer and salt concentration conditions

The quantitative characterization of pore structure of Sartobind Q, a strongly basic membrane anion exchanger that is formed by cross-linked cellulose support and a hydrogel layer on its pore surface, was made combining the results obtained by several experimental techniques: liquid impregnation, batch size-exclusion, inverse size-exclusion chromatography, and permeability. Mercury intrusion and nitrogen sorption porosimetry were carried out for a dry cellulose support membrane in order to get additional information for building a model of the bimodal pore structure. The model incorporated the distribution of the total pore volume between transport and gel-layer pores and the partitioning of solutes of different molecular weights was expressed through the cylindrical pore model for the transport pores and random plane model for the gel layer. The effect of composition of liquid phase on the pore structure was investigated in redistilled water, phosphate and Tris-HCl buffers containing up to 1M NaCl. Evident differences in the bimodal pore structure were observed here when both the specific volume and size of the hydrogel layer pores significantly decreased with the ionic strength of liquid phase.

Influence of ligand density on antibody binding capacity of cation-exchange adsorbents.

Adsorption properties of a set of polymethacrylate-based cation exchangers designed for purification of monoclonal antibodies were investigated. The materials differed significantly in the density of sulphoisobutyl ligand groups. The ligand density had a pronounced effect on the static adsorption capacity of a polyclonal human immunoglobulin G. An optimal ligand density was observed at any pH and NaCl concentration tested when sharp optima were observed at low pH and ionic strength values. This was caused by effective clogging of pore mouth at high ligand densities. An anomalous effect of ionic strength was observed for the adsorbents with the high ligand density when the adsorption capacity increased with the addition of NaCl at low pH.

Design and economics of industrial production of fructooligosaccharides

A process for industrial production of fructooligosaccharides (FOS’s) based on the conversion of sucrose by immobilized fructosyltransferase (FTase) from the cells of Aureobasidium pullulans CCY 27-1-94 was developed. Particular process operations and conditions were designed employing results of laboratory and semi-pilot scale experiments. The process flowsheet comprised three sections: FTase production, which included fermentation, isolation and purification of the enzyme, FTase immobilization and FOS’s production where a product with a high content of FOS’s was prepared by the removal of glucose, fructose and unreacted sucrose from the reaction mixture using simulated moving-bed chromatography. Two alternative process flowsheets were proposed for the annual production of 10 000 t of FOS’s: one for a powdery product and the second one for syrup. The economic analysis provided the costs for the production of immobilized FTase and FOS’s using two different price estimates for sucrose.

Influence of immobilization on the thermal inactivation of yeast invertase

Abstract Different immobilized preparations of yeast invertase were studied for the purpose of refining the mechanism of inactivation of free enzyme. Four immobilization techniques were applied: biospecific adsorption on the concanavalin A-bead cellulose matrix; covalent immobilization on the activated bead cellulose matrix; and cross-linking of the aforementioned preparations with glutaraldehyde. All preparations were assessed with respect to the activity loss during immobilization, thermal stability, and inactivation kinetics. For each preparation, a corresponding inactivation mechanism was verified using the multitemperature data evaluation. The preparations, whose biological activities were strongly modified during immobilization, were shown to be unsuitable for elucidating the inactivation mechanism of free invertase. The biospecifically adsorbed invertase on the concanavalin A-bead cellulose matrix met all criteria for a preparation that should be a convenient model of free enzyme. This preparation was found to inactivate through a three-step series mechanism with the activation energies of individual reactions at 304, 833, and 675 kJ mol −1 . The results obtained in this study complemented an existing mechanism of the inactivation of free invertase.

Determination of effective diffusion coefficient of substrate in gel particles with immobilized biocatalyst

Abstract A method of determining of the effective diffusion coefficient of substrate in a particle, where the diffusion and consumption of substrate by biocatalytic reaction are present simultaneously, was designed and experimentally verified. The method is based on measuring the overall rate of heterogeneous biocatalytic reaction in particles of varying diameter. The effective diffusion coefficient, De, was determined by fitting the measured reaction rates with the solution of the reaction-diffusion equation. The method is tailored for cases where the enzyme reaction is governed by the Michaelis-Menten kinetics. The value of Km required for the solution of the mathematical model was adopted from the measurement of the kinetics of free cells, whereas the rate parameter, k2, was optimized together with De. As an experimental model, the sucrose hydrolysis catalyzed by Ca-alginate-entrapped yeast cells was examined. The particle diameter varied in the range of 1.2–3.9 mm and the initial reaction rates were measured in a batch-stirred reactor at a sucrose concentration of 100 m m . The De of sucrose at 30°C was found to be 2.9 · 10−10 m2s−1.

Effect of pH on protein adsorption capacity of strong cation exchangers with grafted layer

The effect of pH on the static adsorption capacity of immunoglobulin G, human serum albumin, and equine myoglobin was investigated for a set of five strong cation exchangers with the grafted tentacle layer having a different ligand density. A sharp maximum of adsorption capacity with pH was observed for adsorbents with a high ligand density. The results were elucidated using the protein structure and calculations of pK(a) of ionizable groups of surface basic residues. Inverse size-exclusion experiments were carried out to understand the relation between the adsorption capacity and pore accessibility of the investigated proteins.

Chromatographic separation and kinetic properties of fructosyltransferase from Aureobasidium pullulans

Efficient chromatographic separation of fructosyltransferase from Aureobasidium pullulans was achieved on a preparative scale using a weak anion-exchanger Sepabeads FP-DA. The recovery yield was about 70% and the purification factor reached a value of 28. The molecular weight of the enzyme determined by size-exclusion chromatography was 570,000. The enzyme exhibited both hydrolytic and transferase activity when the latter was higher in the whole concentration range and completely dominating at higher sucrose concentrations. It was found that sucrose was the only donor of fructosyl moiety used in the transfer reaction. The initial rate method was used for the investigation of the kinetics of the action of fructosyltransferase on sucrose in the concentration range 30-2,430 mM. The initial transfructosylation rate was well fitted with a linear function of the sucrose activity where the activity coefficient was an exponentially decreasing function of the sucrose concentration.