Vladimír Štefuca

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.

Application of the enzyme thermistor to the direct estimation of intrinsic kinetics using the saccharose-immobilized invertase system

The possibility of using the enzyme thermistor (ET) for the direct determination of kinetic parameters (Km, Ki, Vm) of immobilized enzyme (IME) was evaluated using different preparations of invertase conjugated to bead celluloses. Two different ET columns packed with IME were operated in the mode of a differential enzyme reactor (short length, low substrate conversion). Kinetic parameters of the above IME reactor were computed by a nonlinear curve-fitting procedure. The obtained kinetic parameters were superverified by means of an independent differential reactor (DR) system. This system utilized an indirect postcolumn analytical method based on determination of glucose concentration in the stirred reservoir. Best agreement between the data acquired by direct (ET) and indirect (DR) methods was obtained if the ET column was operated at flow rates within the range of 1.0-1.5 ml min-1 using invertase-cellulose chlorotriazine conjugate. Influence of heat loss and flow nonideality is discussed. The proposed ET method offers a rapid, convenient, and general approach to determination of kinetic constants of IME preparations by omitting postcolumn analytical methods.

Biochemical engineering of biocatalysts immobilized on cellulosic materials

Complete design of the optimum immobilized biocatalyst seems to still be a matter of the future. To be successful, it would require numerical determination of all significant parameters at each enzyme engineering phase, that is at the design of the carriers, immobilized biocatalysts and immobilized reactors. Future research trends should follow this strategy. For processing, cellulosic materials have been considered carriers that fulfill requests to an example model: they represent a unique family of carriers that cover a broad variety of physical and chemical properties, immobilizing techniques, and immobilized reactors as well. The reason for writing this review article was to test the reliability of such a processing and subsequently, to confront theoretical considerations with practical applications of biocatalysts immobilized on cellulose materials.

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.

Gluconobacter in biosensors: applications of whole cells and enzymes isolated from gluconobacter and acetobacter to biosensor construction

Bacteria belonging to the genus Acetobacter and Gluconobacter, and enzymes isolated from them, have been extensively used for biosensor construction in the last decade. Bacteria used as a biocatalyst are easy to prepare and use in amperometric biosensors. They contain multiple enzyme activities otherwise not available commercially. The range of compounds analyzable by Gluconobacter biosensors includes: mono- and poly-alcohols, multiple aldoses and ketoses, several disaccharides, triacylglycerols, and complex parameters like utilizable saccharides or biological O2 demand. Here, the recent trends in Gluconobacter biosensors and current practical applications are summarized.

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.

New approaches for verification of kinetic parameters of immobilized concanavalin A: Invertase preparations investigated by flow microcalorimetry

In our preceding article, we demonstrated a procedure based upon enzymic flow microcalorimetry using an enzyme thermistor (ET) to characterize the microkinetic properties of an immobilized enzyme (IME) and its further application in the screening of IMEs. To consider the ET method (single ET unit, ET system 1) as standard, it was necessary to show that the estimated relative kinetic parameter (ΔTmax) calorimetrically corresponds with the absolute value for the reaction rate within the whole measurement range. This article presents three experimental verification procedures. Two procedures are based on adaptation of the flow‐through ET column to a mini‐differential‐reactor (DR) system with substrate recirculation and post‐ET‐column methods for determination of the concentration change of the product (spectrophotometrically in ET system 2) or the substrate (calorimetrically in ET system 3) with the IME‐catalyzed enzymic hydrolysis. The third procedure is an independently operating DR system which spectrophotometrically estimates the concentration change of the product. The results obtained exhibited good correlation (r = 0.921) between the relative kinetic parameter ΔTmax, as determined calorimetrically by ET system 1, and the absolute value for the reaction rate (rmax) as determined by ET systems 2 and 3. These data proved that, within the whole range of experimental conditions applied in this study, the parameter ΔTmax instead of the true reaction rate could be employed for the IME screening. Moreover, the generality of the detection principle and the standardized configuration of the ET favor ET systems 2 and 3 for normal screening of IMEs and as miniaturized DR systems allowing dual measurements of kinetic parameters.

Polyelectrolyte complex capsules as a material for enzyme immobilization : catalytic properties of encapsulated lactate dehydrogenase

The polyelectrolyte complex (PEC) membrane formed by cellulose sulfate and poly(dimethyldiallylammonium chloride) was used to encapsulate lactate dehydrogenase. The exclusion limit of the membrane was found to be low enough to secure irreversible entrapping of the enzyme. The obtained capsules were checked for their functionality in a stirred-batch reactor by following the kinetics of NADH oxidation. The data were fitted with an isotropic kinetic model including competitive product-inhibition phenomenon. The results of mathematical modeling demonstrated that the anisotropic system, like PEC capsules, could be satisfactorily described by the isotropic model.

Study of porous cellulose beads as an enzyme carrier via simple mathematical models for the hydrolysis of saccharose using immobilized invertase reactors

Abstract Among four conjugates of invertase differently immobilized on porous cellulose beads, the chlorotriazinyl cellulose-invertase (stable catalytic activity in the time interval between 10th and 80th day of storage) was chosen for studies of reaction kinetics in a stirred tank reactor and a packed bed reactor. In the stirred tank reactor, due to localization of invertase on external surface of the beads, the external diffusion does not exert any influence for reaction kinetics. The spherical shape of cellulose beads was partly destroyed particularly when the rate of stirring exceeded 200 rev min−1. In the packed bed reactor, owing to the Sherwood number kept to below 2 (flow rates 0.5–4 ml min−1), deformation of the ideal spherical shape of beads was observed. When the preservation of original shape of beads is required, other types of reactors or reinforcement of beads is recommended.

Biosensors with Immobilised Microbial Cells Using Amperometric and Thermal Detection Principles

Biosensor is a device in which the recognition system utilizes a biological element and this information is further transformed into an analytically useful signal by a transducer (Figure 1). Biosensors can be classified according to the biological element or transducer used. Several transducers were exploited for successful biosensor construction using electrochemical, optical, calorimetric, piezoelectric and others principles of detection []. The most common biocomponents that have been used for the preparation of biosensors are enzymes, antibodies, DNA and cells [1]. The basic requirement for effective performance of the biosensor is a close proximity between transducer and a biological element, which is achieved by a suitable immobilisation strategy.