Regina Hüttl

Calorimetric investigations into enzymatic urea hydrolysis

Abstract Enzymatic urea hydrolysis has been investigated calorimetrically as a function of urea concentration, the buffer system and the pH value. It is shown that the reaction follows a complex mechanism. Unambiguous conclusions on the reaction sequences are made possible by extensive thermodynamic calculations. The most important conclusion from the experimental results and thermodynamic calculations is the absolute necessity of knowledge of the experimental conditions, i.e. urea concentration, pH value and buffer system, for the meaningful discussion of calorimetric data. The molar reaction enthalpy of the enzymatic partial reaction is determined to be ΔRH1 = (−26.5 ± 1.5) kJ per mole urea. Thermokinetic evaluation by means of a simple consecutive reaction model allows the velocity constants to be calculated. For the enzymatic partial reaction, parameters Km and vmax are determined in phosphate buffer and HEPES buffer.

Degradation of selected (bio-)surfactants by bacterial cultures monitored by calorimetric methods

The subjects of the article are investigations concerning the ability of both Rhodococcus opacus 1CP and mixed bacterial cultures to use selected surfactants as sole carbon and energy source. In a comparative manner the biosurfactants rhamnolipid, sophorolipid and trehalose tetraester, and the synthetic surfactant Tween 80 were examined. Particular emphasis was put on a combinatorial approach to determine quantitatively the degree of surfactant degradation by applying calorimetry, thermodynamic calculations and mass spectrometry, HPLC as well as determination of biomass. The pure bacterial strain R. opacus was only able to metabolize a part of the synthetic surfactant Tween 80, whereas the mixed bacterial cultures degraded all of the applied surfactants. Exclusive for the biosurfactant rhamnolipid a complete microbial degradation could be demonstrated. In the case of the other surfactants only primary degradation was observed.

Sequential flow injection analysis based on calorimetric detection

A calorimetrically based sequential flow injection method with enzyme catalyzed reactions is proposed and demonstrated. In contrast to multi-detection devices with immobilized enzymes miniaturized flow-through reaction calorimeters are used as sensing device with independent substrate and toggled enzyme solution flows. From the sequence of the heat power signals information about composition of the substrate is obtainable. A reasonable application of the method requires miniaturized calorimeters. Different constructions of flow-through reaction calorimeters based on silicon chips with integrated thermopiles as heat power transducers have been analyzed with respect to their sensitivities and mixing behavior. As an application the analysis of saccharides containing mixtures is discussed.

Calorimetric investigations of the enzyme catalyzed sucrose hydrolysis

The study presents the results of calorimetric investigations of the invertase catalyzed sucrose hydrolysis. The aim of the investigation is the kinetic interpretation of calorimetric curves with respect to the inhibitor effect of selected heavy metal ions and the calorimetric determination of the invertase activity.

Calorimetric investigations into enzyme catalysed glucose oxidation

Abstract The study presents the results of calorimetric investigations into enzyme catalysed glucose oxidation. It was shown that the convertible amount of glucose is limited by the availability of the oxidant or mediator: O2, K3[Fe(CN)6] and benzoquinone were used as mediators. The measurable glucose concentration range was much expanded by using benzoquinone. Independently of the mediator used, the enthalpy value for the oxidation of glucose in the presence of the enzyme glucose oxidase was found to be ΔRH = −125 ± 2 kJ mol−1.

Calorimetric investigations of urease inhibition by heavy metal ions

Abstract The subject of this publication is calorimetric investigations on the inhibitor effect of heavy metal ions on the enzyme urease. The obtained results allow quantification of the inhibitors (Cd 2+ -, As 3+ -, Zn 2+ -, Pb 2+ -ions) via the initial reaction rate of enzymatically catalysed urea hydrolysis. The interpretation potential of the calorimetric measurements is underlined by the determination of the inhibiting mechanisms for the example of Cd 2+ - and As 3+ -ions, the findings on the time regime of the inhibition process, and the detection of heavy metal ions in the ppm range. The use of several different buffer substance revealed the influence of the latter on the intensity of heavy metal inhibition. This opens the path to both the selective analysis of heavy metals via pattern recognition and to the improvement of detection sensitivity.

Problems associated with using thermal measurement principles in enzymatic reactions

Abstract The primary intention when applying themoanalytical techniques to enzymatic reactions is the derivation of reliable thermochemical and thermokinetic information for selected reactions. The enzymatic oxidation of glucose was selected as a model reaction. Caloric measurements showed that the extrapolated thermal power at t = 0 is proportional to the concentration of the substrate in GOD-catalysed glucose oxidation, so that measurement can be restricted to the determination of the initial power. The range of measurable glucose oxidation was initially limited by the oxygen content of the solution (0–1.4 mmoll −1 ). This concentration range has been expanded considerably by adding mediators. The additional use of mutarotase resulted in an increased reaction rate.

Thermokinetic investigations into enzyme catalysed glucose oxidation

Abstract The kinetic investigation of the enzyme (glucose oxidase) catalysed glucose oxidation was performed by calorimetric monitoring of the reaction. The measurable concentration range was expanded by using benzoquinone as a mediator. A comprehensive kinetic evaluation of the adiabatic calorimetric measurement curves allowed us to determine the Michaelis—Menten constant K M , the enzyme activity, the reaction rate equations with the corresponding velocity constants, and the functional dependence of the reaction rate on enzyme and glucose concentration.

Thermal investigations of enzyme-catalyzed reactions for detection of heavy metals in the case of cadmium

Abstract The present work gives the results of extensive calorimetric measurements to determine the inhibitory effect of cadmium ions on enzymatic urea hydrolysis. Three milligrams of urease and 10 mmol urea (kg solution)−1 were found to be the optimum reaction conditions in 0.05 M HEPES buffer (pH 6.6). A correlation between the initial rate ν0 and the cadmium concentration in the ppm range can be demonstrated by determination of the initial slope of the calorimetric measuring curves. By determination of the enzyme kinetic data, a contribution to detailed information on the inhibitor mechanism of cadmium can be made with these results.

On-line Monitoring of Enzyme Activities using Microreactor Heat Power Meters

To study the influence of the design parameters of miniaturized flow-through calorimeters on their signal generation behavior four different versions of flow-through calorimeters have been prepared and tested. For chemical reactions with low rate constants such as enzyme catalyzed reactions the calorimeter volume needs to be larger than a threshold volume of about 20 μl provided sufficient mixing. The modular arrangement of a highly efficient micromixer with a low-volume calorimeter of 2 μl appears to accomplish a considerable signal increase compared to using an integrated mixer of poor efficiency.