The Classical Model describing the effects of temperature on enzyme activity consists of two processes: the catalytic reaction defined by ΔG cat and irreversible inactivation defined by ΔG inact, this model however, does not account for the observed temperature- dependant behaviour of enzymes. The recent development of the Equilibrium Model is governed not only by ΔG cat and ΔG inact but also by two new intrinsic parameters ΔHeq and Teq, which describe the enthalpy and the temperature of the midpoint, respectively, of a active and reversibly inactive enzyme transition. Teq is central to the physiological adaptation of an enzyme to its environmental temperature and links the molecular, physiological and environmental aspects of life to temperature in a way that has not been previously possible. The Equilibrium Model is therefore a more complete and accurate description of the effects of temperature on enzymes, it has provided new tools for describing and investigating enzyme thermal adaptation and possibly new biotechnological tools. The effects of the incorporating in the new Model of the parameters Teq and ΔH eq yield major differences from the Classical Model, with simulated data calculated according to the Equilibrium Model fitting well to experimental data and showing an initial rate temperature optimum that is independent of assay duration. Simulated data simulated according to the Classical Model can not be fitted to experimental data. All enzymes so far studied (gt30) display behaviour predicted by the Equilibrium Model. The research described here has set out to: experimentally test observations made by Eisenthal et al., on the basis of enzyme reactor data simulated according to the Equilibrium Model; to test the Equilibrium Model using an unusual (rapidly renaturable) enzyme, RNAase; and to test the proposed molecular basis of the Equilibrium Model by examining the effect of a change at the enzymes active site. The experimental results gathered here on the effect of time and temperature on enzyme reactor output confirm the predictions made by Eisenthal et al. (2006) and indicate that the Equilibrium Model can be a useful aid in predicting reactor performance. The Equilibrium Model depends upon the acquisition of data on the variation of the Vmax of an enzyme with time and temperature, and the non-ideal behaviour of RNase A made it impossible to collect such data for this enzyme, as a result the Equilibrium Model could not be applied. The disulfide bond within the active site cleft of A.k 1 protease was cleaved as a probe of the mechanism of the Equilibrium Model, which is proposed to arise from molecular changes at the enzymes active site. Support for the proposed mechanism was gained through the comparison of experimentally determined temperature dependence of the native and reduced forms of the enzyme and application of this data to the Equilibrium Model.

This work describes the integration of a separation capillary for capillary electrophoresis (CE) with an on-column enzyme reactor for selective determination of the enzyme substrate. The enzyme reaction occurs during a capillary separation, allowing selective determination of the substrate in complex samples without the need for pre- or post- separation chemical modification of the analyte. The overall goal of this work is to develop a system in which sample introduction, separation of the analyte/substrate from other biological species, enzymatic conversion of the analyte/substrate into a detectable product, and sensitive detection are all included within a single analysis scheme. Immobilization of the enzyme is achieved by electrostatic assembly of poly(diallydimethylammonium chloride) (PDDA) followed by adsorption of a mixture of the negatively charged enzyme glucose oxidase (GOx) and anionic poly(styrenesulfonate) (PSS). The reaction of glucose with the immobilized glucose oxidase produces H2O2 which migrates the length of the capillary under the influence of electroosmotic flow and is detected amperometrically at the capillary outlet. The optimal response, kinetics, and stability for the enzyme reactor are determined through characterization of several parameters including the concentration ratio of PSS:GOx, applied separation voltage, and the inner diameter of the separation capillary. Various analyte mixtures containing the substrate and other biological species were evaluated to illustrate selective separation and determination of the substrate from other biomolecules. Optimization of this electrostatically assembled capillary enzyme reactor lead to application of these parameters to similar enzymes such as glutamate oxidase. Future application to similar enzymes like L-amino acid oxidase and possible microfluidic systems is a long-term goal of the system. / Ph. D.

This master thesis deals with the use of microfluidics for the purpose of microfluidic enzymatic reactor for drug screening. At first it considers the issue from a theoretical point of view – describes microfluidics as a newly developing and promising field of production of microfluidic devices, materials, biomedical applications and advantages and disadvantages of microfluidics overall. Furthermore, it focuses on an area of analytical utilization of enzymes within enzyme reactors. In the first part of the experimental section, conditions for the testing of enzymes of xenobiotics metabolism in the liver were optimized, namely the model of coumarin metabolism via the spectrofluorimetry method. The second part of the experimental work dealt with optimization of the fabrication conditions of microfluidic chips from OSTE (off-stoichiometry Thiol Ene) via the soft lithography method. Subsequently, the functionality of the produced chips was tested. Based on the results of both parts of the experimental work, an evaluation was carried out to assess the suitability of their interconnection for future research – screening of microsomal enzyme activity and model biotransformation of drugs within the channels of the fabricated devices.

La digestion enzymatique des protéines est une méthode de base pour les études protéomiques ainsi que pour le séquençage en mode « bottom-up ». Les enzymes sont ajoutées soit en solution (phase homogène), soit directement sur le gel polyacrylamide selon la méthode déjà utilisée pour l’isolation de la protéine. Les enzymes protéolytiques immobilisées, c’est-à-dire insolubles, offrent plusieurs avantages tels que la réutilisation de l’enzyme, un rapport élevé d’enzyme-sur-substrat, et une intégration facile avec les systèmes fluidiques. Dans cette étude, la chymotrypsine (CT) a été immobilisée par réticulation avec le glutaraldehyde (GA), ce qui crée des particules insolubles. L’efficacité d’immobilisation, déterminée par spectrophotométrie d’absorbance, était de 96% de la masse totale de la CT ajouté. Plusieurs différentes conditions d’immobilisation (i.e., réticulation) tels que la composition/pH du tampon et la masse de CT durant la réticulation ainsi que les différentes conditions d’entreposage tels que la température, durée et humidité pour les particules GA-CT ont été évaluées par comparaison des cartes peptidiques en électrophorèse capillaire (CE) des protéines standards digérées par les particules. Les particules de GA-CT ont été utilisés pour digérer la BSA comme exemple d’une protéine repliée large qui requit une dénaturation préalable à la digestion, et pour digérer la caséine marquée avec de l’isothiocyanate de fluorescéine (FITC) comme exemple d’un substrat dérivé afin de vérifier l’activité enzymatique du GA-CT dans la présence des groupements fluorescents liés au substrat. La cartographie peptidique des digestions par les particules GA-CT a été réalisée par CE avec la détection par absorbance ultraviolet (UV) ou fluorescence induite par laser. La caséine-FITC a été, en effet, digérée par GA-CT au même degré que par la CT libre (i.e., soluble). Un microréacteur enzymatique (IMER) a été fabriqué par immobilisation de la CT dans un capillaire de silice fondu du diamètre interne de 250 µm prétraité avec du 3-aminopropyltriéthoxysilane afin de fonctionnaliser la paroi interne avec les groupements amines. Le GA a été réagit avec les groupements amine puis la CT a été immobilisée par réticulation avec le GA. Les IMERs à base de GA-CT étaient préparé à l’aide d’un système CE automatisé puis utilisé pour digérer la BSA, la myoglobine, un peptide ayant 9 résidus et un dipeptide comme exemples des substrats ayant taille large, moyenne et petite, respectivement. La comparaison des cartes peptidiques des digestats obtenues par CE-UV ou CE-spectrométrie de masse nous permettent d’étudier les conditions d’immobilisation en fonction de la composition et le pH du tampon et le temps de réaction de la réticulation. Une étude par microscopie de fluorescence, un outil utilisé pour examiner l’étendue et les endroits d’immobilisation GA-CT dans l’IMER, ont montré que l’immobilisation a eu lieu majoritairement sur la paroi et que la réticulation ne s’est étendue pas si loin au centre du capillaire qu’anticipée. / Digesting proteins using proteolytic enzymes is a standard method in proteomic studies and bottom-up protein sequencing. Enzymes can be added in solution or gel phase depending on how the protein has been isolated. Immobilized, i.e., insoluble, proteolytic enzymes offer several advantages such as reusability of enzyme, high enzyme-to-substrate ratio, and integration with fluidic systems. In this study, we prepared glutaraldehyde-crosslinked chymotrypsin (GA-CT), which creates insoluble particles. The immobilization efficiency was determined by absorbance spectrophotometry and found to be 96% of the total amount of chymotrypsin added. Different immobilization (i.e., crosslinking) conditions such as buffer composition/pH and initial mass of CT during crosslinking as well as different storage conditions such as temperature, time and humidity for the GA-CT particles were evaluated by comparing capillary electrophoretic (CE) peptide maps of protein standards digested with the particles. The GA-CT particles were used to digest BSA as an example of a large folded protein that needs denaturation prior to digestion, and casein-fluorescein isothiocyanate (FITC) as an example of a small, labeled substrate to test enzyme activity in the presence of substrate-bound fluorescent groups. Peptide mapping of digests from GA-CT particles was achieved by CE with ultraviolet (UV) absorbance or laser induced fluorescence (LIF) detection. FITC-labeled casein was digested by GA-CT to the same extent as with free (i.e., soluble) CT. An immobilized enzyme microreactor (IMER) was fabricated by immobilizing CT inside a 250 µm i.d. fused-silica capillary tube pre-treated with 3-aminopropyltriethoxysilane to functionalize the inner walls with amine groups. Glutaraldehyde was reacted with the amine groups and then CT was immobilized by crosslinking to the GA. IMERs based on GA-CT were fabricated using an automated CE system and used to digest BSA, myoglobin, a 9-residue peptide and a dipeptide as examples of large, medium and small substrates. Digests were studied by comparing peptide maps obtained by CE coupled to either UV or mass spectrometric (MS) detection in order to evaluate immobilization conditions as a function of buffer composition/pH and reaction times. A separate study, which used fluorescence microscopy to investigate the extent and location of GA-CT immobilization in the IMER, showed that immobilization only takes place primarily near the capillary walls and that crosslinking does not extend as far into the center of the IMER as had been expected.