Academic literature on the topic 'Immobilized enzyme reactor (IMER)'

AbstractOnline enzyme assay based on capillary electrophoresis (CE) offers several advantages for the assay, such as low consumption of samples, easy automation of all steps, and less requirement of sample work-up. As a widely used approach for online enzyme assay, CE-integrated immobilized enzyme microreactor (IMER) has been applied in almost all aspects of enzyme assays during the past two decades, including evaluation of the enzymatic activity and kinetics, screening of inhibitor, investigation of enzyme-mediated metabolic pathways, and proteome analysis. In a CE-integrated IMER, enzyme is bound to the capillary surface or a suitable carrier attached to the capillary and substrates/products of the enzymatic reaction are separated and online detected by CE at downstream of the capillary. Enzymatic reactions can be viewed as interaction between the stationary phase (immobilized enzyme) and the mobile phase (substrate(s)/co-enzyme(s) solution), in analogy to the well-known separation technique, capillary electrochromatography. From this point of view, CE-integrated IMERs can be categorized into open tubular capillary IMER, monolithic IMER, and packed capillary IMER. In this review, we have surveyed, analyzed, and discussed advances on fabrication techniques of the three categories of CE-integrated IMERs for online assays involving various enzymes in the past two decades (1992–2015). Some recent studies using microfluidic-based IMERs for enzyme assays have also been reviewed.

Ca₃(PO₄)₂–ChT hybrid nanoflowers were synthesized by a facile approach. The nanoflowers exhibited an enhanced enzymatic activity and can be used as an immobilized enzyme reactor (IMER) for highly efficient protein digestion.

Hybrid nanoflowers were synthesized by a novel approach. The nanoflowers exhibited an enhanced enzymatic activity and can be used as an immobilized enzyme reactor (IMER) for highly efficient protein digestion.

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) fromTrypanosoma cruziacts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in theTcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gaveKMof 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.

L’analyse de protéines est principalement réalisée selon l’approche bottom-up qui consiste en une digestion enzymatique et l’analyse des peptides obtenus en chromatographie en phase liquide couplée à la spectrométrie de masse en tandem. L’étape de digestion, réalisée en solution, est une procédure longue. L’utilisation de réacteurs à base d’enzymes immobilisées (IMER) ainsi que leur intégration en ligne avec la LC-MS/MS améliore la fiabilité et la sensibilité de la méthode globale. Le premier objectif de cette thèse a été d’évaluer le potentiel des IMER pour la caractérisation de la glycosylation. La complémentarité d’IMER de pepsine et de trypsine, développés en format classique par greffage sur support de Sepharose, a permis d’identifier les N-glycanes sur les 4 sites de glycosylation d’une hormone de grossesse contenue dans 2 médicaments. Le second objectif était la miniaturisation de ces outils. Des monolithes obtenus par voie sol-gel ou par polymérisation radicalaires ont été synthétisés in situ dans des capillaires de 100 µm de diamètre interne. La meilleure répétabilité de synthèse de la voie radicalaire a conduit à la sélection des supports obtenus par cette voie pour leur greffage par de la trypsine ou de la pepsine. Un montage miniaturisé pour l’analyse de digestats a été aussi réalisé. Cela permettra l’inclusion postérieure de l’IMER dans le dispositif analytique
Protein analysis is mainly carried out using the "bottom-up" approach which is based on enzymatic digestion of proteins and analysis of resulting peptides by liquid chromatography coupled with tandem mass spectrometry. Usually, enzymatic digestion is carried out in solution. However, this procedure is long. The use of immobilised enzyme reactors (IMER) and their hyphenation with LC-MS/MS increases the reliability and sensitivity of the overall method. The study conducted during this thesis had two distinct objectives. The first was to evaluate the potential of IMERs for glycosylation characterisation. The complementarity of IMERs of pepsin and trypsin, developed in classical format by grafting the proteases on a Sepharose support, allowed to identify the N-glycans on the 4 glycosylation sites a pregnancy hormone contained in 2 drugs. The second objective was the miniaturization of these tools. To do this, monoliths obtained by sol-gel approach in the presence of organosilanes or by radical polymerization of organic monomers were synthesized in situ in capillaries of 100 µm internal diameter. The better repeatability of synthesis of organic supports led to their selection for functionalization by trypsin or pepsin. In parallel, a miniaturised set-up for the analysis of digests was carried out. This will allow the subsequent inclusion of IMER in the overall analytical device

Notre projet porte sur la mise au point d’un système chromatographique couplant la chromatographie en phase liquide à haute performance (CLHP), un réacteur d’enzyme immobilisée (IMER) et une détection par spectrométrie de masse (SM) destiné à détecter les inhibiteurs de l’acétylcholinestérase (AChE) et de comparer leurs activités inhibitrices respectives. En utilisant ce couplage, les inhibiteurs d’AChE peuvent être séparés en ligne et leurs activités inhibitrices comparées directement par la simple mesure, par SM, de la surface des pics chromatographiques de l’acétylcholine (ACh) résiduelle, dont l’hydrolyse a été inhibée par les composés recherchés. 3 étalons connus d’inhibiteurs d’AChE (la galanthamine-GL, l’huperzine A-HuA et la tacrine-TA), ont été sélectionnés pour mettre au point les conditions analytiques de ce couplage original. Après avoir validé cette méthode avec les 3 étalons inhibiteurs de l’AChE, les extraits alcaloïdiques de différentes plantes ont été injectés dans le dispositif afin de cribler les nouveaux candidats inhibiteurs de l’AChE. Parmi les extraits injectés, il y des inhibiteurs d’AChE ont pu être découvert grâce à la présence des pics d’ACh. Pour compenser la dérive de la ligne de base chromatographique (m/z 146), due au gradient de phase organique (MeOH), une pompe de contre-gradient a été ajoutée à la sortie de la colonne de CLHP. Les résultats montrent que cet ajout d’une pompe de contre-gradient améliore considérablement l’horizontalité de la ligne de base mais relève celle-ci à des valeurs trop importantes et réduit d’autant la sensibilité du dispositif par rapport à la méthode chromatographique réalisée sans contre-gradient. Cette étude a permis la mise au point d’une méthode innovante en couplant la CLHP, un IMER et une détection par SM destinée au criblage les inhibiteurs de l’AChE. Grace à cette méthode, l’activité des différents inhibiteurs peut être facilement évaluée en mesurant l’aire des pics d’ACh correspondant aux composés recherchés. 3 inhibiteurs connus de l’AChE ont été choisis pour valider cette méthode. Cette dernière a permis de mettre clairement en évidence une relation dose-effet croissante qui lie des pics des inhibiteurs de l’ACh en fonction de leurs quantités injectées. Cette méthode CLHP-IMER-SM a été appliquée au criblage les inhibiteurs de l’AChE dans des extraits de plante. Grâce à cette méthode, un nouvel inhibiteur de l’AChE a été découvert dans l’extrait de Lycoris Radiata
The project focuses on the development of a chromatographic system coupling high-performance liquid chromatography (HPLC), immobilized enzyme reactor (IMER) and mass spectrometry (MS) detection to detect acetylcholinesterase (AChE) inhibitors and compare their respective inhibitory activities. Using this device, the AChE inhibitors can be separated on-line and their inhibitory activities can be compared directly by the simple measurement of the chromatographic peaks of the residual acetylcholine (ACh) on MS whose hydrolysis was inhibited by the desired compounds. 3 wildly known standards of AChE inhibitors (Galanthamine-GL, Huperzine A-HuA and Tacrine-TA), were selected to develop the analytical conditions of this coupling. After having validated this method with the 3 AChE inhibiting standards, the alkaloid extracts of different plants were injected into the device in order to screen the new AChE inhibitor candidates. Among the injected extracts, some AChE inhibitors could be discovered thanks to the presence of ACh peaks. To compensate for the drift of the chromatographic baseline (m/z 146), due to the organic phase gradient (MeOH), a counter-gradient pump was added to the outlet of the HPLC column. The results show that this addition of a counter-gradient pump considerably improves the horizontality of the baseline but raises it to high values and reduces correspondingly the sensitivity of the device compared to the chromatographic method performed without counter-gradient. This study led to the development of an innovative method by coupling HPLC, IMER and MS detection for AChE inhibitor screening. Thanks to this method, the activity of the various inhibitors can be easily evaluated by measuring the area of the ACh peaks corresponding to the desired compounds. 3 known AChE inhibitors were chosen to validate this method and have clearly demonstrated a growing dose-response relationship that binds peaks of ACh according to the injected AChE inhibitor amounts. This method CLHP-IMER-SM was applied to screening for AChE inhibitors in plant extracts. Thanks to this method, a new AChE inhibitor has possibily been discovered in the Lycoris Radiata extract

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.

The objective of this work was to develop a novel enzyme immobilization scheme for supported lipase sol-gels and to evaluate the potential of the immobilized biocatalyst for the production of biodiesel in a packed bed reactor. Two sources of lipase (EC 3.1.1.3 triacylglycerol hydrolase) were used in this study and the transesterification of methanol and triolein to produce glycerol and methyl oleate was used as a model reaction of biodiesel production. A commercially available form of immobilized lipase, Novozym® 435, was used as a basis for comparison to the literature. Upon establishing a lipase sol-gel formulation technique, the experimental methodology for the transesterification reaction using Novozyme® 435 was developed. Subsequently, a series of inert materials were considered based on their suitability as supports for immobilized lipase sol-gels and the synthesis of methyl oleate. The value of a supported lipase sol-gel is to improve the activity and stability of the enzyme and develop an immobilized biocatalyst that is practical for use under packed bed reactor conditions. Of the six support materials considered (6-12 mesh silica gel, Celite® R633, Celite® R632, Celite® R647, anion exchange resin, and Quartzel® felt), the diatomaceous earth supports (Celite® R633, R632 and R647) exhibited high enzymatic activity, were thermally stable, and possessed high sol-gel adhesion. From the three types of diatomaceous earth considered, Celite® R632 supported lipase sol-gels were identified as the most promising supported lipase sol-gels for methyl oleate production via transesterification. Upon further evaluation, the Celite® R632 lipase sol-gels were found to achieve high methyl oleate percent conversions, glycerol-water absorption was only significant at glycerol levels higher than 75%, and the immobilized lipase had high stability upon storage at 4°C for 1.5 years. To determine the effects of methanol and glycerol inhibition as well as temperature on the reaction kinetics, a ping-pong bi-bi kinetic model was developed and validated over a range of methanol concentrations and temperatures. The optimal methanol concentration for the conditions tested was in the range of 1.3 M to 2.0 M, and increased with increasing temperature. The model developed was consistent with the experimental data and confirmed that glycerol inhibition and the presence of products had significant effects on the reaction kinetics. The methyl oleate production capabilities of the Celite® supported lipase sol-gel were investigated using a packed bed reactor and compared with Novozym® 435 under similar operating conditions. A kinetic and mass transfer based model was developed for the reactor system using a novel efficiency correlation to account for the effect of glycerol on the enzymatic activity. Increasing the flow rate (1.4 mL/min to 20 mL/min) increased the reaction rate, presumably due to the reduction of the glycerol inhibition effect on the immobilized biocatalyst. The Celite® supported lipase sol-gel was found to have superior performance over Novozym® 435 both under batch stirred tank reaction conditions and in a packed bed reactor (83% conversion for Celite® sol-gel vs. 59% conversion for Novozym® 435 at 20 mL/min in the packed bed reactor). Based on the results obtained, Celite® supported lipase sol-gels exhibited good performance for the transesterification of triolein with methanol to produce methyl oleate in both batch and packed bed reactors, and warrant further exploration for the enzymatic production of biodiesel.

We propose an improved micro reactor design for a scalable microfluidic device, in which enzymes are immobilized in a hydrogel matrix. Furthermore, fluid flow is controlled by means of hydrogel-based micro-valves. In this work, computational flow simulations will be compared to experimental results to highlight new design ideas and to improve wetting and concentration distribution through the entire chamber volume, even for high aspect ratios. Additionally, modelling concepts will be introduced to efficiently describe multi-domain problems like enzyme reactions. With the help of a computer-aided design process which is capable to simulate hydrogel-based microfluidic systems it is possible to better understand, predict and visualize the behavior of micro-reactors and support the development of highly integrated hydrogel-based microfluidic circuits.