Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β-secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β-secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.

• Klíčová slova
• capillary electrophoresis, immobilized enzyme reactor, inhibition, magnetic particles, secretase,
• MeSH
• Alzheimerova nemoc farmakoterapie   metabolismus   MeSH
• bioreaktory * MeSH
• elektroforéza kapilární MeSH
• enzymy imobilizované antagonisté a inhibitory   chemie   metabolismus   MeSH
• HEK293 buňky MeSH
• inhibitory enzymů chemie   farmakologie   MeSH
• kinetika MeSH
• lidé MeSH
• peptidy chemie   farmakologie   MeSH
• sekretasy antagonisté a inhibitory   chemie   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy imobilizované MeSH
• inhibitory enzymů MeSH
• peptidy MeSH
• sekretasy MeSH

Capillary electrophoresis is a modern separation technique characterized by many benefits, which qualify it also for enzyme assays and the study of enzyme kinetics during drug development. Homogeneous or heterogeneous approaches can be followed for the enzymatic incubation. In this study, an immobilization procedure of aldehyde oxidase on magnetic particles was developed considering their integration with capillary electrophoresis. A number of magnetic nano/microparticle types were tested for this purpose, showing that aldehyde oxidase was most active when immobilized on bare silica magnetic nanoparticles. Primarily, the reusability of the enzyme immobilized on bare silica nanoparticles was tested. Three consecutive incubations with substrate could be performed, but the activity considerably dropped after the first incubation. One reason could be an enzyme detachment from the nanoparticles, but no release was detected neither at 4°C nor at 37°C during 5 h. The drop in enzymatic activity observed in consecutive incubations, could also be due to inactivation of the enzyme over time at given temperature. For the immobilized enzyme stored at 4°C, the activity decreased to 83% after 5 h, in contrast with a steep decrease at 37°C to 37%.

• Klíčová slova
• aldehyde oxidase, capillary electrophoresis, enzyme immobilization, magnetic nanoparticles, micellar electrokinetic chromatography,
• MeSH
• aldehydoxidasa analýza   metabolismus   MeSH
• elektroforéza kapilární MeSH
• enzymatické testy * MeSH
• enzymy imobilizované analýza   metabolismus   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aldehydoxidasa MeSH
• enzymy imobilizované MeSH

Enzymes play an essential role in many aspects of pharmaceutical research as drug targets, drug metabolizers, enzyme drugs and more. In this specific field, enzyme assays are required to meet a number of specific requirements, such as low cost, easy automation, and high reliability. The integration of an immobilized-enzyme reactor to capillary electrophoresis represents a unique approach to fulfilling these criteria by combining the benefits of enzyme immobilization, that is, increased stability and repeated use, as well as the minute sample consumption, short analysis time, and efficient analysis provided by capillary electrophoresis. In this review, we summarize, analyze, and discuss published works where pharmaceutically relevant enzymes were used to prepare capillary electrophoresis-integrated immobilized-enzyme reactors in an online manner. The presented assays are divided into three distinct groups based on the drug-enzyme relationship. The first, more extensively studied group employs enzymes that are considered to be therapeutic targets, the second group of assays present tools to assess drug metabolism and the third group assesses enzyme drugs. Furthermore, we examine various methods of enzyme immobilization and their implications for assay properties.

• Klíčová slova
• capillary electrophoresis, drug metabolism, druggable enzymes, immobilized-enzyme reactors,
• MeSH
• design vybavení MeSH
• elektroforéza kapilární * MeSH
• enzymatické testy MeSH
• enzymy imobilizované chemie   MeSH
• farmaceutický výzkum MeSH
• genom lidský MeSH
• kinetika MeSH
• léčivé přípravky MeSH
• lidé MeSH
• magnetismus MeSH
• proteomika MeSH
• reprodukovatelnost výsledků MeSH
• statická elektřina MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• enzymy imobilizované MeSH
• léčivé přípravky MeSH

Liquid chromatography coupled with mass spectrometry is widely used in the field of proteomic analysis after off-line protein digestion. On-line digestion with chromatographic column connected in a series with immobilized enzymatic reactor is not often used approach. In this work we investigated the impact of chromatographic conditions on the protein digestion efficiency. The investigation of trypsin reactor activity was performed by on-line digestion of N-α-benzoyl-L-arginine 4-nitroanilide hydrochloride (BAPNA), followed by separation of the digests on the mixed-mode column. Two trypsin column reactors with the different trypsin coverage on the bridged ethylene hybrid particles were evaluated. To ensure optimal trypsin activity, the separation temperature was set at 37.0 °C and the pH of the mobile phase buffer was maintained at 8.5. The on-line digestion itself ongoing during the initial state of gradient was carried out at a low flow rate using a mobile phase that was free of organic modifiers. Proteins such as cytochrome C, enolase, and myoglobin were successfully digested on-line without prior reduction or alkylation, and the resulting peptides were separated using a mixed-mode column. Additionally, proteins that contain multiple cysteines, such as α-lactalbumin, albumin, β-lactoglobulin A, and conalbumin, were also successfully digested on-line (after reduction and alkylation). Moreover, trypsin immobilized enzymatic reactors were utilized for over 300 injections without any noticeable loss of digestion activity.

• Klíčová slova
• Immobilized enzymatic reactor, Mass spectrometry, Mixed-mode column, On-line protein digestion, Trypsin digestion,
• MeSH
• alkylace MeSH
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• proteolýza MeSH
• proteomika * MeSH
• trypsin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy imobilizované MeSH
• laktalbumin * MeSH
• trypsin MeSH

In our ongoing study focused on Corydalis cava (Fumariaceae), used in folk medicine in the treatment of memory dysfunctions, we have investigated fifteen previously isolated alkaloids for their potential multifunctional activity on Alzheimer's disease (AD) targets. Determination of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition was carried out using a BACE1-Immobilized Enzyme Reactor (IMER) by validating the assay with a multi-well plate format Fluorescence Resonance Energy Transfer (FRET) assay. Seven alkaloids out of fifteen were found to be active, with (-)-corycavamine (3) and (+)-corynoline (5) demonstrating the highest BACE1 inhibition activity, in the micromolar range, in a concentration dependent manner. BACE1-IMER was found to be a valid device for the fast screening of inhibitors and the determination of their potency. In a permeation assay (PAMPA) for the prediction of blood-brain barrier (BBB) penetration, the most active compounds, (-)-corycavamine (3) and (+)-corynoline (5), were found to be able to cross the BBB. Not all compounds showed activity against glycogen synthase kinase-3β (GSK-3β) and casein kinase-1δ (CK-1δ). On the basis of the reported results, we found that some C. cava alkaloids have multifunctional activity against AD targets (prolyl oligopeptidase, cholinesterases and BACE1). Moreover, we tried to elucidate the treatment effectivity (rational use) of its extract in memory dysfunction in folk medicine.

• Klíčová slova
• Allocryptopine (PubChem CID: 98570), BACE1 inhibitors, Canadaline (PubChem CID: 321459), Canadine (PubChem CID: 443422), Corycavamine (PubChem CID: 90478581), Corycavidine (PubChem CID: 12304033), Corydalis cava alkaloids, Corynoline (PubChem CID: 177014), Corypalmine (PubChem CID: 12304090), Immobilized enzyme reactor, Isocorypalmine (PubChem CID: 10220), PAMPA assay, Scoulerine (PubChem CID: 22955),
• MeSH
• alkaloidy chemie   izolace a purifikace   MeSH
• Alzheimerova nemoc MeSH
• aspartátové endopeptidasy antagonisté a inhibitory   MeSH
• berberinové alkaloidy chemie   izolace a purifikace   MeSH
• Corydalis chemie   MeSH
• enzymy imobilizované antagonisté a inhibitory   MeSH
• hematoencefalická bariéra MeSH
• lidé MeSH
• rekombinantní proteiny MeSH
• sekretasy antagonisté a inhibitory   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkaloidy MeSH
• aspartátové endopeptidasy MeSH
• BACE1 protein, human MeSH Prohlížeč
• berberinové alkaloidy MeSH
• corycavamine MeSH Prohlížeč
• corynoline MeSH Prohlížeč
• enzymy imobilizované MeSH
• rekombinantní proteiny MeSH
• sekretasy MeSH

Penicillin G (2%, w/v in phosphate buffer, pH 8) was hydrolysed in a flow-through, miniature electro-membrane reactor with the penicillin G acylase immobilized in 5% (w/v) polyacrylamide (diam. 10 mm, thickness 2.6 mm, enzyme activity 24 U ml(-1)). The conversion of penicillin G increased from 0.15 to almost 0.5 when the electric current applied to the reactor was changed from -600 to +600 A/m2 with a substrate residency of 1 h.

• MeSH
• aktivace enzymů MeSH
• bioreaktory * MeSH
• design vybavení MeSH
• elektrochemie přístrojové vybavení   metody   MeSH
• elektromagnetická pole MeSH
• enzymy imobilizované chemie   MeSH
• hydrolýza MeSH
• kyselina penicilanová analogy a deriváty   chemická syntéza   MeSH
• membrány umělé * MeSH
• penicilin G chemie   MeSH
• penicilinamidasa chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminopenicillanic acid MeSH Prohlížeč
• enzymy imobilizované MeSH
• kyselina penicilanová MeSH
• membrány umělé * MeSH
• penicilin G MeSH
• penicilinamidasa MeSH

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.

• MeSH
• biosenzitivní techniky * MeSH
• enzymy imobilizované metabolismus   MeSH
• glykosidhydrolasy metabolismus   MeSH
• invertasa MeSH
• kinetika MeSH
• matematika MeSH
• Saccharomyces cerevisiae enzymologie   MeSH
• sacharosa MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• enzymy imobilizované MeSH
• glykosidhydrolasy MeSH
• invertasa MeSH
• sacharosa MeSH

The use of enzymes for cleavage, synthesis or chemical modification represents one of the most common processes used in biochemical and molecular biology laboratories. The continuing progress in medical research, genomics, proteomics, and related emerging biotechnology fields leads to exponential growth of the applications of enzymes and the development of modified or new enzymes with specific activities. Concurrently, new technologies are being developed to improve reaction rates and specificity or perform the reaction in a specific environment. Besides large-scale industrial applications, where typically a large processing capacity is required, there are other, much lower-scale applications, benefiting form the new developments in enzymology. One such technology is microfluidics with the potential to revolutionize analytical instrumentation for the analyses of very small sample amounts, single cells or even subcellular assemblies. This article aims at reviewing the current status of the development of the immobilized microfluidic enzymatic reactors (IMERs) technology.

• MeSH
• biosenzitivní techniky metody   MeSH
• chemické techniky analytické přístrojové vybavení   metody   MeSH
• enzymy imobilizované * MeSH
• klinická chemie přístrojové vybavení   metody   MeSH
• lidé MeSH
• mikrofluidní analytické techniky * MeSH
• proteiny analýza   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• enzymy imobilizované * MeSH
• proteiny MeSH

In this work, the combination of an immobilized enzyme microreactor (IMER) based on the clinically important isoform cytochrome P450 2C9 (CYP2C9) with capillary electrophoresis (CE) is presented. The CYP2C9 was attached to magnetic SiMAG-carboxyl microparticles using the carbodiimide method. The formation of an IMER in the inlet part of the separation capillary was ensured by two permanent magnets fixed in a cassette from the CE apparatus in the repulsive arrangement. The resulting on-line system provides an integration of enzyme reaction mixing and incubation, reaction products separation, detection and quantification into a single fully automated procedure with the possibility of repetitive use of the enzyme and minuscule amounts of reactant consumption. The on-line kinetic and inhibition studies of CYP2C9's reaction with diclofenac as a model substrate and sulfaphenazole as a model inhibitor were conducted in order to demonstrate its practical applicability. Values of the apparent Michalis-Menten constant, apparent maximum reaction velocity, Hill coefficient, apparent inhibition constant and half-maximal inhibition concentration were determined on the basis of the calculation of the effective substrate and inhibitor concentrations inside the capillary IMER using a model described by the Hagen-Poisseulle law and a novel enhanced model that reflects the influence of the reactants' diffusion during the injection process.

• Klíčová slova
• Capillary electrophoresis, Cytochrome P450, Drug metabolism, Immobilized enzyme reactor,
• MeSH
• bioreaktory MeSH
• difuze MeSH
• diklofenak chemie   MeSH
• elektroforéza kapilární * MeSH
• enzymy imobilizované metabolismus   MeSH
• kinetika MeSH
• objevování léků přístrojové vybavení   metody   MeSH
• systém (enzymů) cytochromů P-450 chemie   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• diklofenak MeSH
• enzymy imobilizované MeSH
• systém (enzymů) cytochromů P-450 MeSH

Open tubular capillary enzyme reactors were studied for rapid protein digestion and possible on-line integration into a CE/ESI/MS system. The need to minimize the time of the analyte molecules to diffuse towards the surface immobilized enzyme and to maximize the surface-to-volume (S/V) ratio of the open tubular reactors dictated the use of very narrow bore capillaries. Extremely small protein amounts (atto-femtomoles loaded) could be digested with enzymes immobilized directly on the inside wall of a 10 microm I.D. capillary. Covalently immobilized L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK)-trypsin and pepsin A were tested for the surface immobilization. The enzymatic activity was characterized in the flow-through mode with on-line coupling to electrospray ionization-time of flight-mass spectrometer (ESI/TOF-MS) under a range of protein concentrations, buffer pH's, temperatures and reaction times. The optimized reactors were tested as the nanospray needles for fast identification of proteins using CE-ESI/TOF-MS.

• MeSH
• biokompatibilní potahované materiály MeSH
• elektroforéza kapilární metody   MeSH
• elektroforéza mikročipová MeSH
• elektrolyty MeSH
• enzymy imobilizované chemická syntéza   klasifikace   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací metody   MeSH
• mikrochemie MeSH
• on-line systémy MeSH
• pepsin A metabolismus   MeSH
• proteiny analýza   chemie   MeSH
• sekvenční analýza proteinů metody   MeSH
• senzitivita a specificita MeSH
• studie proveditelnosti MeSH
• trypsin metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biokompatibilní potahované materiály MeSH
• elektrolyty MeSH
• enzymy imobilizované MeSH
• pepsin A MeSH
• proteiny MeSH
• trypsin MeSH