Dynamic modeling of an immobilized cell reactor

A mathematical model has been developed to describe the dynamic aerobic reaction occurring in a semibatch type of mixed flow reactor, containing cells immobilized in gel beads. This modeling is an extension of that developed in our previous study, for an immobilized cell reactor involving ethanol fermentation. In contrast to anaerobic reactions such as ethanol fermentation, (wherein the influent substrate concentration can be set at any desired level), aeration becomes necessary to provide additional substrate (oxygen) for most aerobic reactions occurring in immobilized cell reactors. Tobacco cell cultivation was chosen as a representative aerobic reaction, and the effect of aeration was assessed in terms of the volumetric coefficient of oxygen from gas to liquid phases.     

A Micro Planar Ampehometric Glucose Sensor Using an Isfet as a Reference Electrode

Abstract

A very small glucose sensor has been realized, which consists of a gold working electrode with a glucose oxidase immobilized membrane on it, and a gold counter electrode, all made on a sapphire substrate. By using the pH sensitive ISFET as a reference electrode, the potential for a solution, whose pH is constant, can be measured and irreversible metal electrodes, such as gold or platinum, can be used as working electrode and counter electrode. The sensor is very suitable for miniaturizing and mass production, because the Integrated Circuit (IC) fabrication process can be applied. The glucose oxidase immobilized membrane was also deposited by a lift off method, one of the IC processes. A glucose concentration, from 1 to 100 mg/dl, was measured with good linear current output.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

Immobilized capillary enzyme reactor based on layer-by-layer assembling acetylcholinesterase for inhibitor screening by CE

A method for creating an immobilized capillary acetylcholinesterase (AChE) reactor based on a layer-by-layer (LBL) assembly for inhibitor screening is described. The unique capillary AChE reactor was easily prepared by the instrument in three steps: first, a 0.5 cm long plug of a solution of the cationic polyelectrolyte polydiallyldimethylammonium (PDDA) was injected into the capillary to produce a positively charged coating on the surface of the capillary; subsequently, the enzyme solution with the same plug length was injected into the capillary and incubated for 10 min to immobilize the enzyme on the capillary wall via electrostatic interaction; third, PDDA solution with the same plug length was injected again into the capillary to cover the immobilized enzyme by forming PDDA-AChE-PDDA sandwich-like structure. The enzyme reactor can be easily renewed after removing the immobilized enzyme by flushing the column with 1 M NaCl solution. Activity of the immobilized enzyme can be assayed simply by carrying out an electrophoretic separation, i.e., the substrate solution was injected and incubated for a short time, followed by applying a voltage to separate the product from the unreacted substrate. The measured peak area of the product then represented the enzyme activity. For enzyme inhibitor screening, the mixture solution of the substrate and the inhibitor was injected and assayed the reduction of the enzyme activity. The immobilized enzyme could withstand 100 consecutive assays by only losing 10% activity. The reproducibility in terms of time-to-time, day-to-day, and batch-to-batch was measured with RSD% less than 4.7%. Furthermore, the screening system was validated by a known inhibitor. Finally, screening a small compound library containing four known AChE inhibitors and 42 natural extracts was demonstrated, and species with inhibition activity can be straightforwardly identified with the system.     

High amplification rates from the association of two enzymes confined within a nanometric layer immobilized on an electrode: modeling and illustrating example

Electrochemical responses (e.g., chronoamperometric) obtained with an immobilized enzyme that produces an electroactive species may be used to quantitate the amount of enzyme or the concentration of its substrate. It is shown, on theoretical and experimental bases, that product-to-substrate coupling with a second enzyme co-immobilized with the first within one or within a small number of monolayers, allows high amplification rates (higher than 1000), avoids membrane transport limitations, and lends itself to precise kinetic analyses that provide guidelines for optimization of the analytical sensitivity. Very large amplification factors, as large as several thousands, can be reached experimentally, in agreement with appropriately derived theoretical predictions, thus opening the route to the rational design of high-performance substrate sensing or affinity assays applications.     

Bioluminescence-based fibre-optic sensor with entrapped co-reactant: an approach for designing a self-contained biosensor

The possibility of designing a self-contained fibre-optic biosensor, i.e., that can be used without renewing reagents in the probe, was investigated. A probe specific for NADH involves immobilized bioluminescent enzymes which require the presence of two co-reactants [a flavin substrate (FMN) and an aldehyde] to catalyse light emission. The FMN was non-covalently immobilized in a synthetic film and was internally released in the vicinity of the bound enzymes, at the sensing tip of the bioprobe. Release of FMN was achieved from the two different matrices tested: a collagen film and a poly(vinyl alcohol) (PVA) network. Continuous-flow assays of NADH could be performed over a linear dynamic range from 10 pmol to 1 nmol. A PVA matrix appears to be a promising support for designing a self-contained biosensor, as 30–35 reliable measurements (R.S.D.=5%) could be achieved without a decrease in the sensor signal, compared with only 10–15 assays with a collagen film.     

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.     

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.     

Amperometric response to reducing carbohydrates of an enzyme electrode based on oligosaccharide dehydrogenase. Detection of lactose and α-amylase

A mediated amperometric enzyme electrode, which was constructed by immobilizing oligosacharide dehydrogenase behind a dialysis membrane on the surface of a carbon paste electrode containing p-benzoquinone, showed a current response to d-xylose, d-galactose, d-mannose, lactose, maltose, maltotriose, maltopentaose and maltohexaose. The sensitivity of the current response to these carbohydrates was dependent on the kinetics of the immobilized enzyme reaction and/or the permeation rate of the substrate through the dialysis membrane. Hence the sensitivity could be varied by controlling the amount of the immobilized enzyme and the thickness of the dialysis membrane. The time dependence of the current response ofthe enzyme electrode with a large amount of the immobilized enzyme and a thicker dialysis membrane could be explained by an equation describing diffusion of the substrate in the membrane. The enzyme electrode was used to measure lactose in milk and to assay α-amylase in standard serum.     

A flow-injection system for assay of the activity of an immobilized enzyme chemically-modified electrode

Chymotrypsin, covalently immobilized to the surface of an IrO₂-coated titanium electrode, responds potentiometrically to various substrates. A flow-injection system is described for assay of the activity of the immobilized enzyme with N-benzoyl-l-tyrosine ethyl ester as substrate and an ultraviolet detector. Least-squares fits of peak height vs. time typically yield correlation coefficients of 0.999 and standard errors of estimate of 0.0043 absorbance for a total absorbance change of about 0.130. Slopes of such plots vary linearly with enzyme activity.     

The Use of PAMAM Dendrimers as a Platform for Laccase Immobilization: Kinetic Characterization of the Enzyme

The kinetic behavior of the enzyme laccase in solution and immobilized onto carbon platforms using poly(amido amine) (PAMAM) dendrimers has been investigated. The results with the immobilized enzymes have demonstrated that almost ten times more enzyme on the carbon support is required for satisfactory kinetic rates to be achieved. Furthermore, the study as a function of the substrate concentration revealed that the kinetic behavior of the enzyme in solution fits the Michaelis?CMenten model. However, when the enzyme is immobilized onto the carbon surface, the catalyzed reaction follows a particular kinetic behavior with apparent positive cooperativity. The highest activity with laccase (in solution or immobilized) is achieved around pH?4.5, and the substrate conversion rate clearly diminishes with rising pH. The optimum temperature lies around 60?°C. The enzyme displays good catalytic activity in a wide range of pH and temperature values. The stability tests evidenced that there is no appreciable reduction in the enzymatic activity after immobilization within the first 30?days. Taking into account both the kinetic and stability tests, one can infer that the use of PAMAM dendrimers seems to be a very attractive approach for the immobilization of enzymes, as well as a feasible and useful methodology for the anchoring of enzymes with potential application in many biotechnological areas.     

Lactose hydrolysis and formation of galactooligosaccharides by a novel immobilized β-galactosidase from the thermophilic fungus Talaromyces thermophilus

β-Galactosidase from the fungus Talaromyces thermophilus CBS 236.58 was immobilized by covalent attachment onto the insoluble carrier Eupergit C with a high binding efficiency of 95%. Immobilization increased both activity and stability at higher pH values and temperature when compared with the free enzyme. Especially the effect of immobilization on thermostability is notable. This is expressed by the half-lifetime of the activity at 50°C, which was determined to be 8 and 27 h for the free and immobilized enzymes, respectively. Although immobilization did not significantly change kinetic parameters for the substrate lactose, a considerable decrease in the maximum reaction velocity V _max was observed for the artificial substrate o-nitrophenyl-β-d-galactopyranoside (oNPG). The hydrolysis of both oNPG and lactose is competitively inhibited by the end products glucose and galactose. However, this inhibition is only very moderate as judged from kinetic analysis with glucose exerting a more pronounced inhibitory effect. It was evident from bioconversion experiments with 20% lactose as substrate, that the immobilized enzyme showed a strong transgalactosylation reaction, resulting in the formation of galactooligosaccharides (GalOS). The maximum yield of GalOS of 34% was obtained when the degree of lactose conversion was roughly 80%. Hence, this immobilized enzyme can be useful both for the cleavage of lactose at elevated temperatures, and the formation of GalOS, prebiotic sugars that have a number of interesting properties for food applications.     

Ein Thermistordetektor zur Bestimmung von Substraten an Tr?ger-gebundenen Enzymen in Durchflu?-Systemen

Ohne Zusammenfassung

---

A thermistor detector for substrate determination with immobilized enzymes in flow systems

     

Carbohydrate microarrays by microcontact "click" chemistry

Carbohydrate microarrays can be prepared by microcontact printing of carbohydrate alkyne conjugates on azide self-assembled monolayers (SAMs). The carbohydrates are immobilized by a "click" reaction in the contact area between the stamp and the substrate. The immobilized carbohydrates retain their characteristic selectivity toward lectins.     

Selection and microencapsulation of an “NADH-oxidizing” bacterium and its use for NAD regeneration

An alternative approach to the regeneration of coenzymes is described here using immobilized microorganisms possessing “NADH-oxidase” function. Bacteria containing NADH-oxidase activity are immobilized by microencapsulation within artificial cells. In this form, the microencapsulated bacteria can recycle NADH back to NAD in the presence of molecular oxygen as an electron acceptor. The only byproduct of the recycling reaction is water. In order to perform the biological regeneration of NAD, the activity of NADH-oxidase was investigated in 13 strains of aerobic bacteria and yeast. The NADH-oxidizing bacteriaLeuconostoc mesenteroides exhibited the highest activity among the microorganisms tested. The permeabilized bacteria showed 10% of their initial activity after microencapsulation. Light and electron microscopy studies of bacteria loaded microcapsules have been done. Enzymatic properties of microcapsule-immobilized bacteria were investigated in comparison with those of the free enzyme complex.Leuconostoc mesenteroides, containing NADH-oxidase, has been microencapsulated together with 3α-hydroxysteroid dehydrogenase (3α-HSDH) for stereospecific steroid oxidation. In a batch reactor, 2 mg of NAD, with recycling, allowed the same substrate consumption as 4.4 mg of NAD without recycling. The microencapsulated system can be used repeatedly. The system is functional for 10 h, during which time each molecule of NAD has been used 7.6 times.     

Monitoring of methanogenic processes with an immobilized mixed culture in combination with a gas-flow meter

A sensitive gas-flow meter is described for use in combination with a preparation of immobilized methanogenic bacteria. Upon administration of substrate, the gas formed is quantified by using this gas-flow meter. When a high density of the extremely slow growing and metabolizing methanogenic bacteria was added, it was possible to quantify and correlate samples with respect to their content of biodegradable matter.     

High temporal resolution monitoring of enzyme reaction and inhibition using optically gated vacancy capillary electrophoresis and immobilized enzyme

A novel method for monitoring of enzyme reaction and inhibition with high temporal resolution was developed by using optically gated vacancy capillary electrophoresis (OGVCE) with laser-induced fluorescence (LIF) detection and immobilized enzyme. Trypsin cleavage reaction and inhibition were investigated by the presented OGVCE-LIF assay, using carboxyfluorescein (FAM) end-labeled Angiotensin as the substrate and commercially available immobilized trypsin. The substrate and the product were continuously loaded into the capillary by the electroosmotic flow while the immobilized enzyme remained in the sample vial. Substrate consumption and product formation were monitored simultaneously at 5 s interval during the whole reaction time. The enzymatic reaction rates obtained from the substrate and the product were highly consistent. The enzyme activity and the Michaelis constants of trypsin cleavage reaction, as well as the inhibition constant (for reversible competitive inhibitor) and the inhibition fraction (for irreversible inhibitor), were obtained. It was showed that the reported OGVCE-LIF method can perform fast, accurate, sensitive and reproducible CE enzyme assay with high temporal resolution, thus has great potential in application of the enzyme-substrate systems with fast reaction rate and the fluorescent substrate and products.     

Structural studies of immobilized ethylenediamine as a preconcentrating agent for molybdate and tungstate

The structure of the complex formed between molybdate and the diamine functional group of N-β-aminoethyl-γ-aminopropylsilane (Dow Corning 2-6020) immobilized on a silica substrate is determined and compared with the analogous complex precipitated from solution. Infrared spectroscopy and photoacoustic spectroscopy were applied to establish the nature of the complex. The precipitate formed with ethylenediamine contains a polymeric molybdate species, whereas the molybdate retained by the immobilized ethylenediamine group is monomeric. Trace levels of tungstate and molybdate can be extracted from aqueous concentrated sodium chloride solutions on a column packed with controlled-pore glass beads treated with Z-6020.     

Development of a silica monolith microbioreactor entrapping highly activated lipase and an experiment toward integration with chromatographic separation of chiral esters

Microbioreactors are effective for high-throughput production of expensive products from small amounts of substrates. Lipases are versatile enzymes for chiral syntheses, and are highly activated when immobilized in alkyl-substituted silicates by the sol-gel method. For practical application of sol-gel immobilized lipases to a flow system, a microbioreactor loaded with a macroporous silica monolith is well suited, because it can be easily integrated with a chromatographic separator for optical resolution. We attempted to develop a microbioreactor containing a silica monolith-immobilized lipase. A nonshrinkable silica monolith was first formed from a 4:1 mixture of methyltrimethoxysilane (MTMS) and tetramethoxysilane (TMOS). It was then coated with silica precipitates entrapping lipase, derived from a 4:1 mixture of n-butyltrimethoxysilane (BTMS) and TMOS. As a result, monolith treated with the BTMS-based silicate entrapping lipase exhibited approximately ten times higher activity than nontreated monolith-immobilized lipase derived from the MTMS-based silicate, in transesterification between glycidol and vinyl n-butyrate in isooctane. A commercially available chiral column was connected in series to the monolith microbioreactor, and a pulse of substrate solution was supplied at the inlet of the reactor. Successful resolution of the racemic ester produced was achieved in the chromatographic column.     

Immobilized glucose-6-phosphate dehydrogenase as a substrate for solubilized epidermal growth factor receptor tyrosine kinase

Based on our previously reported solution assay protocol, a solid-phase assay for the tyrosine kinase activity of the epidermal growth factor receptor has been developed. Glucose-6-phosphate dehydrogenase, immobilized noncovalently on microtiter plates, was used as the substrate in the solid-phase assay. Phosphorylation of the immobilized substrate takes place in the presence of ATP and a solubilized epidermal growth factor receptor preparation. After washing off the soluble reaction mixture, the phosphotyrosine-containing dehydrogenase produced on the well surface is quantitated by an ELISA method using a polyclonal antiphosphotyrosine antibody, a second antibody conjugated with horseradish peroxidase, and finally theo-phenylenediamine reaction. The absorbance at 492 nm developed in the wells is a measure of the kinase activity of the solubilized receptor preparation. Putative inhibitors of receptor kinase can be conveniently incorporated in this assay system to test for potential inhibitory activity. This assay, being rapid and convenient, is useful in drug screening programs where a high through-put rate is required.