Enzyme Entrapment Research Articles

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (±0.1) and 7.2 (±0.1) nA/mM, respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.

Disposable interference-free glucose biosensor based on an electropolymerised poly(pyrrole) permselective film

Miniaturised disposable amperometric biosensors for glucose determination in serum are described. A commercially available three-electrode system created on a planar corundum ceramic base was used as biosensor substrate and the working Pt electrode was modified by an electrogenerated overoxidised poly(pyrrole) film (PPYox). After the optimisation of the PPy layer synthesis, two different approaches were investigated for glucose oxidase (GOD) immobilisation: enzyme entrapment into the electropolymerised film by an ‘all-electrochemical’ procedure (PPYox/GOD) and gel-entrapment over the PPYox modified electrode by co-crosslinking with glutaraldehyde/bovine serum albumin (PPYox/GOD-gel). A comparison of the sensitivities to glucose, performed by dropping 50 μl of the sample solutions onto the relevant electrode systems, showed that both sensors have a linearity range extending up to 10 mM, though Pt/PPYox/GOD-gel sensor is more sensitive (168±15 versus 53±7 nAm/M). The two sensors showed remarkable anti-interferent selectivity, moreover the PPYox/GOD-gel sensor had also a good stability and could be used for flow injection analysis of glucose, with a linearity range extending up to 25 mM.

A membrane based reactor with an enzyme immobilized by an avidin–biotin molecular recognition in a polymer matrix

Glucose oxidase (GOD) and peroxidase (POD) have successfully been immobilized in a polypyrrole matrix by an avidin–biotin molecular recognition process. A biotin labeled pyrrole was the precursor of the electropolymerized polymer. The amount of enzyme entrapped in the polymeric matrix was 60% of the initial amount for GOD and 83% for POD; these values are higher than those previously reported for enzyme entrapment in polymer matrixes. These reactive membranes performed a chemical transformation during the permeation of the substrates; the GOD membrane catalyzed the oxidation of glucose in the presence of oxygen, whereas the peroxidase membrane catalyzed the reduction of hydrogen peroxide in the presence of the oxidant pyrrogallol. The catalytic activity of the membranes has been assessed. A turnover number of 900 in 1 min was determined for the POD membrane. The loss of activity of the enzyme during the membrane formation (42%) by the avidin–biotin method of immobilization was weak. Moreover the enzymatic POD membrane was remarkably stable after a 80-day storage (less than 6% decrease of activity). These performances point to the interest of such enzymatic membranes for the treatment of aqueous media.

Entrapment of enzyme in water-restricted microenvironment — amyloglucosidase in reverse micelles

Amyloglucosidase (E.C. 3.2.1.3) was entrapped into reverse micelles of Triton X-100–xylene–hexanol. The observed activity in reverse micelles was influenced by the water content. Activity was studied at different water–surfactant ratios (Wo) and pH. The effects of Wo, glycerol and temperature on stability were analysed. Amyloglucosidase was active in the above mentioned system, although the overall activity was lower than in the aqueous system, it showed higher stability in reverse micelles up to 50°C.

Proteases Entrapped in Hydrogels Based on Poly(N-Vinyl Caprolactam) as Promising Biocatalysts in Water/Organic Systems

Chemico-enzymatic methods are a promising approach to obtain various biologically active compounds, including enantiomerically pure substances. Entrapment in gels is one of the most convenient methods to stabilize enzymes for their application in water/organic media. In this study, proteases (trypsin and chymotrypsin) were entrapped into a composite poly (N-vinyl caprolactam)-calcium alginate (PVCL-CaAlg) hydrogel, and properties of the biocatalysts obtained were studied. Both entrapped trypsin and α-chymotrypsin retained high activity (up to 85% of the initial value for the immobilized trypsin) and displayed high storage stability. The entrapped proteases were active over broad temperature ranges, and their heat tolerance was approximately 20-25°C higher than that of the soluble enzymes. The dependence of the activities of the soluble and entrapped enzymes on the organic solvent concentration was studied. It appeared that gel entrapment provided retention of enzyme activity in the media with high concentrations of organic solvents (up to 0.5% water content) and ensured high operational stability of α-chymotrypsin in a cyclic process (270 h in total). The entrapped α-chymotrypsin was used as a biocatalyst for obtaining optically pure L-phenylalanine (e.e. 87.8%) by enantioselective hydrolysis of Schiff's base of amino acid ethyl ester in an acetonitrile/water system.

Sol-Gel Entrapment of Enzymes

ABSTRACTTwo enzymes, lipase and β-galactosidase, have been encapsulated within sol-gel matrices. Enzymatic activity of encapsulated lipase for hydrolysis and trans-esterification reactions is maintained. Encapsulation yields depend not only on the sol-gel porous texture but also on the water amount added for the sol-gel synthesis and the hydratation history of the enzyme. When the water amount is low, the highly active enzyme conformation generated by the phase separation is frozen during gelation. Escherichia Coli have been also encapsulated. The cellular organization appears to be well preserved. Their β-galactosidase activity seems to be better in wet gels but decreases dramatically upon drying.

Enzyme immobilization in silica-hardened organogels.

In this study we describe a novel method for immobilizing enzymes in a solid nanocomposite matrix based on gelatin gels, which are subsequently hardened by in situ polymerization of tetraethoxysilane (TEOS). Chromobacterium viscosum lipase is taken as the example. This immobilization method possesses the advantages of enzyme entrapment in microemulsions, together with newly beneficial qualities, such as transparency, which permits direct spectroscopic investigation, and considerable mechanical stability in both aqueous and organic solvents, which results in the maintenance of enzymatic activity for several months. The first step is enzyme solubilization in AOT reverse micelles, followed by transformation of this solution into an organogel by the addition of gelatin. The enzyme-containing gel, is then hardened by the formation of silicate polymer. A glassy nanocomposite is obtained, which is optically transparent, so that the protein can be studied directly spectroscopically. Circular dichroic spectra of cytochrome-c are shown as an example. The nanocomposite material can be dried and ground, yielding a powder that is stable in both aqueous and organic solvents. After extensive washing with water, the enzyme-containing nanocomposite showed good activity in cyclohexane. The synthesis of water-insoluble fatty acid esters was carried out in this solvent with yields close to 90%. In this case, the enzyme preparations can be used over a period of several months without loss of activity or chemical yield.

A glucose biosensor based on enzyme entrapment within polypyrrole films electrodeposited on mesoporous titanium dioxide

Abstract The electrochemical immobilization of glucose oxidase (GOD) within polypyrrole films electrogenerated on mesoporous TiO 2 films is described. The porosity of anatase TiO 2 layers allows the diffusion of amphiphilic pyrrole ammonium tetrafluoroborate ( 1 ) and its electrochemical oxidation at the underlying SnO 2 surface. The determination of glucose is carried out at−0.15 V (vs. Ag ∣ AgCl) via the selective reduction of H 2 O 2 at the TiO 2 surface. The analytical characteristics of different poly 1 –GOD coatings have been compared. In addition, the influence of the thickness of the TiO 2 layer on the performance of the biosensor for H 2 O 2 and glucose detection has been determined.

Influence of PEGylation on the Release of Low and High Molecular-Weight Proteins from PVA Matrices

Model proteins of therapeutic interest, ribonuclease, superoxide dismutase, catalase and albumin, extensively PEGylated with linear 5,000 Mw PEG, were entrapped in PVA hydrogels (31,000-50,000 Mw) by means of a cryogel procedure and the rate of release was evaluated. All of the PEGylated proteins were released from the gel, but at a lower rate than the unmodified proteins. The rate for both native and PEGylated species decreases as the molecular weight of the protein increased. Moreover, for both native and PEGylated proteins, the release rate was reduced when the gel was lyophilized before the release evaluation. Release was also dependent on the PVA Mw, in fact, for Mw's of 124,000-186,000, permanent entrapment was achieved. This behaviour was verified with PEGylated glucose oxidase and coline oxidase which were permanently retained in high Mw PVA. This study indicates the potential for the combination of PEGylation and cryogel entrapment of enzymes for the biotechnological and therapeutic applications.

Fabrication of amperometric biosensors by entrapment of enzymes in functionalized polypyrrole films

AbstractThe concept and potentialities of an original electrochemical procedure of enzyme immobilization are described. This two‐step method consists of the adsorption of an aqueous amphiphilic pyrrole monomer‐enzyme mixture on an electrode surface followed by the electropolymerization of the adsorbed monomers. This procedure is compared with the conventional electrochemical methods of biosensor construction. Several bienzyme electrodes devoted to the improvement of the biosensor selectivity or to the amplification of the biosensor sensitivity are described. In addition, examples of organic phase enzyme electrode and the electrical wiring of immobilized enzymes are presented.

A Low-Volume Electrochemical Cell for the Deposition of Conducting Polymers and Entrapment of Enzymes

A new small-volume electrochemical cell for the entrapment of biological recognition elements in electrochemically deposited conducting polymer films has been developed. It allows one to save significant amounts of the monomer and especially of the used biological recognition element, and hence permits one to choose very expensive enzymes for the development of respected biosensors. Since the conductivity, morphology and film thickness of the obtained conducting polymer films are dependent on the presence of molecular oxygen, the electrochemical cell allows a facilitated handling of the electrode and electrolyte solution under strict exclusion of any oxygen. To prove the applicability of the new electrochemical cell, the entrapment of glucose oxidase into polypyrrole films has been performed as an example.

A Low-Volume Electrochemical Cell for the Deposition of Conducting Polymers and Entrapment of Enzymes

A Low-Volume Electrochemical Cell for the Deposition of Conducting Polymers and Entrapment of Enzymes

1H NMR Self-Diffusion in Polymer–Surfactant Nanocapsules and Cryogels with Enzyme

The multicomponent self-diffusion in nanocapsules and cryogel biocatalytic systems containing α-chymotrypsin has been studied with the NMR-PGSE method at various temperatures and compared with the diffusion of such systems without enzyme. Unilamellar vesicles have been formed in water after “coating” with Brij-97 of the poly-(N,N-diallyl-N,N-didodecyl ammonium bromide), poly-DDAB, nanocapsules. The latter have been obtained by UV-irradiation of reversed hydrated micelles from DDAB in cyclohexane. Cryogels were made from poly(vinyl alcohol), PVA, aqueous solutions by a freezing–thawing cyclic process. Both compartmented systems were used as vehicles of the enzyme entrapped in inner aqueous cavities. The activation energies of self-diffusion for both these systems have been calculated. These data contain information concerning morphology and molecular packing. Encapsulation of α-chymotrypsin in the poly-DDAB/Brij-97 vesicles and the PVA cryogel lowers theDsvalues for all molecules and shifts the cloud point toward the lower temperature. On the contrary, the syneresis point for the PVA cryogel is shifted for 8° toward the higher temperature by the entrapment of the enzyme. Besides, entrapment of α-chymotrypsin in the cryogel promotes the increase of theEavalues for the PVA chain on 1.5 kJ/mol below the syneresis point. Such a difference indicates the influence of the H-bond system of PVA hydroxyl groups and water molecules on the interference of the protein globule. Entrapment of α-chymotrypsin leads to consolidation of this H-bond system.

Sol−Gel-Entrapped Cholinesterases: A Microtiter Plate Method for Monitoring Anti-cholinesterase Compounds

Herein is reported the successful entrapment of three esterase enzymes within silica, by means of the sol−gel process. The enzymes were acetylcholinesterase (AChE) from electric eel (ee), AChE from bovine erythrocytes (er), and butyrylcholinesterase (BChE) from horse serum. Enzyme entrapment was carried out in a novel configuration by casting the doped sol−gel material in 96-well microtiter plates. The study determined the apparent kinetics of the activity and inhibition of the three entrapped enzymes and elucidated the optimal sol−gel matrix composition and preparation procedure. The enzyme mode of action within the sol−gel matrix was compared with that obtained in solution, under various experimental conditions. The activity of entrapped ee-AChE was found to depend on the concentration of the entrapped enzyme and on the sol−gel preparation procedure and composition. It was found that whereas the activity of ee-AChE was 3−4-times lower than that in solution, one gained significantly higher stability. The...

Kinetic studies on the electron transfer between bacterial c-type cytochromes and metal oxides

Cyclic voltammetry was used to investigate the kinetics of the electron transfer between various soluble or solid metal oxides, and polyheme c-type cytochromes from Desulfuromonas acetoxidans and Desulfovibrio. The second order rate constant for the catalytic reduction of soluble chromate ions by Desulfuromonas acetoxidans cytochrome c7 was found to be 6×105 M−1 s−1. By using the membrane electrode technology, it has been shown that the catalytic process for Cr(VI) reduction is efficient even when the cytochrome is entrapped in the close vicinity of the electrode surface. Moreover, this proceeding allowed the catalytic reduction of solid metal oxides such as manganese(IV), vanadium(V) and iron(III) oxides to be performed. Results suggest that the metal reductase activity of a microorganism is governed by its c-type cytochrome content. Furthermore, only cytochromes with bishistidinyl heme iron coordination act as metal reducers whereas mitochondrial c-type cytochromes do not. This approach opens new pathways for the use of sulfur or sulfate bacteria in the bioremediation of metal contaminated waters and waste streams. Processes involving the use of entrapped enzymes reactors could be developed according to the metal reducing activity of their polyheme c-type cytochromes.

Kinetic studies on the electron transfer between various c-type cytochromes and iron (III) using a voltammetric approach

Cyclic voltammetry was used here to investigate the kinetics of the electron transfer between a soluble iron(III) complex and several cytochromes isolated from Desulfovibrio and Desulfuromonas species. The second order rate constants were calculated and found to be in the 10 5–10 6 M −1 s −1 range. Their values are discussed from the point of view of the redox potentials, heme content and pI value of the proteins. The ability of poly(ester–sulfonic acid) ionomer (Eastman AQ-29D) to entrap positively charged cytochromes made it possible to study the immobilized proteins. When the cytochromes are incorporated into this polymer matrix, diffusion limitations lead to a 50 times lower catalytic efficiency as compared to catalysis with protein in solution. The results of this study demonstrate that only cytochromes with bishistidinyl heme iron coordination act as metal reducers whereas mitochondrial c-type cytochromes do not. This approach opens new pathways for the use of sulfur and sulfate reducing bacteria in the bioremediation of metal contaminated waters and waste streams. Processes involving the use of entrapped enzymes reactors could be developed according to the metal reducing activity of their polyhemic c-type cytochromes.

Method for construction of a simple laboratory-scale nonwoven filament biocatalytic filter

A method is described for constructing a simple laboratory-scale nonwoven filament biocatalytic filter (FBF) from multilayer acrylic vinyl acetate copolymer coated thread (yarn) potentially useful for investigation of biotransformations using entrapped enzymes or viable microorganisms. The porous structure of a commercial 128 &mgr;m, 279 denier, 100% polyester thread was sealed with a latex coat (precoat), then coated with a latex + sulfanilamide-azocasein mixture, and finally coated with a latex top coat to generate the multilayer coated thread. The FBF was constructed by drawing the thread across a 4 cm diameter rubber O-ring in the form of multiple layers and sealing the layers with silicone sealer. The FBF is combatible with a commercially available filter housing containing a flow distrtibutor. A method for characterizing the permeability of the sealant (top) coat of the FBF using azocasein release was also developed.

Characterization of microbial endo‐β‐glucanase immobilized in alginate beads

AbstractEndo‐β‐glucanase (endo‐β‐1,4‐glucano‐glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH‐activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a Km value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent Vmax of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half‐life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β‐glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β‐glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co‐immobilization of both types of enzymes reduced by hydrolytic efficiency to half.

Comparison of β-galactosidase immobilization by entrapment in and adsorption on poly(2-hydroxyethylmethacrylate) membranes

β-Galactosidase was immobilized in/on poly(2-hydroxyethyl methacrylate) (pHEMA) membranes by two different methods: adsorption on Cibacron F3GA derivatized pHEMA membranes (pHEMA-CB), and entrapment in the bulk of the pHEMA membranes. The maximum β-galactosidase adsorption on pHEMA-CB membranes was obtained as 95·6μgcm-2 in 2·0mgcm-3 enzyme solution. The adsorption phenomena appeared to follow a typical Langmuir isotherm. In the entrapment, an increase in β-galactosidase loading resulted in a consistent increase in membrane activity from 3·3×10-2 to 17·8×10-2Ucm-2 pHEMA membranes. The Km values for both immobilized β-galactosidase (adsorbed 0·32mM and entrapped 0·81mM) were higher than that of the free enzyme (0·26mM). The optimum reaction temperature of the adsorbed enzyme was 5°C higher than that of both the free and the entrapped enzyme. The optimum reaction pH was 7·5 for free and both immobilized preparations. After 15 successive uses the retained activity of the adsorbed and the entrapped enzymes was 80% and 95%, respectively. The storage stability of the enzyme was found to increase upon immobilization. ©1997 SCI

Pulse technique for the electrochemical deposition of polymer films on electrode surfaces

Often, electrochemically-induced deposition of conducting polymer films on electrode surfaces fails using potentiostatic, galvanostatic or multisweep deposition procedures if bulky substituents at the monomer, nucleophilic attack at intermediate radical cations, hindered diffusional mass transport of the monomer to the electrode surface or the copolymerization of monomers with different oxidation potentials prevent fast chain propagation. A pulse profile for the electrochemical deposition of conducting polymer films has been developed based on the rationalization of the limiting steps and the concentration profiles in front of the electrode surface. The pulse deposition method could be advantageously applied for the localized deposition of conducting polymers using scanning electrochemical microscopy, for the copolymerization of pyrrole/[Os(2,2′-bipyridine) 2(3-{pyrrole-1-ylmethyl}pyridine)Cl] + and for the entrapment of enzymes within the growing ramified network of the polymer.