Showing papers on "Immobilized enzyme published in 1997"

Immobilization of lysozyme on food contact polymers as potential antimicrobial films

Abstract: The objective of this work was to investigate the feasibility of incorporating an antimicrobial enzyme (lysozyme) into polymers which are suitable for food contact. Hen egg white lysozyme was immobilized on polyvinyl alcohol (PVOH) beads, nylon, 6,6 pellets and cellulose triacetate (CTA) films. Polyvinyl alcohol and nylon 6,6 yielded low activity against a suspension of dried Micrococcus lysodeikticus cells, while CTA yielded the highest activity; 1.25 cm2/ml (CTA film area to substrate volume ratio) fully hydrolyzed a 0.015% (w/v) suspension of dried cells in 30 min. The activity retention of lysozyme immobilized on CTA was 90% after one use and 60% after 20 repeated uses. The amount of enzyme added to the film during immobilization affected the activity of the immobilized lysozyme; highest activities were obtained when CTA films were formed by adding 150–250 mg lysozyme per g polymer. No significant effect of CTA film thickness on lysozyme activity was observed. Viability of M. lysodeikticus grown on tripticase soy broth (TSB) at 30°C was decreased in the presence of CTA film containing lysozyme. The film (0.01 cm2/ml TSB) was inhibitory and bactericidal against 103 and 108 c.f.u./ml M. lysodeikticus respectively. © 1997 John Wiley & Sons, Ltd.

Generation of biotin/avidin/enzyme nanostructures with maskless photolithography.

Abstract: Micrometer-sized domains of a carbon surface are modified to allow derivatization to attach redox enzymes with biotin/avidin technology. These sites are spatially segregated from and directly adjacent to electron transfer sites on the same electrode surface. The distance between these electron transfer sites and enzyme-loaded domains must be kept to a minimum (e.g., less than 5 μm) to maintain the fast response time and high sensitivity required for the measurement of neurotransmitter dynamics. This is accomplished through the use of photolithographic attachment of photobiotin using an interference pattern from a UV laser generated at the electrode surface. This will allow the construction of microscopic arrays of active enzyme sites on a carbon fiber substrate while leaving other sites underivatized to facilitate electron transfer reactions of redox mediators, thus maximizing enzyme activity and detection of the enzyme mediator. The ultimate sensitivity of these sensors will be realized only through care...

Esterification of phenolic acids from green coffee with an immobilized lipase from Candida antarctica in solvent-free medium

Abstract: Phenolic acid esters were synthesized by direct esterification catalysed by an immobilized lipase obtained from Candida antarctica. Esterification yields ranged from 3 to 98%; yields seem to be linked to the electronic distribution of phenolic acids which affects the reactivity of the carboxylic function. The carbon chain length of the alcohols also plays an important role. It was observed that even for high yields, reaction times were two days or more.

Direct binding of protein to magnetic particles

Abstract: A new method of binding bovine serum albumin on to freshly precipitated magnetic particles is reported. The binding was confirmed by electron micrograph studies, magnetic measurements and FTIR spectroscopy. Under optimum conditions more than 90% of the protein was bound to the magnetic particles. When alkaline phosphatase was immo-bilised using this method, it retained 75% enzyme activity. The method may prove to be applicable to radio-immuno assays (binding of antibodies to magnetic particles), cell and enzyme immobilisation and in affinity chromatography

Characterization of immobilization of an enzyme in a modified Y zeolite matrix and its application to an amperometric glucose biosensor.

Abstract: A new approach to construct an amperometric biosensor is described. Without using bovine serum albumin−glutaraldehyde, glucose oxidase (GOx) was immobilized on a dealuminized Y zeolite (DAY)-modified platinum electrode to construct a glucose sensor. The large specific surface area of the zeolite substrate resulted in high enzyme loading. The immobilized GOx in this manner was stable and could maintain its high activity for at least 3 months. The interactions between the zeolite and the enzyme were investigated by means of Fourier transform infrared spectra, and the pore distribution and the surface acid property of DAY were preliminarily studied. The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization. This resulting biosensor exhibited good reproducibility and selectivity, owing to the uniform pore structure and unique ion-exchange property of the zeolite. The biosensor responded rapidly to glucose in the linear range from 2.0...

Biosensor for Neurotransmitter L-Glutamic Acid Designed for Efficient Use of L-Glutamate Oxidase and Effective Rejection of Interference

Abstract: An amperometric biosensor for L-glutamic acid (Glu) was constructed by the adsorption and dip coating of L-glutamate oxidase (GluOx, 200 U ml–1 phosphate buffer, pH 7.4) onto 60-µm radius Teflon-coated Pt wire (1 mm exposed length). The enzyme was then trapped on the surface by electropolymerisation of o-phenylenediamine that also served to block electroactive interference. This procedure afforded electrodes with similar substrate sensitivity compared with the classical approach of immobilising enzyme from a solution of monomer, and represents an approximately 10000-fold increase in the yield of biosensors from a batch of enzyme. A number of strategies were examined to enhance the sensitivity and selectivity of the Pt/PPD/GluOx sensors operating at 0.7 V versus SCE. Pre-coating the Pt with lipid and incorporation of the protein bovine serum albumin into the polymer matrix were found to improve the performance of the electrode. The sensors had a fast response time, high sensitivity to Glu, with an LOD of about 0.3 µmol l–1, and possessed selectivity characteristics suggesting that monitoring Glu in biological tissues in vivo may be feasible.

Dramatically stabilized phosphotriesterase-polymers for nerve agent degradation.

Abstract: Phosphotriesterase (EC 3.1.8.1) was immobilized within a polyurethane foam matrix during polymer synthesis using a prepolymer synthesis strategy. In addition to retaining greater than 50% of the enzyme specific activity, numerous benefits were incurred upon immobilization. Orders of magnitude increases in storage and thermal stability (net stabilization energy = 12.5 kJ/mol) were observed without the need for enzyme premodification. The immobilized enzyme system was protease resistant and seemed to display no adverse effects from immobilization, such as an alteration of enzyme function. The organic solvent, dimethyl sulfoxide, also exhibited a stabilizing effect on phosphotriesterase enzyme systems over a range of intermediate concentrations. We attribute these effects in part to direct interaction between the aprotic solvent and metal containing residues present at the enzyme's active site. Our data demonstrate that just 2.5 kg of immobilized enzyme may be sufficient to degrade 30,000 tons of nerve agent in just 1 year. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 105–114, 1997.

Ph-isfet based urea biosensor

Abstract: A new method of direct immobilization of urease on a hydrated silicon nitride surface with the use of glutaraldehyde is presented. The main parameters characterizing the developed urea biosensor based on pH-ion-selective field effect transistor (pH-ISFET) with directly immobilized urease are: (1) maximal analytical signal, 120–140 mV in 10 mM phosphate buffer solution; (2) linear range of ΔUgs=f(log Curea):pCurea(2–3.5) in 10 mM phosphate buffer; (3) response time, 80 s; and (4) lifetime of 35 days with a stable analytical signal. The biosensor was used for urea determination in blood serum and in hemodialysis fluid with satisfactory results.

Polarity Index: The Guiding Solvent Parameter for Enzyme Stability in Aqueous-Organic Cosolvent Mixtures

Abstract: Enzyme catalysis in aqueous–organic cosolvent mixtures has wide applications. However, inadequate attention has been paid to the issue of stability of enzymes in such media. The results with polyphenol oxidase, peroxidase, acid phosphatase, and trypsin show that solvents with polarity indexes of 5.8 and above are “good” solvents. These solvents when used as cosolvents in aqueous–organic solvent media do not denature the enzymes irreversibly. Enzyme(s) exposed to these solvents retain most of their activity even after 48 h of exposure, whereas solvents with polarity indexes of <5.1 denature the enzyme completely within 0–4 h in most of the cases studied. It appears that at higher concentrations (50% and above) cosolvents effectively compete with the water layer around the enzyme. Fluorescence spectroscopy shows that, although the presence of all the organic cosolvents cause conformational changes in the enzyme molecule at a concentration of 50% (v/v), these changes were completely reversible (when the concentration of organic solvent is diluted with aqueous buffer) in case of solvents having polarity indexes of 5.8 and above. In cases of the solvents having polarity indexes of 5.0 and below, the exposure at 50% concentration changed the conformation of the enzymes irreversibly. Thus, a simple parameter, viz. polarity index, may help in medium engineering of enzyme catalysis in nonaqueous surroundings.

How do additives affect enzyme activity and stability in nonaqueous media

Abstract: The catalytic activities of lyophilized powders of alpha-chymotrypsin and Candida antarctica lipase were found to increase 4- to 8-fold with increasing amounts of either buffer salts or potassium chloride in the enzyme preparation. Increasing amounts of sorbitol in the chymotrypsin preparation produced a modest increase in activity. The additives are basically thought to serve as immobilization matrices, the sorbitol being inferior because of its poor mechanical properties. Besides their role as supports, the buffer species were indispensable for the transesterification activity of chymotrypsin because they prevented perturbations of the pH during the course of the reaction. Hence, increasing amounts of buffer species yielded a 100-fold increase in transesterification activity. Effects of pH changes were not as predominant in the peptide synthesis and the lipase-catalyzed reactions. Immobilization of the protease on celite resulted in a remarkable improvement of transesterification activity as compared to the suspended protease, even in the absence of buffer species. Immobilization of the lipase caused a small improvement of activity. The activity of the immobilized enzymes was further enhanced 3-4 times by including increasing amounts of buffer salts in the preparation.The inclusion of increasing amounts of sodium phosphate or sorbitol to chymotrypsin rendered the catalyst more labile against thermal inactivation. The denaturation temperature decreased with 7 degrees C at the highest content of sodium phosphate, as compared to the temperature obtained for the denaturation of the pure protein. The apparent enthalpy of denaturation increased with increasing contents of the additives. The enhancement of hydration level and flexibility of the macromolecule upon addition of the compounds partly provides the explanation for the observed results.

New enzyme potentiometric sensor for hypochlorite species detection

Abstract: Biochemical assay for hypochlorite couple (HOC1/OC1−) concentration in water solutions was developed. Acetylcholine sensitive enzyme sensor based on ion sensitive field effect transistors (ENFET) was prepared by cross-linking acetylcholinesterase (AChE) with bovine serum albumin in saturated glutaraldehyde vapour on the sensor chip and used for such purpose. The corresponding detection limit was at least about 10−5 M of HOC1/OC1− at pH 6.0-6.5. At these pH from 90% up to 100% of hypochlorite species were under the form of hypochlorous acid. Sensitivity of the developed biosensor to hypochlorite species depends on the concentration of acetylcholine and inhibition time. This phenomenon can be used to increase the sensitivity of such biosensor to hypochlorite species concentration and detection limit for inhibitor analysis can be decreased. The use of a pyridine-2-aldoxime methyliodide (PAM-2), a well known restoration activity agent for cholinesterases, did not give positive effect. So it can be concluded that the inhibition of AChE by HOCl/OCl− is due to another mechanism than those involved in the case of an inhibition by organophosphorous pesticides.

Reagentless mediated laccase electrode for the detection of enzyme modulators.

Abstract: We have investigated aerobic mediation of electron transfer to a laccase enzyme by the solution redox couples [Os(bpy)2Cl2]+/0 and [Os(bpy)2(MeIm)Cl]2+/+, where bpy is 2,2-bipyridine and MeIm is N-methylimidazole. The factors that influence the homogeneous mediation reaction are investigated and discussed. Investigation of ionic strength, pH, and temperature effects on the kinetics of intermolecular electron transfer elucidates the governing factors in the mediator−enzyme reactions. Coimmobilization of both enzyme and an osmium redox mediator in a hydrogel on glassy carbon electrodes results in a biosensor for the reagentless addressing of enzyme activity, consuming only oxygen present in solution. Thus, these immobilized enzyme biosensors can be utilized for the detection of modulators of laccase activity, such as the inhibitor sodium azide. The enzyme inhibition biosensor can detect levels of azide as low as 2.5 × 10-6 mol dm-3 in solution.

Properties of free and immobilized lipase from Pseudomonas cepacia.

Abstract: The purified lipase from Pseudomonas cepacia (PS, Amano) was immobilized on a commercially available microporous polypropylene support. The enzyme was rapidly and completely adsorbed on the support. Special attention was devoted to the demonstration of the lack of diffusional limitations, either internal or external, when a soluble substrate (p-nitrophenylacetate, pNPA) was used. The activity yield was high (100%) with pNPA and very low (0.4%) with p-nitrophenylpalmitate (pNPP). These values clearly showed that the immobilized enzyme was fully active as soon as activity was assayed on a soluble substrate rather than an insoluble one. With the latter one, the low activity was due mainly to a slow rate of substrate diffusion inside the porous support. The same diffusional phenomenon could explain the complete change of fatty acid specificity of the immobilized lipase. After immobilization, the lipase was mainly specific for short chain fatty acid esters, whereas the free enzyme was mainly specific for long chain esters. The activity-versus-temperature profiles were not greatly affected by immobilization with maximal reaction rates in the range 45 degrees to 50 degrees C for both enzyme preparations. However, immobilization increased enzyme stability mainly by decreasing the sensitivity to temperature of the inactivation reaction. Half-lives at 80 degrees C were 11 and 4 min for the immobilized and free enzymes, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 181-189, 1997.

The role of silica gel in lipase-catalyzed esterification reactions of high-polar substrates

Abstract: The crucial role of silica gel in lipase-catalyzed esterification reactions using adsorbed high-polar substates was established. It was found that in these kinds of reactions, the presence of silica gel-adsorbed substrates did not alter the kinetic mechanisms of the synthesis of acylglycerides but improved significantly the conversion yields. An explanation for the critical role of silica gel in these kinds of reactions was proposed whereby the silica gel behaves as a “polar substrate reservoir” and plays a protective role for the immobilized enzyme avoiding its blockage. In this way it was shown that the use of silica gel facilitates reactions at high glycerol concentrations without alteration of reaction rates or conversion yields. It also was demonstrated that highly hydrophilic polyols enhanced blockage of the enzyme. The importance of the presence of an organic solvent in the reaction medium was shown to have a critical role in the conversion yields of the reactions tested. Finally, the influence of different supports used for adsorption of the substrates was compared with respect to their efficiency in protecting the enzymatic activity.

Optical Biosensing of Nitrate Ions Using a Sol–Gel Immobilized Nitrate Reductase

Abstract: The coupling of enzymes with sol–gel technology creates exciting possibilities for biosensing. Enzymes can be highly selective and will only respond to specific analytes. Sol–gels provide a unique matrix in which various biomaterials can be immobilized without any loss of enzyme activity. These two components have been combined for the optical biosensing of nitrate ions. The periplasmic nitrate reductase (Nap) extracted from the denitrifying bacterium Thiosphaera pantotropha reacts specifically with the nitrate (NO 3 - ) anion. The encapsulation of this enzyme in a sol–gel structure for the optical biosensing of nitrate ions is reported. The reduction of nitrate by periplasmic nitrate reductase results in a characteristic change in the UV/VIS absorption spectrum of the nitrate reductase. This spectroscopic change has been quantitatively calibrated against nitrate concentration. The nitrate biosensing system is fully reversible and is highly sensitive and selective to nitrate ions. The results obtained show that the activity of the enzyme is not affected by the sol–gel matrix, even after a storage period of up to six months. As no leaching of the Nap from the sol–gel matrix was observed, it is clear that the encapsulation of this nitrate sensitive enzyme in a sol–gel medium represents an ideal anionic recognition element of an optical biosensor for the detection of nitrate ions in the µmol l - 1 range.

An Electrochemical Method for Making Enzyme Microsensors. Application to the Detection of Dopamine and Glutamate

Abstract: A novel method of microbiosensor fabrication is described. It is based on the electrochemical polymerization of an enzyme−amphiphilic pyrroleammonium solution on the surface of a microelectrode in the absence of supporting electrolyte. By trapping glutamate oxidase (GMO) or polyphenol oxidase (PPO) in such polypyrrole films, we made microbiosensors for the amperometric determination of glutamate or dopamine, respectively. The response of the GMO microelectrode to glutamate was based on the amperometric detection of the enzymically generated hydrogen peroxide at 0.6 V vs SCE. The detection limit and sensitivity of this microbiosensor were 1 μM and 32 mA M-1 cm-2, respectively. The response of the PPO microelectrode to dopamine was based on the amperometric detection of the enzymically generated quinoid product at −0.2 V. The calibration range for dopamine measurement was 5 × 10-8−8 × 10-5 M and the detection limit and sensitivity were 5 × 10-8 M and 59 mA M-1 cm-2, respectively.

Thermal Stabilization of Enzymes Immobilized within Carbon Paste Electrodes.

Abstract: In this note we report on the remarkable thermal stabilization of enzymes immobilized in carbon paste electrodes. Amperometric biosensors are shown for the first time to withstand a prolonged high-temperature (>50 °C) stress. Nearly full activity of glucose oxidase is retained over periods of up to 4 months of thermal stress at 60-80 °C. Dramatic improvements in the thermostability are observed for polyphenol oxidase, lactate oxidase, alcohol oxidase, horseradish peroxidase, and amino acid oxidase. Such resistance to heat-induced denaturation is attributed to the conformational rigidity of these biocatalysts within the highly hydrophobic (mineral oil or silicone grease) pasting liquid. While no chemical stabilizer is needed for attaining such protective action, it appears that low humidity (i.e., low water content) is essential for minimizing the protein mobility. Besides their implications for electrochemical biosensors, such observations should lead to a new generation of thermoresistant enzyme reactors based on nonpolar semisolid supports.

Immobilization of Xylanase in Chitosan−Xanthan Hydrogels

Abstract: endo-1,4-β-Xylanase (E.C. 3.2.1.8) has been immobilized in hydrogel beads prepared by complexation between chitosan and xanthan. The enzyme immobilization efficiency is between 88 and 98% within a wide range of concentrations of xylanase. The optimum xylanase activity requires a pH between 5.0 and 5.5. The immobilized enzymes show 60-70% higher activity than free enzymes. The K m of the immobilized xylanase increases with the concentration of the enzyme in the beads. The latter show, by electron microscopy, a predominantly fibrilar microstructure within which there are regions having globular shapes where the enzymes are lodged.

Biosensor properties of glucose oxidase immobilized within SiO2 gels

Abstract: The sol-gel immobilization of enzymes within metal oxides results in thin composite layers which are useful for biosensors, e.g., in combination with optical or electrochemical transducers. The activity of glucose oxidase (GOD) immobilized within SiO2 gels is mainly determined by the chemical and physical parameters of the sol-gel process. So, the type of the sol, the conditions of the SiO2 formation, the GOD concentration and the addition of penetration agents are studied to optimize the enzyme activity and the response of the complete amperometric biosensors. Both optical and electrochemical methods are used to measure activities of immobilized GOD. The applications of such GOD-SiO2 composites (i) directly on the electrode surface of a flat sensor, (ii) indirectly within Cuprophan® membranes of an amperometric hydrogen peroxide sensor or (iii) on zeolite beads in a cartridge followed by an H2O2 sensor are compared with regard to sensitivity and selectivity of the respective sensor systems. The best membrane sensor properties are obtained for silica hydrogel composites containing large amounts of GOD produced with sols from acidic hydrolysis of a 25% 3-aminopropyltrimethoxysilane-75% tetraethoxysilane precursor. Xerogels from acidic hydrolysed tetraethoxysilane sols within aprotic solvents like dioxane are favoured for direct application on Pt working electrodes of flat sensor structures.

Methods for the immobilization of lipases and their use for ester synthesis

Abstract: The lipase from Pseudomonas fluorescens was immobilized onto five different carriers: celite, octyl-silica, aminopropyl-silica, gluterdialdehyde-activated silica and Eupergit C250L. Activities and operational stabilities of the prepared catalysts were compared using the enantioselective acylation of (R,S)-1-phenylethanol by vinyl acetate as acyl donor and t -butylmethyl ether with variable water content (0.038-0.97% v/v) as reaction medium. The above carriers provide catalysts with widely different specific activities ranging from excellent 25 mmol/h mg protein (celite) to 0.07 mmol/h mg protein (glutardialdehyde-activated silica) on the lower end. The lipase immobilized onto Eupergit C250L exhibited the best operational stability among the catalysts studied. It retained 30% of its initial activity after 11 cycles of application, each with a duration between 2 and 6 h.

Improving the Activity of Immobilized Subtilisin by Site-Specific Attachment to Surfaces

Abstract: Understanding the properties of immobilized proteins is critical to the optimal design of biosensors, bioseparations, and bioreactors. The protease subtilisin BPN' was used as a model protein to study how the orientation of immobilized enzyme molecules on surfaces affects their catalytic properties. To achieve this goal, a single cysteine residue was introduced into the cysteine-free enzyme by site-directed mutagenesis. This cysteine residue was designed to be away from the active site of the enzyme. The enzyme molecules were immobilized through the side-chain sulfhydryl group of the cysteine residue on several supports. This site-specific immobilization method leads to ordered two-dimensional arrays of enzyme molecules on the support surface with the active sites of the enzyme oriented toward the solution phase. Such oriented immobilized subtilisin demonstrated a higher catalytic efficiency compared to subtilisin that was immobilized by a conventional method that leads to random immobilization.

Plant seed oil-bodies as an immobilization matrix for a recombinant xylanase from the rumen fungus Neocallimastix patriciarum

Abstract: Canola seed oil-bodies were investigated as a production vehicle and immobilization matrix for xylanases. A recombinant xynC gene from Neocallimastix patriciarum encoding a xylanase (XynC) was fused to an oleosin coding sequence suitable for targeting the xylanase to the oil-body membrane. This fusion gene was introduced into Brassica napus using Agrobacterium-mediated transformation. Transgenic Canola plants were obtained expressing xylanase which was targeted to the oil-bodies of seeds as shown by analysis with XynC-specific antibodies. Oil-bodies extracted from transgenic seeds exhibited xylanase activity, indicating the immobilization of XynC on the surface of oil bodies and the functioning of the xylanase as a fusion protein. The immobilized XynC retained its optimal temperature, Km value and specificity. However, it exhibited reduced sensitivity to pH. Furthermore, it was shown that the enzyme immobilized on oil-bodies could be recycled by flotation several times without loss of activity.

Glassy State and Thermal Inactivation of Invertase and Lactase in Dried Amorphous Matrices

Abstract: The thermal stability of enzymes lactase and invertase in dried, amorphous matrices of sugars (trehalose, maltose, lactose, sucrose, raffinose) and some other selected systems (casein, PVP, milk) was studied. The glass transition temperature (T g ) was limited as a threshold parameter for predicting enzyme inactivation because (a) enzyme inactivation was observed in glassy matrices, (b) a specific effect of enzyme stabilization by certain matrices particularly trehalose was observed, and (c) enzyme stability appeared to depend on heating temperature (T) per se rather than (T - T g ). For these reasons, a protective mechanism by sugars related to the maintenance of the tertiary structure of the enzyme was favored. A rapid loss of enzyme (lactase) activity was observed in heated sucrose systems at T > T g , and this was attributed to sucrose crystallization since it is known that upon crystallization the protective effect of sugars is lost. Thus, the stabilizing effect could be indirectly affected by the T g of the matrix, since crystallization of sugars only occurs above T g . Trehalose model systems (with added invertase) showed an exceptional stability toward darkening (e.g., non-enzymatic browning) when heated in the dried state to elevated temperatures and for long periods of time.