Showing papers on "Immobilized enzyme published in 1991"

Amperometric glucose microelectrodes prepared through immobilization of glucose oxidase in redox hydrogels.

Abstract: Glucose microelectrodes have been formed with glucose oxidase immobilized in poly[(vinylpyridine)Os(bipyridine)2Cl] derivative-based redox hydrogels on beveled carbon-fiber microdisk (7 microns diameter) electrodes. In the resulting microelectrode, the steady-state glucose electrooxidation current density is 0.3 mA cm-2 and the sensitivity is 20 mA cm-2 M-1. The current density and sensitivity are 10 times higher than in macroelectrodes made with the same hydrogel. Furthermore, the current is less affected by a change in the partial pressure of oxygen. The higher current density and lower oxygen sensitivity point to the efficient collection of electrons through their diffusion in the redox hydrogel to the electrode surface. These results contrast with those observed for enzyme electrodes based on diffusing mediators, where loss of the enzyme-reduced mediator by radial diffusion to the solution decreases the current densities of microelectrodes relative to similar macroelectrodes.

Protein immobilization : fundamentals and applications

Abstract: For those who carry out basic R&D in protein immobilization, as well as those who utilize protein immobilization technologies in their processes and products, this volume considers the role of immobilized proteins, including immobilized enzymes, antibodies, structural proteins, and macromolecular co

On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity.

Abstract: Enzymes were deposited on different porous support materials and these preparations were used to catalyze reactions in organic media. Reactions were carried out at specific water activities, achieved by equilibrating both the enzyme preparation and the substrate solution at the desired water activity before mixing them and thereby starting the reactions. The reaction rates obtained at the same water activity with different supports differed greatly, indicating a direct influence of the support on the enzyme. For horse liver alcohol dehydrogenase, Celite was the best support, and the reaction rate increased with increasing water activity. In the alpha-chymotrypsin-catalyzed alcoholysis of N-acetyl-L-phenylalanine ethyl ester with 1-butanol, high rates were again obtained with Celite, but with this support only about one third of the ethyl ester was converted to butyl ester, the rest was hydrolyzed. With the polyamide support, Accurel PA6, alcoholysis was the dominating reaction, and by using a low water activity (0.33), hydrolysis was completely suppressed while still maintaining a high alcoholysis activity. Controlled pore glass (CPG), derivatized with either hexyl or glucosyl groups, had quite different properties as enzyme supports. For horse liver alcohol dehydrogenase, glucose-CPG was a much better support than hexyl-CPG, and in the alpha-chymotrypsin-catalyzed reactions, glucose-CPG favored hydrolysis, and hexyl-CPG alcoholysis, at water activities exceeding 0.8. The results are discussed considering the absorption of water on the enzymes, on the supports and the solubility of water in the reaction media; all these parameters were measured separately.

Immobilization‐stabilization of α‐chymotrypsin by covalent attachment to aldehyde‐agarose gels

Abstract: We have developed a strategy for immobilization-stabilization of alpha-chymotrypsin by multipoint covalent attachment of the enzyme, through its amino groups, to agarosealdehyde gels. We have studied the role of the main variables that control the intensity of these enzyme-support multi-interaction processes (surface density of aldehyde groups in the activated gel, contact time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives, etc.). In this way, we have prepared a number of very different chymotrypsinagarose derivatives. Our best derivatives, with the most intense multipoint attachment, were more stable than one-point attached derivatives and were more than 60,000-fold more stable than soluble enzyme in the absence of autolysis phenomena. In spite of the dramatic stabilization, the catalytic activity of these derivatives is little changed (they only lose 35% of intrinsic activity after this intense enzyme-support multi-interaction process). In addition, we have also demonstrated the very high capacity of 6% aldehyde-agarose gels to immobilize pure chymotrypsin (40 mg enzyme/mL catalyst). Furthermore, we have been able to establish a clear correlation between enzyme-support multipoint covalent attachment, stabilization against very different denaturing agents (heat, urea, organic cosolvents), and insensitivity of those immobilized chymotrypsin molecules to some activating agents.

Enzyme and microbial technology: biotechnology of fats and oils.

Abstract: To develop an enzyme technology industry, a sizeable enzyme bank is a priority requirement. A group of enzymes of interest to us and currently attracting attention of researchers throughout the world are the lipases. Apart from their usual hydrolytic properties, these enzymes can perform synthetic reactions under controlled conditions. Our group have meticulously carried out a screening programme for microbes that can produce these enzymes. So far we have isolated over 50 microbial isolates exhibiting lipolytic activities on plate and managed to study in detail 3 fungi and 2 bacteria. To improve the versatility of these enzymes we are redesigning the enzymes with the hope of obtaining an analogue with better solubility in organic solvents. We have covalently attached aldehydes of various sizes and hydrophobicity onto the enzyme lipase, and the enzyme thus derivatised showed. enhanced catalytic activity and thermal stability compared with the native unmodified enzyme. Another approach is to immobilise the enzyme. The enzyme molecule is still, in principle, to another molecule. In most cases, the molecule attached yo the enzyme is usually insoluble, hence confering the derivatised enzyme a new property of insolubility. We have shown that enzymes can be immobilised by simple adsorption onto readily available resins.

Short-chain flavour ester synthesis by immobilized lipase in organic media

Abstract: Mucor miehei lipase has been adsorbed on Celite and covalently bound to nylon. The obtained derivatives have been studied regarding their ability for synthetize several flavouring esters in biphasic aqueous/organic media. The influence of the immobilization procedure on the synthetic activity of the derivatives was considered. Solvent hydrophobicity and water content in the biphasic system influenced both enzyme stability and equilibrium displacements. In this way, solvents with log P>3.5 and less than 1% water were optimal. It was important to consider pH effects on enzyme microenvironment when using acidic substrates. Optimum temperature and reuse of the catalyst were also checked.

Enzymatic resolution of (S)-(+)-naproxen in a continuous reactor.

Abstract: An enzymatic method for the continuous production of (S)−(+)−2−(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen ethoxyethyl ester catalyzed by Candida cylindracea lipase. The reaction has been carried out in a continuous-flow closed-loop column bioreactor packed with Amberlite XAD−7, a slightly polor resin on which the lipase has been immobilized by adsorption. Various immobilization conditions as well as the properties of the immobilized lipase have been studied. The performance and the productivity of the bioreactor were evaluated as a function of the critical reaction parameters such as temperature, substrate concentration, and product inhibition. By using a 500-mL column bioreactor, 1.8 kg of optically pure (S)-(+)-Naproxen were produced after 1200 h of continuous operation with a slight loss of the enzymatic activity.

Enzymatic synthesis of esters using an immobilized lipase.

Abstract: Various esters were synthesized in nearly anhydrous hexane from alcohols and carboxylic acids using a lipase from Candida cylindracea. The enzyme was immobilized on a nylon support and protein loadings as high as 10 mg/g were obtained. The activity of the immobilized enzyme was maximum in a range of temperatures from 25 to 37°C. Ethylpropionate was formed from ethanol and propionic acid at a rate of 0.017 mol/h g immobilized protein. Different esters were formed at comparable rates and equilibrium conversions could generally be approached in less than 10 h in a batch reaction system. The immobilized lipase catalyst was quite stable and retained about one third of the initial activity after repeated experiments during the course of 72 days. A stirred tank continuous flow reactor was used successfully for the continuous production of esters.

Detoxification of organophosphate pesticides using an immobilized phosphotriesterase from Pseudomonas diminuta.

Abstract: A purified phosphotriesterase was successfully immobilized onto trityl agarose in a fixed bed reactor. A total of up to 9200 units of enzyme activity was immobilized onto 2.0 mL of trityl agarose (65 micromol trityl groups/mL agarose), where one unit is the amount of enzyme required to catalyze the hydrolysis of one micromole of paraoxon in one min. The immobilized enzyme was shown to behave chemically and kinetically similar to the free enzyme when paraoxon was utilized as a substrate. Several organophosphate pesticides, methyl parathion, ethyl parathion, diazinon, and coumaphos were also hydrolyzed by the immobilized phosphotriesterase. However, all substrates exhibited an affinity for the trityl agarose matrix. For increased solubility and reduction in the affinity of these pesticides for the trityl agarose matrix, methanol/water mixtures were utilized. The effect of methanol was not deleterious when concentrations of less than 20% were present. However, higher concentrations resulted in elution of enzyme from the reactor. With a 10-unit reactor, a 1.0 mM paraoxon solution was hydrolyzed completely at a flow rate of 45 mL/h. Kinetic parameters were measured with a 0.1-unit reactor with paraoxon as a substrate at a flow rate of 22 mL/h. The apparent K(m) for the immobilized enzyme was 3-4 times greater than the K(m) (0.1 mM) for the soluble enzyme. Immobilization limited the maximum rate of substrate hydrolysis to 40% of the value observed for the soluble enzyme. The pH-rate profiles of the soluble and immobilized enzymes were very similar. The immobilization of phosphotriesterase onto trityl agarose provides an effective method esterase onto trityl agarose provides an effective method for hydrolyzing and thus detoxifyuing organophosphate pesticides and mammalian acetylcholinesterase inhinbitors.

Resolution of (±)-trans-sobrerol by lipase PS-catalyzed transesterification and effects of organic solvents on enantioselectivity

Abstract: Resolution of the mucolytic drug (±)- trans -sobrerol ( 1 ) was achieved by transesterification with vinyl acetate in organic media, catalyzed by free or immobilized Lipase PS. The enantioselectivity of the enzyme was markedly influenced by the nature of the organic solvent, but there was no correlation between enantiomeric ratio values (70–500) and either the hydrophobicity or the dielectric constant of the medium. With the enzyme immobilized onto Hyflo Super Cell and t -amyl alcohol as the solvent, the selectivity of Lipase PS for (−)- 1 was extremely high and, at 50 % conversion both (−)- trans -sobrerol and (+)- trans -sobrerol monoacetate were obtained in practically 100 % optical purity

Enzymic methods in preparative carbohydrate chemistry.

Abstract: Publisher Summary This chapter discusses the use of enzymic methods in preparative carbohydrate chemistry. Natural sugars are very active metabolites in cells, and so it can be expected that enzymes are useful at some steps in their in vitro processing. However, enzymes are rare, costly, or need to be isolated by unfamiliar techniques. Coenzymes, which are often necessary, are in most cases complicated and costly molecules and their preparation on a large scale may be exceedingly tedious. An inconvenience of enzymic reactions appears in glycosylation with stoichiometric amounts of “nucleotidesugars.” The result of the coupling reaction is the accumulation, in the medium, of the corresponding free nucleotide that may prove inhibitory to transferases at millimolar concentrations. The now-classical solution to these problems is to attach the enzyme to a suitable polymer that is used as an aqueous suspension. When the reaction is finished, the enzyme is separated from the products by filtration and, not infrequently, may be used again many times. This chapter describes the concepts related to immobilization process. It provides details about aldol additions and other C–C, bond-forming reactions. It also explains the concepts related to phosphorylation and transfer reactions catalyzed by glycosidases.

Determination of Organophosphorus and Carbamic Pesticides with a Choline and Acetylcholine Electrochemical Biosensor

Abstract: Pesticides as paraoxon and aldicarb have been determined with an amperometric hydrogen peroxide based choline sensor. These pesticides inhibit the enzyme acetylcholinesterase which in presence of its substrate, acetylcholine, produces choline. When these pesticides are in presence of acetylcholinesterase, the activity of this enzyme decreases; this causes a decrease of choline production which is monitored by a choline sensor and correlated to the concentration of pesticide in solution. Two different procedures were followed: one with both the enzymes acetylcholinesterase and choline oxidase immobilized, the second one with the acetylcholinesterase in solution and the choline oxidase immobilized. Parameters as pH, buffer, enzyme concentration, substrate concentration and reaction and incubation times were optimized. Results showed that these compounds can be detected in the range 10 – 100 ppb. The use of the enzyme in solution gave the best results with a detection limit of 2 ppb pesticide.

Preparation of fine magnetic particles and application for enzyme immobilization

Abstract: Fine magnetic particles (ferrofluid) were prepared from a co-precipitation method by oxidation of Fe2+ with nitrite. The particles were activated with (3-aminopropyl)triethoxysilane in toluene and the activated particles were combined with some enzymes by using glutaraldehyde. Enzyme-immobilized magnetic particles were between 4-70 nm and the size could be changed corresponding to the ratio of the amount of Fe2+ to that of nitrite. In the immobilization of β-glucosidase, activity yield was 83% and 168 mg protein was immobilized per g magnetite. Other enzymes or proteins could be immobilized at the level between about 70 and 200mg/g support. Immobilized β-glucosidase was stable at 4°C. Magnetic particles immobilized with β-glucosidase responded quickly to the magnetic field and “ON-OFF” control of the enzyme reaction was possible.

Detoxification of organophosphate pesticides using a nylon based immobilized phosphotriesterase from Pseudomonas diminuta

Abstract: A partially purified phophostriesterase was successfully immobilized onto nylon 6 and 66 membranes, nylon 11 powder, and nylon tubing. Up to 9000 U of enzyme activity was immobilized onto 2000 cm2 of a nylon 6 membrane where 1 U is the amount of enzyme necessary to catalyze the hydrolysis of 1.0 μmol of paraoxon/min at 25°C. The nylon 66 membrane-bound phosphotriesterase was characterized kinetically where the apparentK m value for the immobilized enzyme was 0.35 mM. This is 5-6 times higher than that observed for the soluble enzyme. However, nylon immobilization limited the maximum rate of paraoxon hydrolysis to less than 10% of the value measured for the soluble enzyme. The addition of the cosolvent, methanol, resulted in an increase in the apparentK m value for paraoxon hydrolysis but concentrations up to 40% had no negative effect on the catalytic effectiveness with the soluble or immobilized phosphotriesterase. Based on the kinetic analysis, methanol appears to be a competitive inhibitor for both forms of enzyme. The nylon powder immobilized enzyme was shown to be stable for at least 20 mo. The immobilization of the phosphotriesterase onto nylon provides a practical method for the detoxification of organophosphate pesticides.

Characterization of the biochemical behavior of glucose oxidase entrapped in a polypyrrole film

Abstract: This article reports the characterization of the biochemical behavior of glucose oxidase entrapped in polypyrrole. The immobilization of glucose oxidase in a polypyrrole film was performed by entrapment during the electropolymerization of pyrrole at a platinum electrode poised at 0.65 V vs. SCE in aqueous solution in a one-compartment electrochemical cell. Thin films of polypyrrole (0.11 μm) were obtained and the entrapped enzyme obeyed Michaelis kinetics, indicating no diffusional constraints of the substrate. Our results indicate that the entrapped glucose oxidase is more resistant to denaturation conditions such as alkaline pH and temperature (50 and 60°C) than the soluble form of the enzyme. The autoinactivation constant for the entrapped enzyme was also determined in presence of 0.25M of glucose and was 6.19 × 10−4 min−1, i.e., corresponding to a half-life value of 20 h. The results reported here show clearly that polypyrrole matrix has a strong stabilizing effect on the stucture and on the activity of glucose oxidase.

Activity and flexibility of alcohol dehydrogenase in organic solvents

Abstract: The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.

Protease immobilization onto porous chitosan beads

Abstract: Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L-arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant Km and the maximum reaction velocity Vm for free and immobilized proteases on the porous chitosan beads are estimated. The apparent Km values were larger for immobilized proteases than for the free ones, while Vm values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.

Continuous glycerolysis of olive oil by Chromobacterium viscosum lipase immobilized in liposome in reversed micelles.

Abstract: Chromobacterium viscosum lipase which has adsorbed on liposome and solubilized in microemulsion droplets of glycerol containing a little amount of water could catalyze the glycerolysis of olive oil. Studies on the continuous glycerolysis of olive oil by the immobilized enzyme was done at 37 degrees C in continuous stirred vessel bioreactor with polysulfone membrane. The effect of the flow rate of substrate (olive oil) in isooctane on the conversion and composition of the outlet was investigated using high-performance liquid chromatography (HPLC). The conversion increased with decrease in the flow rate. And we studied the effect of water content in the glycerol-water-lipase solution on the glycerolysis reaction. The conversion to desirable products, mono- and di-olein, was improved without a substantial production of oleic acid at lower water concentrations, i.e., below 8.0% (w/v) which corresponds to a w(o) value of 0.97. At water concentration higher than 8.0% (w/v), the amount of free fatty acid was dramatically increased. Higher operational stability of the enzyme reactor, and the half-line of the enzyme continuous reaction was about 7 weeks.