Showing papers on "Immobilized enzyme published in 1989"

Immobilization of glucose oxidase with Bombyx mori silk fibroin by only stretching treatment and its application to glucose sensor.

Abstract: Glucose oxidase (GOD) was immobilized in Bombyx mori silk fibroin membrane by only physical treatment, i.e., stretching without any chemical reagents. This is due to the structural transition of the silk fibroin membrane from random coil to antiparallel beta-sheet (Silk II) induced by the stretching treatment. Permeability coefficients of glucose and oxygen through the fibroin membrane were determined; the permeability of glucose decreased with increasing degree of stretching. The immobilized enzyme activity was characterized with apparent Michaelis constant K(m) (app) and maximal activity V(m). Optimum pH of the activity of the immobilized enzyme was shifted to the value around neutrality, and the activity was maintained to the higher values on both sides of the optimum pH compared with the case of free enzymes. Thermal stability was scarcely lost even at 50 degrees C, although the free enzyme lost about 70% of the original activity. Thus, the stabilities of the enzyme vs. pH and heat were much improved by the immobilization with silk. Glucose sensor prepared with this GOD-immobilized fibroin membrane was developed; the capabilities such as the response time, calibration curve, and repeating usage were determined.

Immobilization of enzymes on clays and soils

Abstract: Various enzymes such as glucose oxidase, β- d -glucosidase, acid phosphatase. tyrosinase and laccases were immobilized on clays and soils activated with 3-aminopropyltriethoxysilane and glutaraldehyde. After immobilization the enzymes retained a large amount of their original activities. The retention of laccase activity increased with the increase of clay content in soils, whereas the activity of β- d -glucosidase, tyrosinase and acid phosphatase decreased. The immobilized enzymes showed varying degrees of resistance to proteolysis and storage at high temperatures. After addition to soil suspensions, soluble laccase and glucose oxidase were rapidly inactivated (100 and 85% loss of activity in 15 days for laccase and glucose oxidase, respectively) whereas after immobilization, these enzymes were extraordinarily stable (12 and 25% loss of activity in 15 days for laccase and glucose oxidase, respectively). The possibility of incorporating the stabilized enzymes into soil for the improvement of numerous desired biochemical processes is discussed.

Covalent immobilization of lipase in organic solvents.

Abstract: Lipase from Rhizopus sp. has been immobilized covalently on tresyl activated silica. Three different coupling media were evaluated: aqueous buffer, n-hexane, and a microemulsion based on n-hexane, aqueous buffer, and the nonionic surfactant triethylene glycol monododecyl ether. In addition, coupling via a very long, hydrophilic spacer arm, polyethylene glycol 1500 (PEG 1500), was compared with attachment to the silica via a short silane bridge only. The enzyme preparations were tested in hydrolysis and transesterification reactions. In the hydrolysis no marked differences in activity were found between the coupling media used. In the transesterification, on the other hand, the choice of immobilization medium had a very large effect on lipase activity, the preparation from microemulsion being the most active one. The use of the hydrophilic spacer had a large effect on activity in the hydrolysis reaction. Whereas direct coupling gave an activity of immobilized lipase of 26-34% of that of free enzyme, depending on the reaction medium, lipase bound via the spacer exhibited 56-67% activity. The latter values are considerably higher than previously reported in the literature for covalently immobilized lipase. The hydrophilic spacer had no effect on enzyme activity in the transesterification, however, a fact which is attributed to the hydrophobic medium of this reaction. The spacer is incompatible with the reaction medium and will, therefore, adsorb on the particles rather than stretch out into the bulk phase. The stability of the bound lipase was extremely good, no loss in activity being observed after a period of three weeks in aqueous solution of 37 degrees C.

Enzyme immobilization using the cellulose-binding domain of a Cellulomonas fimi exoglucanase

Abstract: We have constructed a fusion between the gene (abg) for a β-glucosidase (Abg) of an Agrobacterium sp. and part of the gene (cex) for an exoglucanase (Cex) of Cellulomonas fimi. The abg-cex fusion encodes a fusion protein (Abg-CBDCex) with the cellulose-binding properties of Cex and β-glucosidase activity of Abg. The fusion protein retained 42 percent of its β-glucosidase activity when bound to cellulose. The binding was stable enough to allow continuous hydrolysis of substrate by the enzyme without detectable leaching of the enzyme from the cellulose. We believe that the cellulose-binding domain (CBDCex) of Cex offers a simple, inexpensive and widely applicable method of enzyme immobilization.

Avidin-biotin coupling as a general method for preparing enzyme-based fiber-optic sensors

Abstract: A general method for preparing enzyme-based fiber-optic sensors is described. Three different sensors, capable of detecting penicillin, organic esters, and urea, were developed based on this method

Properties of a reversible soluble-insoluble cellulase and its application to repeated hydrolysis of crystalline cellulose.

Abstract: Cellulase was covalently immobilized on an enteric coating polymer, Eudragit L, that is reversibly soluble and insoluble depending on the pH of the medium. The hydrolysis of solid cellulose with the immobilized enzyme can take advantage of the soluble property of the immobilized enzyme itself at the most reactive pH value; on the other hand, recovery of the enzyme can take advantage of the insoluble property of the enzyme at other pH values. It was experimentally confirmed that 100% of immobilized enzyme activity in solution can be recovered by precipitation and by dissolving it again by alternative change of pH. After a period of hydrolysis, immobilized enzyme and unreacted cellulose were precipitated together to remove the product-the soluble sugar solution-by changing pH. Following this, a new buffer solution was added to the precipitate to dissolve it and resume the reaction. This was repeated several times. The hydrolysis rate of this process increased significantly compared with that of a batch process. Utilization of the reversible soluble-insoluble carrier for immobilizing enzyme is promising, not only for cellulose-cellulase systems, but also for other heterogeneous reaction systems.

Immobilized enzymes as chromatographic phases for HPLC: the chromatography of free and derivatized amino acids on immobilized trypsin

Abstract: A new HPLC stationary phase was synthesized by the covalent immobilization of the enzyme trypsin (TRYP) on silica. The hydrolytic activity of the immobilized enzyme was 72% of the activity of an equivalent molar amount of free TRYP. The initial chromatographic studies indicate that this phase can be used for chiral separations of enantiomeric O- and N,O-derivatized amino acids which are natural substrates on TRYP and that the stereochemical resolutions are a result of the activity of the enzyme.

Enzymatic electrode and its preparation method

Abstract: An enzymatic electrode, having a homogeneous composition throughout, consists essentially of a uniform intimate admixture of a conducting powder with at least one immobilized enzyme, or with an immobilized enzyme and a mediator agent or a co-enzyme, in a matrix. The electrode is one which has a surface available for direct contact with a substrate without a permeable or semi-permeable intermediate membrane or membranes. The enzymatic electrode is prepared, e.g., by intimately admixing (a) a homogeneous paste or matrix (resulting from an intimate admixture of a conducting powder with a binder), (b) at least one enzyme and (c) an enzyme crosslinking solution, and then incorporating therein a mediator agent and/or a co-enzyme.

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation.

Abstract: Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.

Cellulase immobilization on Fe3O4 and characterization.

Abstract: A successful procedure for attaching cellulase to 45 microm iron oxide particles with a high-molecular-weight (1000) ligand has been developed. Mass and activity balances were calculated for immobilized enzymes with different loadings. The highest specific activity of the immobilized enzyme was 5.9 mmol glucose/g bound protein/h. The efficacy of retaining enzymatic activity was 128%. The optimum pH was 5.5 compared to 4.0 of the free enzyme. The half-life of the IMC was extended to 272 h compared to 0.77 h of the free enzyme.

Immobilization of a lactase onto a magnetic support by covalent attachment to polyethyleneimine-glutaraldehyde-activated magnetite.

Abstract: A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalently coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 °C. The immobilized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 °C. Galactose competitively inhibited the immobilized lactase preparation (Ki 20 mM). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4–6 h in a stirred batch reactor at 55 °C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the free enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Abstract: Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.

Conductometric transducers for enzyme-based biosensors.

Abstract: The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.

Mechanism of thermoinactivation of immobilized glucose isomerase

Abstract: Irreversible thermoinactivation of immobilized glucose isomerase from Streptomyces olivochromogenes has been mechanistically investigated at the pH-optimum of enzymatic activity (pH 8.0). Ligands (high fructose corn syrup and the competitive inhibitor xylitol) greatly stabilize the immobilized enzyme at high temperatures. At 90 degrees C in the presence of 2M xylitol, irreversible inactivation of immobilized glucose isomerase is caused by deamidation of its asparagine/glutamine residues. On the basis of the data obtained, it appears that the time-dependent decay of glucose isomerase activity in industrial bioreactors is brought about by oxidation of the enzyme's cysteine residue and/or heat-induced deleterious reactions with high fructose corn syrup or its impurities.

Device for liquid chromatography or immobilized enzyme reaction

Abstract: A device for separating molecular components from liquids. A multi-layer microporous membrane assembly is located within the housing (1) with edges of each membrane within the assembly lying adjacent to a barrier. Means (5,6) are provided for substantially preventing flow between the membrane edges and the barrier, so that flow is through the membranes in series. Inlet means (2) direct incoming liquid on to a first surface of the membrane assembly, and outlet means (3) gather and direct outgoing liquid from a second surface of the membrane assembly. The membrane material of the assembly has a ligand or an immobilized enzyme bound thereto for effecting chromatographic separation or an immobilized enzyme reaction.

Detoxification Of 2,4-dichlorophenol By A Laccase Immobilized On Soil Or Clay

Abstract: We investigated the ability of an immobilized fungal laccase from Trametes versicolor to transform 2,4-dichlorophenol (2,4-DCP), an intermediate of pesticide degradation. Immobilization supports were a silt loam soil and several clays, kaolinite, and two types of smectite, referred to as montmorillonite 1 and montmorillonite 2. The majority of the added laccase activity was immobilized to these supports, using 3-aminopropyltriethoxysilane and glutaraldehyde. The catalytic properties of these laccase-clay and laccase-soil complexes toward the substrate, 2,4-DCP, were assessed using radiolabeled tracer and chromatographic techniques. Incubation of 2,4-DCP with laccase caused the formation of oligomers, which are insoluble in aqueous solutions and can, therefore, be removed. The efficiency of the laccase immobilized on kaolinite and soil to remove 2,4-DCP from solution was similar to that of the free enzyme (approximately 95% of the added 14C-2,4-DCP was removed) and greater than that of the enzyme immobilized on montmorillonites 1 and 2 (69 and 42%, respectively). For these last two supports, however, enzymatic removal may not have been responsible for the entire loss of 2,4-DCP, as adsorption on montmorillonites 1 and 2 also contributed to the observed decreases in 2,4-DCP. A noteworthy advantage of the immobilized laccase over the free enzyme was that it could be separated from the reaction mixture and used repeatedly. After repeated 2-h incubation cycles of the immobilized enzyme with 14C-2,4-DCP, the laccase-kaolinite, laccase-montmorillonite 1, and laccase-soil complexes exhibited only a slight decrease in the ability to remove 2,4-DCP. The possibility of nonenzymatic removal of 2,4-DCP was excluded in experiments performed under anaerobic conditions, in which laccase activity was completely inhibited. The effect of a proteolytic enzyme on the free and immobilized laccase was also tested, and, as a result of their resistance to proteolysis, the immobilized enzyme catalyzed the removal of greater amounts of 2,4-DCP than the free enzyme. This stability and certain other characteristics, such as their reusability and their ability to transform xenobiotics as efficiently as the soluble enzyme, should make these laccase complexes useful catalysts of detoxification reactions in soil.

Characteristics of immobilized lipase-catalyzed hydrolysis of olive oil of high concentration in reverse phase system.

Abstract: Candida rugosa lipase immobilized by adsorption on swollen Sephadex LH-20 could almost completely hydrolyze 60% (v/v) olive oil in isooctane. Kinetic analysis of the lipase-catalyzed hydrolysis reaction was found to be possible in this system. Amount of fatty acids produced was linearly proportional to the enzyme concentration of 720 microg/g wet gel. The specific enzyme activity was 217 units/mg protein at 60% (v/v) olive oil concentration. When the initial rate is plotted versus concentration of olive oil, this system did not follow Michaelis-Menten kinetics. Maximum activity was obtained at pH 7, but optimum temperature shifted towards higher one with the increase of olive oil concentration. Among the various chemical compounds tested, Hg(2+) and Fe(2+) inhibited the lipase seriously. As the concentration of olive oil increased, the rate of the hydrolysis also increased, but degree of the hydrolysis was observed to decrease. The supply of water from the inside of the gel to the surface of the gel was the main factor for the control of the rate of hydrolysis in batch hydrolysis. The immobilized lipase was used to hydrolyze olive oil two times. Achievement of chemical equilibrium took a longer time with the addition of water and the degree of hydrolysis decreased in the second consecutive trial. After the second hydrolysis trial, the gels were regenerated in a packed column first by eluting out both residual fatty acids around the gel particles and the accumulated glycerol with ethanol and then with 0.05 M phosphate buffer, pH 7. The immobilized lipase on the regenerated gel showed the same hydrolysis activity as the original one.