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Showing papers on "Immobilized enzyme published in 1984"


Book
01 May 1984
TL;DR: The specificity of enzymes and their ability to catalyze reactions of substrates at low concentrations is of great use in chemical analysis and has been used for analytical purposes for a long time.
Abstract: Although soluble enzymes can be used as excellent reagents for the analysis of inorganic and organic compounds, they face a serious challenge when attempts are made to utilize them in complex matrices, like blood or crude water. Problems center about the effect of activators, inhibitors, other substrates, pH, and temperature on the soluble enzyme. However, upon immobilization most of these effects can be eliminated or minimized. For example, an enzyme with a narrow pH range of 4–6 can be transformed upon insolubilization to a more viable reagent with a broad pH range of 4–10. Also, following immobilization the enzymes are much more stable; they can be heated to 37, 40 or 50°C, with little loss of activity; and the activity persists after several thousand analysis are performed. However, the biggest advantage, analytically speaking, of immobilization, is that the insolubilized reagent becomes a much more selective reagent. No longer do many activators and inhibitors have an effect; only the most powerful can actually attack the enzyme.

242 citations


Journal ArticleDOI
TL;DR: Dans cette electrode, les enzymes sont immobilisees a l'interieur d'une membrane attachee a un detecteur electrochimique de l'oxygene, cosubstrat de the reaction enzymatique.
Abstract: Dans cette electrode, les enzymes sont immobilisees a l'interieur d'une membrane attachee a un detecteur electrochimique de l'oxygene, cosubstrat de la reaction enzymatique. Le modele mathematique presente concerne les phenomenes de reaction et de diffusion ayant lieu a l'interieur de la membrane

166 citations



Journal ArticleDOI
TL;DR: Choline oxidase and cholinesterase were found to retain their activity for 1-2 weeks at room temperature while adsorbed to a commercially available anion-exchange cartridge.

100 citations


Journal ArticleDOI
TL;DR: The immobilized enzyme was tested for its ability to synthesize soluble peptides from N‐acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water–water‐miscible organic solvent mixtures and it was found that the yield of peptide increased with increasing concentration of organic cosolvent.
Abstract: alpha-Chymotrypsin was immobilized with a high coupling yield (up to 80%) to tresyl chloride activated Sepharose CL-4B.The immobilized enzyme was tested for its ability to synthesize soluble peptides from N-acetylated amino acid esters as acyl donors and amino acid amides as acceptor amines in water-water-miscible organic solvent mixtures. It was found that the yield of peptide increased with increasing concentration of organic cosolvent. Almost complete synthesis (97%) of Ac-Phe-Ala-NH(2) was obtained from Ac-Phe-OMe using a sixfold excess of Ala-NH(2). The rate of peptide formation in aqueous-organic solvent mixtures was good. Thus, 0.1M peptide was formed in less than 2 h in 50 vol% DMF with 0.1 mg immobilized chymotrypsin/mL reaction mixture. The immobilized enzyme distinguished between the L and D configurations of acceptor amino acid amides even in high concentration of nonaqueous component (90% 1,4-butanediol). The effect of temperature was studied. It was found that both the yield of peptide and the stability of immobilized enzyme increased when the temperature was lowered. Experiments could be performed at subzero temperatures in the aqueous-organic solvent mixtures resulting in very high yield of peptide. After three weeks continuous operation at 4 degrees C in 50% DMF, the immobilized enzyme retained 66%of its original synthetic activity. The activity of the immobilized enzyme was better conserved with a preparation made from agarose with a higher tresyl group content compared to a preparation made from a lower activated agarose, indicating that multiple point of attachment has a favorable effect on the stability of the enzyme in aqueous-organic solvent mixtures. The major advantage of using water-miscible instead of water-immiscible organic solvents to promote peptide syntheses appears to be the increased solubility of substrates and products, making continuous operation possible.

89 citations


Journal ArticleDOI
TL;DR: Commercially available lactate oxidase from Mycobacterium smegmatis is immobilized on a nylon net which is fixed on an oxygen probe to provide a simple l -lactate sensor and effects of pH, temperature and different buffers are described.

83 citations


Journal ArticleDOI
TL;DR: In this paper, a crude mixture of N-acetylmannosamine and Nacetylglucosamine has been used to synthesize N -acetylneuraminic acid on 2.8 millimoles scale.

82 citations


Journal ArticleDOI
TL;DR: In this paper, a hybrid sensor for inorganic phosphate and fluoride detection was developed by coupling a potato (Solanum tuberosum) tissue slice and immobilized glucose oxidase with a Clark oxygen electrode, based on the inhibition by either ion of potato acid phosphates catalyzed glucose 6phosphate hydrolysis.
Abstract: A biosensor for inorganic phosphate and fluoride has been developed by coupling a potato (Solanum tuberosum) tissue slice and immobilized glucose oxidase with a Clark oxygen electrode. Measurement is based on the inhibition by either ion of potato acid phosphates catalyzed glucose 6-phosphate hydrolysis. The precision is 1.7% and 6.5% and the lower detection limit 2.5 X 10/sup -5/ M and 1 X 10/sup -4/ M for phosphate and fluoride, respectively. For phosphate determination the hybrid sensor is stable for 28 days or 300 assays. With a higher limit of detection the sensor can be applied in a commercial enzyme electrode based device. Its application for phosphate determination in fertilizer and urine samples is described.

77 citations


Journal ArticleDOI
TL;DR: The construction and use of a filtration system with milliseconds time resolution is described here and the use of this rapid-filtration system is illustrated for time-resolved measurements of calcium binding and transport by sarcoplasmic reticulum Ca-ATPase and of the initial phase of ADP transport by the ADP/ATP carrier of intact mitochondria.

70 citations



Book ChapterDOI
TL;DR: Histone H1, a potent activator of automodification of soluble enzymes, inhibits markedly the automodization of the immobilized enzyme in the presence of Mg2+.
Abstract: Publisher Summary This chapter focuses on assay, purification, and properties of immobilized poly(ADP-ribose) synthetase. It is a nuclear enzyme that catalyzes the transfer of ADP-ribose moiety of NAD to an acceptor protein and then to this protein-bound ADP-ribose; the former reaction (initiation) produces an ADP-ribosyl carboxylate ester of protein, whereas the latter reaction (elongation) produces a long polymer of ADP-ribose linked by ribosyl-ribose glycosidic bonds. The enzyme undergoes automodification, serving not only as a catalyst but also as an acceptor. The assay is based on the quantification of [14C]ADP-ribose incorporated into acid(20% CCl3COOH)-insoluble material from [adenine-14C]NAD. Immobilized enzyme has a specific activity of about one-fourth to one tenth of that of the original enzyme; the value fluctuates among the preparations as well as with the assay conditions used. DNA is almost absolutely required for the activity. Histone H1, a potent activator of automodification of soluble enzymes, inhibits markedly the automodification of the immobilized enzyme in the presence of Mg2+. Also the intrinsic NAD glycohydrolase activity is higher in the immobilized enzyme compared with soluble enzymes.

Journal ArticleDOI
TL;DR: Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed, and titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.

Journal ArticleDOI
TL;DR: In this article, an electrocatalytic steady-state current for the oxidation of D-glucose was observed using this electrode in the presence of p-benzoquinone as an electron transfer mediator.
Abstract: Glucose oxidase was immobilized on the surface of a graphite electrode by irreversible adsorption. An electrocatalytic steady-state current for the oxidation of D-glucose was observed using this electrode in the presence of p-benzoquinone as an electron transfer mediator. The electrocatalytic current at 0.5 V vs. SCE was analyzed as a function of the concentrations of D-glucose and p-benzoquinone, and the maximum current, Ismax, and the Michaelis constants (K1 and K2 for D-glucose and p-benzoquinone, respectively) of the electrocatalysis were determined. The dependence of the current on the electrode potential, pH, and temperature was also investigated. The results indicate that the kinetics of the immobilized enzyme are essentially the same as those of the enzyme in the solubilized state. The effect of various electron transfer mediators on the electrocatalytic current was also examined and evaluated in terms of Ismax, K1, and K2 values.

Journal ArticleDOI
TL;DR: In this article, the dispersion of turnover rates in reacting immobilized enzyme gel spheres of a continuously stirred tank reactor is evaluated based on flow-through microfluorimetry and is exemplified with β-galactosidase immobilized on Sepharose 4B, with resorufin-β- d -galactopyranoside as a new fluorogenic substrate.

Journal ArticleDOI
TL;DR: In this article, a polyenzymatique immobilisation of pepsine, trypsine, chymotrypsine and peptidase intestinale de porc is presented.
Abstract: Developpement d'un systeme polyenzymatique immobilise sur lit de verre poreux et comprenant pepsine, trypsine, chymotrypsine et peptidase intestinale de porc. Application a l'evaluation de la valeur nutritive des proteines alimentaires

Journal ArticleDOI
TL;DR: A novel biosensor, biophotodiode, is proposed for the determination of substances of biochemical and clinical importance that is a unique combination of a photodiode and matrix-bound peroxidase.
Abstract: A novel biosensor, biophotodiode, is proposed for the determination of substances of biochemical and clinical importance. The biophotodiode is a unique combination of a photodiode and matrix-bound peroxidase. Since the enzyme catalyzes the luminescent reaction of the luminol-H2O2 system, this assembly is effective on the determination of hydrogen peroxide. The photocurrent of the sensor is correlated with the hydrogen peroxide concentration ranging from 1mM to 10 mM. A biophotodiode for glucose is also constructed by assembling a glucose oxidase-peroxidase bienzyme membrane and a photodiode.

Journal ArticleDOI
TL;DR: The results showed a noticeable improvement over previous reports in which tyrosinase was immobilized for L‐dopa production, and the stabilization was enhanced by the presence of ascorbate.
Abstract: Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.


Journal ArticleDOI
TL;DR: Several β- d -glucosidase-phenolic copolymers were synthesized and three examined in detail: those containing l -tyrosine, pyrogallol and resorcinol as discussed by the authors.
Abstract: Several β- d -glucosidase-phenolic copolymers were synthesized and three examined in detail: those containing l -tyrosine, pyrogallol and resorcinol. These copolymers were similar to naturallyoccurring soil humic-enzyme complexes in many ways: E 4 /E 6 ratios, C, H, N and S content and IR spectra. The enzyme activity of the copolymers showed varying degrees of resistance to proteolysis, organic solvents, and storage at high temperatures. All immobilized enzymes had increased K m values and decreased V max values in comparison with soluble β- d -glucosidase (9.3 mM, 190μmol p -nitrophenol mg −1 h −1 ); the β- d -glucosidase-resorcinol copolymer was the most active (10.5 mM, 104μmol p -nitrophenol mg −1 h −1 ). β- d -Glucosidase activity was completely resistant to protease when the copolymer was fixed to bentonite clay but V max values were reduced further (e.g. β- d -glucosidase-resorcinol-bentonite complex, 58.5μmol p -nitrophenol mg −1 h −1 ). On addition to soil, soluble β- d -glucosidase was rapidly inactivated (38% loss in 3 days, 80% loss in 21 days) whereas β- d -glucosidase-resorcinol/pyrogallol and β- d -glucosidase-L-tyrosine copolymers were comparatively stable (no loss in 9 days, 25% and 44% loss in 21 days). It is suggested that the copolymerization of enzyme during humic matter formation is a major factor leading to the stabilisation of soil enzymes and that adsorption and entrapment are comparatively insignificant.

Journal ArticleDOI
TL;DR: Direct microscopic examination of FITC‐labelled protein in sectioned Sepharose particles and indirect activity–loading studies with activated carbon–enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates.
Abstract: Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.

Journal ArticleDOI
TL;DR: A simple method is described to obtain pure prunin in high yield from naringin with the help of this immobilizate.
Abstract: Immobilized naringinase can be converted to a preparation showing only rhamnosidase activity by treatment with 0.1M glycine-NaOH buffer, pH 12. A simple method is described to obtain pure prunin in high yield from naringin with the help of this immobilizate.

Journal Article
TL;DR: The results on glucose and uric acid correlated satisfactorily with those obtained by other well-established methods, and the present method was hardly affected by ascorbic acid, while the peroxidase-linked colorimetric method is usually influenced significantly by as Corbenic acid.
Abstract: A method for the flow injection analysis of glucose and uric acid in serum using immobilized enzymes in column form and chemiluminescence detection is described. The method is based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide which is produced by the action of the respective oxidases on glucose and uric acid. Glucose or uric acid in serum were determined with 1 microliter of the sample at a speed of 120 samples/h without carryover and at an assay time of approximately 10 s. The immobilized glucose oxidase column measured only 1.0 X 5 mm, and the immobilized uricase column 1.0 X 20 mm. The present method gave perfect linearity of the data up to 4.0 g glucose per liter or 0.10 g uric acid per liter with satisfactory precision, reproducibility, and accurate reaction recoveries. Furthermore, the present method was hardly affected by ascorbic acid, while the peroxidase-linked colorimetric method is usually influenced significantly by ascorbic acid. Both column reactors showed good operational stability for a 2-month period, during which time they were repeatedly used for analyses over 2000 times. The results on glucose and uric acid correlated satisfactorily with those obtained by other well-established methods.

Journal ArticleDOI
TL;DR: The coimmobilized preparation was superior to a combination of separately immobilized biocatalysts, however, in this preparation, one‐half the enzyme activity was lost within a week when incubated at the operational temperature in the absence of substrate.
Abstract: The efficiency of ..beta..-glucosidase and Saccharomyces cerevisiae in directly converting cellobiose to ethanol was studied for various combinations of the two catalytic species, both free and immobilized, in order to elucidate the advantages of using a coimmobilized system. The coimmobilized preparation was superior to a combination of separately immobilized biocatalysts. However, in this preparation, one-half the enzyme activity was lost within a week when incubated at the operational temperature in the absence of substrate. In continuous experiments, an 80% conversion of cellobiose to ethanol was obtained using the coimmobilized preparation, compared to 40% using separately immobilized biocatalysts when applying a dilution rate of 0.1 h/sup -1/ in a packed bed reactor. The immobilized biocatalysts showed no decline in productivity during two weeks of continous operation.

Journal ArticleDOI
TL;DR: Under controlled laboratory conditions, it was possible to correlate inhibition of cholinesterase activity with intramembranal pH shifts induced by substrate hydrolysis and this type of sensor could be used for screening purposes in the detection of pesticides in water pollution control.
Abstract: A simple and reliable method based on cholinsetersee inhibition Is proposed to detect organophosphate pesticides in water. The potenflometrlc method to measure enzymatic activity was applied to an immobilized chollneeteraae film coupled directly to a flat glass pH electrode. Under controlled laboratory conditions, It was possible to correlate inhibition of chollnseterese activity with intramembranal pH shifts Induced by subetrate hydrolysis. Measurements can be performed with such an enzyme electrode system In real time by monitoring the inhibition process, or after Incubation of the enzymatic film. Technical grade compounds of methylparathlon, azinphosethyl, and mevinphos were used as examples, and detected from ppm to several ppb after oxidative treatment. The sensitivity of the enzyme sensor depends on the Inhibitory power of the pesticide molecules, and therefore determines additional toxicity Information unavailable from other physico-chemlcal methods. This type of sensor could be used for screening purposes in the detection of pesticides in water pollution control and Is intended to be complementary to existing analytical methods. methods by developing automatic systems (6) and rapid determination (7), or by testing different cholinesterase sources to enhance selectivity and sensitivity to pesticides (8). This is because cholinesterases possess some unique characteristics: a relative specificity for pesticides and a great amplifying capacity related to the high enzyme turnover. The cholinesterase inhibition method can be considered an indirect biological assay in which it is not the pesticide itself that is detected (as in the other physicochemical methods), but rather the toxicological effect through the inhibitory power of the pesticide. The cholinesterase inhibition method can be related to, as well as complement, toxicological tests such as the LD,o or CL~0. The purpose of this study was to develop a simple and reliable method of pesticide detection that could be used for quality control tests in water treatment processes. The objective was to place an easily operated device for pesticide monitoring at the disposal of water distributors. This work was made possible by the availability of an acetylcholine electrode described previously (9), which consisted of an immobilized cholinesterase membrane coupled to a pH sensor.

Journal Article
TL;DR: The results suggest that using a combination of immobilized proteases in concert gives essentially total hydrolysis of protein substrates in a time period comparable to conventional acid Hydrolysis methods.
Abstract: Pronase, proteinase K, carboxypeptidases A and B, aminopeptidase M, intestinal mucosa exopeptidases and prolidase, immobilized to derivatized controlled-pore glass beads, were used in a study of total enzymic hydrolysis of proteins. The combined use of immobilized enzymatic and acid hydrolysis, for assessment of protein quality, will give a more accurate chemical score than that afforded by acid hydrolysis alone. Amino acid analysis of enzymic hydrolysates of native protein substrates (beta-lactoglobulin and insulin) yielded 92% of the theoretical values and 103% of the values observed for standard acid hydrolysates. These results suggest that using a combination of immobilized proteases in concert gives essentially total hydrolysis of protein substrates in a time period (18-24 h) comparable to conventional acid hydrolysis methods.

Journal ArticleDOI
TL;DR: Optimal conditions were found, for enzymatic synthesis of the dipeptide, N-acetyl-L-tryptophanyl- L-leucine amide in the biphasic system water - ethyl acetate, and the enzyme practically did not inactivate and may be used repeatedly.

Journal ArticleDOI
TL;DR: In this article, a chemically modified reticulated vitreous carbon electrode substrate was constructed and compared to a saturated calomel reference electrode, and the response was shown to be linear with concentration, with a sensitivity of about 400 nA mM−1.

Journal ArticleDOI
TL;DR: The enzyme converted starch to cyclodextrins without significant loss of activity under the conditions of continuous reaction for about two weeks by using the column system and the optimum pH of immobilized enzyme was 9.0.
Abstract: Cyclomaltodextrin glucanotransferase [1,4-alpha-D-glucan-4-alpha-D-(1,4-alpha-D-glucano)-transferase (cyclizing), E.C.-2.4.1.19] of an alkalophilic Bacillus sp. No. 38-2 (ATCC 21783), which contains three types of enzymes (acid, neutral, and alkaline enzymes), was immobilized on synthetic adsorption resin. No distinguishing changes in pH or thermal stabilities of enzyme were observed due to the immobilization. Since acid-enzyme activity had disappeared, the optimum pH of immobilized enzyme was 9.0. Optimum temperature for the enzyme activity changed from 50 to 55 degrees C. The enzyme converted starch to cyclodextrins without significant loss of activity under the conditions of continuous reaction for about two weeks by using the column system (60 degrees C at pH 8.0). About 63% of soluble starch solution [4% (w/v)] was changed to cyclodextrins, as tested so far.

Journal ArticleDOI
TL;DR: In this article, a new method was developed which uses α-chymotrypsin (E.C.21.4.1) immobilized in a liquid surfactant membrane emulsion.

Journal ArticleDOI
TL;DR: Direct evidence was obtained for the existence of two distinct forms of active α‐chymotrypsin immobilized on CNBr‐activated Sepharose 4B and the specific activity of the more constricted immobilized enzyme active form was shown to be approximately 15 times smaller than that of the other class of immobilized enzymes molecules with an indole EPR spectrum similar to that of chymotypsin in solution.
Abstract: Direct evidence was obtained for the existence of two distinct forms of active alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectra of five different spin-labeled immobilized enzyme formulations in the presence of indole were all resolved into the same two spectral components. Both subpopulation spectra were approximately identified experimentally, and the subpopulation exhibiting greatly restricted spin-label motion was shown also to be relatively inaccessible to solvent. Using overall specific activity data and subpopulation fractions from EPR spectral analysis, the specific activity of the more constricted immobilized enzyme active form was shown to be approximately 15 times smaller than that of the other class of immobilized enzyme molecules with an indole EPR spectrum similar to that of chymotrypsin in solution. Variations in overall specific activity of formulations with different loadings and different supports results entirely from changes in the proportions of the same two subpopulations of immobilized enzyme molecules.