Showing papers on "Immobilized enzyme published in 1982"

Enzyme activity in soil: Location and a possible role in microbial ecology

Abstract: The activity of any particular enzyme in soil is a composite of activities associated with various biotic and abiotic components, e.g. proliferating cells, latent cells, cell debris, clay minerals, humic colloids and the soil aqueous phase. The location of the enzyme is at least partially determined by such factors as the size and solubility of its substrate, the species of microorganism, and the physical and chemical nature of the soil colloids. However, enzymes may change location with time, for example, many hydrolases are intracellular sensu stricto but are also found associated with cell debris and clay and organic colloids. There are difficulties in quantifying the various activities, but this may be possible by employing different types of assays, the prudent use of controls and the study of crude enzyme extracts from soil. Enzymes bound to clay and humic colloids (the immobilized or accumulated enzyme fraction) have a residual activity not found in enzymes free in the soil aqueous phase. However, the mere adsorption of enzymes to soil surfaces does not guarantee subsequent activity, and it appears that some mechanism of association with the humic polymer offers the best form of protection, yet permits the retention of enzyme activity. The catalytic activity of extracellular enzymes is discussed and a possible relationship between soil microorganisms, exogenous substrates and immobilized enzymes is suggested.

Electrochemical measuring apparatus comprising enzyme electrode

Abstract: An immobilized enzyme electrode effective in measurement of the substrate concentration of the enzyme and in conversion from enzyme reaction energies into electric energies. The immobilized enzyme, of an oxidase system, such as glucose oxidase, amino acid oxidase, xanthine oxidase or the like and a metal oxide capable of constituting a redox system which is reduced through coupling with these enzyme reactions and is electrochemically oxidized (anodic oxidation) are combined with each other. The use of the enzyme electrode allows the determination quantity of the enzyme substrate as extremely low as approximately 10 -5 to 10 -6 mole/l in concentration.

Immobilization of strictosidine synthase from Catharanthus cell cultures and preparative synthesis of strictosidine.

Abstract: Strictosidine synthase was partially purified from Catharanthus roseus cell suspension cultures and immobilized on CNBr-activated Sepharose. The immobilized enzyme exhibits a thermostability increased 300 fold over that of the soluble enzyme and catalyses exclusively the formation of the 3alpha(S)-isomer, strictosidine. Gram quantities of this biologically active glucoalkaloid, which hitherto had been difficult to synthesize and purify, were prepared.

Theoretical considerations of membrane fouling and its treatment with immobilized enzymes for protein ultrafiltration

Abstract: The ultrafiltration process was modelled in three separate stages with distinctive time constants. It was shown that in the first stage lasting less than 5 s a quasi-steady-state concentration profile is reached on the membrane/solution interface. In the second stage of 1–10-min solute adsorption on the membrane surface including the pores controls the permeation rate. The third stage is governed by a reaction mechanism which produces a surface gel causing flux decline at a slower rate than in the previous adsorption step. This polymerization of the protein to a gel on the membrane was shown to be second order in the interface protein concentrations. A reproducible and inexpensive method has been developed to attach food-grade proteases onto UF membranes by producing a primary adsorbed layer of enzyme which then retards the rate of gel formation on the ultrafilter. This resulted in 25–78% improvement in cumulative permeate yield in a standard 22-h run when processing 0.5% albumin or hemoglobin. The enhanced fluxes with self-cleaning membranes were modelled by incorporating an enzyme activity term to counteract the deposition of gel on the membrane surface and altering the apparent order of the gelation reaction.

Modeling enzyme immobilization in porous solid supports

Abstract: Enzymes are often immobilized on the internal surfaces of porous solid by immersing enzyme‐free particles in a well mixed solution of enzyme. The ensuing impregnation process involves coupled transient mass transfer and surface attachment of enzyme. A mathematical model is employed to explore the influences of process parameters on the amount of enzyme loaded and the distribution of immobilized enzyme within the support particles. Nonuniform loading of the support occurs under some conditions. This is significant since the distribution of enzyme within the support particle influences the overall activity and stability of the immobilized enzyme catalyst. The model developed here may also be used to describe removal of reversibly immobilized enzyme during washing or utilization of the immobilized enzyme catalyst.

Studies on the properties of cellulase enzyme from Aspergillus terreus GN1

Abstract: The cellulase enzyme is produced by cellulolytic microorganisms and is able to degrade cellulose materials. It has assumed greater importance due to its potential use in modifying low-grade roughages, the production of syrups, powering alcohol from cellulosic wastes, and other applications such as single-cell protein production. This paper studies the properties of a cellulase enzyme system from Aspergillus terreus GN1 and the effect of various modulators on its activity. (Refs. 18).

Diffusion resistance and enzyme activity decay in a pellet.

Abstract: The effective enzyme activity decay can be decreased by diffusion limitation in the immobilized pellet. Thiele modulus changes and/or poisoning of various enzyme forms are two phenomena which are influenced by diffusion limitation. This article considers these effects on enzyme decay as applied to glucose isomerase.

Properties of bacterial luciferase/NADH : FMN oxidoreductase and firefly luciferase immobilized onto sepharose.

Abstract: NADH : FMN oxidoreductase and bacterial luciferase have been efficiently coimmobilized onto Sepharose 4B. This luminescent immobilized enzyme system can be used to assay NADH. The assay is rapid and sensitive with a lower limit of detection of 0.2 pmol/assay tube. The intra-assay precision was 3.5% at 2 × 10-5 M and 5.8% at 2 × 10-6 M NADH. Light intensity was proportional to NADH concentration from 0.2 to 1000 pmol. Added serum and certain dehydrogenases were found to be inhibitory; however, inhibition could be eliminated by a combination of heat treatment and dilution. Firefly luciferase has also been immobilized onto both Sepharose 4B and CL 6B. The detection limit for ATP using this immobilized enzyme was 0.2 pmol and the assay was linear from 0.2 to 2000 pmol. The intra-assay precision was 4.8% at 2 × 10-4 M and 3.2% at 1 × 10-5 M ATP. The immobilized enzymes remained fully active when rapidly frozen in the presence of glycerol and DTT. Such preparations could be stored for at least two months with no loss of activity. A variety of different compounds were used to block any remaining reactive groups on the Sepharose following immobilization of the enzymes. Glycine, 2-aminoethanol, and ethylenediamine were examined. The preparations where ethylenediamine was used as a blocking agent exhibited better activity and stability than the others.

The effect of Hofmeister anions and protein concentration on the activity and stability of some immobilized NAD‐dependent dehydrogenases

Abstract: The effect of several factors on the activity and stability of alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and 20-beta-hydroxysteroid dehydrogenase, both free and immobilized on CNBr-activated Sepharose 4B, was investigated. Enzymes were immobilized under different conditions including various degrees of matrix activation, variable amounts of protein, in the presence, or in the absence of, additives (coenzymes, dithiothreitol, salts). Activity recovery was in general satisfactorily high with 20-beta-hydroxysteroid dehydrogenase, low with glyceraldehyde-3-phosphate dehydrogenase, and markedly linked to the concentration of immobilized protein with alcohol dehydrogenase. In the latter case the advantageous stabilizing effect of high enzyme concentrations was notably diminished by the paralled decrease of the effectiveness factor. The effect of high concentrations of anions of the Hofmeister series was examined. It was found that 1M phosphate and 0.5M sulfate dramatically stabilize both free and immobilized enzymes against inactivation by temperature and urea. Km values of apolar substrates were considerably lowered by the two anions while Km values of polar substrates were not affected. In some cases Vmax values also were influenced by high concentrations of these anions. The present results appear of interest particularly in view of enzyme utilization for analytical as well as for preparative purposes. (Refs. 13).

The enzyme thermistor

Abstract: This is a brief review of some applications and a description of the enzyme thermistor, which in effect is a flow-calorimeter designed for routine analysis in clinical chemistry, biochemistry, environmental control, and biotechnology. The heat produced in a small column filled with immobilized enzyme is measured with a thermistor, hence the name enzyme thermistor (1, 2).

The effect of methylacrylate on the activity of glucomylase immobilized on granular polyacrylonitrile.

Abstract: Glucoamylase was immobilized on granular polyacrylonitrile (PAN) and the optimum condition in its immobilization reaction was determined. The effect of the ratio of the imidoester and methylester to the total cyanogen on the activity of the immobilized enzyme was studied. The activity of the immobilized enzyme increased in proportion to the molar number of imidoester and decreased with that of methylester. The K(m) and V(m) values of immobilized glucoamylase which were prepared at various conditions of immobilization were determined. There were opposite trends in K(m)S between glucoamylase immobilized on imidoester-rich support and immobilized on methylester in the support, evidenced as functions of temperature. This suggests that opposite charges in the support, produced by heat deformation of PAN by hydrolysis of methylester, were an influence on the apparent K(m) of immobilized glucoamylase, besides the diffusional limitation.

Characterization and evaluation of Aspergillus oryzae lactase coupled to a regenerable support.

Abstract: A derivative of crosslinked Sepharose, p-(N-acetyl-L-tyrosine azo) benzamidoethyl-CL-Sepharose 4B, was synthesized and used for the selective immobilization of thermostable lactase from Aspergillus oryzae.Preparations of soluble and immobilized lactase were evaluated under initial velocity conditions in a batch process. Immobilization had no significant effect on the pH optimum at 50 degrees C or kinetic parameters at pH 4.5 or pH 6.5 and 50 degrees C. At pH 4.5, the soluble enzyme possessed maximum activity at 60 degrees C and the immobilized at 55 degrees C; at pH 6.5 both showed maximum activity at 55 degrees C. The activation energy, entropy, and enthalpy decreased significantly with immobilization at pH 4.5 but not at pH 6.5. When the immobilized enzyme was placed in a packed-bed reactor, the effect of temperature on activity was altered as reflected by a marked decrease in the thermodynamic parameters of activation at both pH levels. Upon immobilization there was also a dramatic increase in the apparent thermal stability of the lactase, and the mean half-life at 50 degrees C was increased from 7.2 to 13 days at pH 4.5 and from 3.8 to 16 days at pH 6.5.

The production of 6‐aminopenicillanic acid by a multistage tubular reactor packed with immobilized penicillin amidase

Abstract: A theoretical model equation was derived to find the correlation between the conversion and the amount of immobilized penicillin amidase in column. The theoretical values of the conversion were predicted form this correlation and compared with experimental results. It was observed in a column reactor that the pH drop along the column path was linear versus the enzyme loading and that the enzyme activity was also linearly dependent on pH up to 8.0. In order to diminish the effect of pH drop, a continuous two-stage plug-flow reactor (PFR) with pH adjustment between the two columns was used was used in the experiments, and two- and three-stage PFRs were simulated by computer. In the case of the two-stage PFR, the maximum productivity was demonstrated experimentally and theoretically as well. when an equal amount of the immobilized enzyme was packed in both columns. It was also predicted in the tree-stage PFR system that the optimal distributions of enzyme loading in three columns were found to be 1:1:1. It was demonstrated that the increased number of reactors in series could enhance the level of the maximum productivity with a given amount of enzyme loading.

Diffusion and the limiting substrate in two-substrate immobilized enzyme systems.

Abstract: The effects of mass transport resistances on two-substrate immobilized enzyme systems are investigated theoretically. It is shown that the effects of mass transport resistances on the overall reaction rate are related mainly to the transport of the limiting substrate. In the absence of external mass transport resistances, the limiting substrate can be identified by knowing only the ratio of the bulk substrate concentrations, the permeability of the support to the two substrates, and the stoichiometry of the reaction. However, a combination of internal and external mass transport resistances may result in the other substrate becoming limiting. These effects are most significant when the mass transport resistances are high. Applications in the design of enzyme electrodes and chemical reactors are discussed.

Immobilized enzymes as analytical reagents.

Abstract: Immobilized enzymes are becoming increasingly popular as analytical reagents because of their reusability, stability, and sensitivity to many inhibitors that would seriously interfere in assays using soluble enzymes. In this article, some of the kinetic and catalytic effects of immobilized enzymes in analysis will be discussed. The shift of the activity-pH profile curves on immobilization, the changes in temperature dependence. the inhibitor constants (K1). Michaelis constants (Km), and the maximum velocity (Vmax). plus others, will be discussed. Finally, the use of these immobilized enzymes in fluorometric and electrochemical monitoring systems will be shown, and the future of these reagents in various areas will be discussed. A survey of enzyme electrodes will be presented as an example of the use of immobilized enzymes. Application of immobilized enzyme technology to the assay of BUN, glucose, uric acid, amino acids, ethanol. and other metabolites will be discussed.

Fibrous Support for Immobilization of Enzymes

Abstract: There are methods, such as adsorption, trapping, cross-linking and covalent-bonding, available for the immobilization of enzymes. No one method seems to have a clear advantage over the others. Therefore, an attempt was made to develop a poly(vinyl alcohol) (PVA) fibrous support for enzyme immobilization.