Showing papers on "Immobilized enzyme published in 1976"

Interfacial Enzyme Kinetics of Lipolysis

Abstract: The growing interest in the mechanisms controlling membrane-bound enzymes has incited a number of biochemists to study in more detail certain aspects of heterogeneous catalysis. In the biological membrane, "immobilized" enzymes act on water-soluble or water-insoluble substrates. Because of difficulties encountered in the purification of these proteins, our knowledge of their kinetic behavior is still very scanty. This explains why most experimental approaches used so far deal with model systems based on the combination either of (a) an immobilized enzyme + soluble substrate or (b) a soluble enzyme + insoluble substrate. The former system ami its kinetic implications have been discussed in reviews by McLaren & Packer ( 1) and Thomas (2, 2a), as well as in the proceedings of a symposium devoted to this subject (3). In this review, we limit ourselves to studies based on approach b. It will come as no surprise that most of the work reported so far deals with lipolysis. Theoretically, heterogeneous catalysis might be studied also using polyamino acids, polynucIeotides, or polysaccharides as substrates in insoluble form. These compounds are of biological interest and soluble enzymes hydrolyzing them are available in a pure state. Some progress has been made using mono­ molecular surface films of proteins; these polymers are more difficult to manage in vitro than lipids. The naturally occurring (phospho) lipids are important building stones of the biological membranes. They are water insoluble and spontaneously form lipid-water interfaces such as monomolecular films, bilayers, emulsions, liposomes, or micelles. In addition, a number of soluble enzymes that play an important role in biological events, such as digestion, membrane and cell fusion, lipid biosynthesis, pinocytoses, etc., are known and have been isolated in pure form. The fact that it is the

[29] Kinetic behavior of immobilized enzyme systems

Abstract: Publisher Summary This chapter discusses the kinetic behavior of immobilized enzyme systems. In the case of immobilized enzymes, the kinetic behavior can be controlled by both microenvironmental and mass-transfer effects. It is useful to distinguish between intrinsic rate parameters of the enzymic reaction—that is, the kinetic parameters characteristic of the native enzyme in solution. The techniques commonly employed for the characterization of diffusional resistances and the evaluation of the intrinsic or inherent kinetic parameters of immobilized enzyme systems are classified in three main groups: direct determination of kinetic and transport parameters, variation of substrate concentration, and variation of characteristic support dimensions.

Immobilization of enzymes to agar, agarose, and Sephadex supports.

Abstract: Publisher Summary This chapter discusses the immobilization of enzymes to agar, agarose, and Sephadex supports From a purely economic point of view, cellulose and starch are perhaps the most attractive starting materials for immobilization Their chemistry is well understood In spite of these facts, they have certain disadvantages that make them less suitable for granular immobilized enzymes, the most serious of which are an improper macroporous structure and a susceptibility to microbial disintegration Dextran in cross-linked form, Sephadex, under certain conditions is superior to cellulose and starch Enzymes fixed to Sephadex exhibit higher relative activity than the corresponding cellulose-bound enzymes Presumably, the microenvironment within Sephadex gels is less disruptive for the exertion of catalytic action than in fibrous cellulose with its microcrystalline regions Starch is more easily attacked by microorganisms than is Sephadex Agarose has been found to be very well suited as a matrix for the production of biospecific adsorbents, although less extensively purified agar may be a better choice for economic reasons in technical applications

Reversibly precipitable immobilized enzyme complex and a method for its use

Abstract: A process for using and preparing a reversibly soluble enzymatically active polymer enzyme product which consists of an enzyme covalently bonded to a water soluble organic polymer selected from polyacrylic acid, dextran, carboxy methyl cellulose, and polyethylene glycol which have carboxyl or amino side groups that impart to the complex its reversible solubility

5-Aminolaevulinic acid dehydratase: structure, function, and mechanism

Abstract: delta-Aminolaevulinic acid dehydratase catalyses the synthesis of porphobilinogen. The enzyme has a molecular mass of 285000 and is composed of eight similar subunits of molecular mass 35000. The N-terminal amino acid is acylated, and the number of peptides found on tryptic digestion equals the number of lysine and arginine residues per mass of 35000. The eight subunits are apparently arranged at the corners of a cube and therefore have dihedral (D4) symmetry. The bovine liver enzyme which has been cystallized contains 4--6 atoms of zinc per mole of enzyme. The apo-enzyme obtained on prolonged hydrolysis can be reactivated by the addition of zinc or cadmium ions. The dialysed enzyme must be first treated with dithiothreitol. There are two very active SH groups in a total of 6--7-SH groups per subunit. The substrate forms a Schiff base with the epsilon-amino group of a lysine residue. Reduction of the Schiff base with NaBH4 should reveal the number of active sites per mole of enzyme. It appears that only four of the eight subunits form a Schiff base with the substrate indicating that the enzyme exhibits the phenomenon of either half-site reactivity or negative cooperativity. The enzyme appears to have a strong subunit-subunit interaction for an immobilized preparation remained stable for at least a month. An immobilized enzyme preparation was treated in a manner so that it dissociated into tetramers. Both the eluate and protein still attached to the Sepharose on a column were enzymically active. The bound enzyme could not reassociate under assay conditions but still contained about 50% of the original enzyme activity. It would seem that the enzyme is active when composed with less than eight subunits.

Preparation of pharmaceutical compounds by immobilized enzymes and cells.

Abstract: Publisher Summary This chapter discusses the preparation of pharmaceutical compounds by immobilized enzymes and cells Immobilized enzymes and cells can be used to prepare many pharmaceutical compounds Industrial exploitation of enzymes and microorganisms traditionally has been accomplished by using intact microorganisms or soluble cell free enzyme preparations These processes are not very efficient because the catalysts (enzymes or microorganisms) are used for just one batch reaction or fermentation Additional uses are not feasible because: enzymes and cells are relatively unstable and may lose activity during a fermentation or reaction; and conventional recovery methods are either expensive or cause denaturation and loss of catalytic activity If enzymes and microorganisms are to be reused effectively, their stability must be improved, and inexpensive non-destructive recovery methods must be developed Immobilization offers a means of achieving both objectives

Glucose isomerase immobolized on porous glass

Abstract: Partially purified glucose isomerase from a Streptomyces species was immobilized on porous glass particles and studied for various characteristics concerning its use as an industrial catalyst. The activities were investigated in relation to the reaction parameters and the enzyme deactivation was studied systematically under various reaction conditions. The half-life of the immobilized enzyme was found to exceed 200 days at 50°C. The rate equation of the reversible glucose ⇄ fructose reaction was derived and the kinetic constants were determined. The rate equation was found to be in good agreement with experimental data for both forward and reverse reactions. The degree of diffusional effects was experimentally measured and theoretically analyzed.

Pilot plant production of glucose with glucoamylase immobilized to porous silica.

Abstract: Glucoamylase was immobilized to porous silica and its kinetics and stability were observed with acid- and alpha-amylase-hydrolyzed dextrin as feed. The enzyme was found to be extremely stable in both laboratory and pilot plant operations. When the feed had been previously only lightly hydrolyzed, pore diffusion limitation caused appreciable decreases in glucose production rate. The severity of starch hydrolysis to dextrin markedly affected ultimate glucose yields. The diffusional gradients present in the carrier pores caused the immobilized enzyme to yield lower glucose concentrations than the free enzyme at similar feed conditions.

Enzyme Thermistor Assay of Cholesterol, Glucose, Lactose and Uric Acid in Standard Solutions as Well as In Biological Samples

Abstract: A special enzyme thermistor device with a probe and a reference thermistor is described. Linear relationships between the temperature changes, At, measured with this device and the concentration of substrates applied were obtained. The substrates tested were cholesterol, glucose, lactose and uric acid in standard solutions as well as in biological samples.

Immobilized enzymes on chitosan columns: alpha-chymotrypsin and acid phosphatase.

Abstract: alpha-Chymotrypsin and acid phosphatase have been immobilized on chitosan, a polyaminosaccharide, without using any intermediate reagent; the immobilized enzymes are active and their activity is much higher than for chitin-immobilized enzymes. The best pH conditions for operating chitosan columns have been determined and columns have been used to transform substrates in large amounts, with no decrease of activity or enzyme losses. Due to the nonconvalent interaction between chitosan and enzymes, the pure and active enzymes can be eventually recovered from the columns. The effects of metal ions, aldehydes, and salts are reported and discussed. Applications are foreseen in the food and biomedical sciences and industries.

Immobilization of proteins with polyurethane polymers

Abstract: A protein which can be an enzyme is immobilized by: (a) admixing the protein and an isocyanate-capped liquid polyurethane prepolymer in the absence of water to form a resulting mixture (an intermediate product); and (b) forming the intermediate product by reacting it with water to form a polyurethane foam comprising the immobilized enzyme. When certain proteins in sufficient amount are mixed with the prepolymer in the absence of water the resultant protein prepolymer mixture will solidify to produce a solid non-foamed product containing a protein immobilized therein. Initially mixing the protein and prepolymer in the absence of water results in immobilization of a substantially greater amount of protein than when water is present.

Physical characteristics of porous cellulose beads as supporting material for immobilized enzymes

Abstract: Cellulose beads prepared in this report have high porosity (75-80%) and evenly distributed pores. The pore size is about 1000 A. The cellulose beads are physically strong and contain large amounts of reactive groups, making them suitable for use as carriers for immobilized enzymes.

[11] Adsorption and inorganic bridge formations

Abstract: Publisher Summary This chapter describes the adsorption and inorganic bridge formations. Adsorption is the adhesion of an enzyme to the surface of a carrier that has not been specifically functionalized for covalent attachment. Adsorption appears in the author's opinion to be the most economical procedure for immobilizing an enzyme on a carrier (on hydrophobic adsorption of enzymes and on aminoacylase ad sorbed to DEAE-Sephadex.) Although the immobilization procedure may be simple, the reactions involved in adsorption are complex and involve multiple types of bond formations. The stability of an adsorbed enzyme will depend upon the additive strength of those bonds formed under the conditions of immobilization and those bonds maintained under the application conditions that are finally employed for the immobilized enzyme. It is discussed that there are three major points to be considered in the optimization of a carrier for adsorption of enzymes: (1) preconditioning the surface with respect to pH, (2) preconditioning the surface with respect to cofactors, and (3) selection of the optimum pore diameter for immobilizing the enzyme.

[25] Assay procedures for immobilized enzymes

Abstract: Publisher Summary This chapter presents assay procedures for immobilized enzymes The most common and often the easiest way of following an enzyme reaction is to use spectrophotometry, thereby studying changes in absorbance caused by consumption of substrate or generation of product The systems to which spectrophotometric measurements can be applied may be conveniently divided into two main groups: that in which the enzyme matrix is in the light path and that in which it is not Second method discussed is titrimetric methods; the assay is easy to handle, accurate, and reproducible Because, however, it is sensitive to exchange of carbon dioxide with the surroundings, the reaction should preferentially be run under nitrogen In the presence of strong buffering substances, for example, charged matrices or buffers, titration is impossible The sensitivity is lower than, for example that obtained from using spectrophotometric methods when a suitable chromophore is present Titrimetry is a realistic alternative in cases with excess of substrate and good enzyme activities, provided the reaction consumes or produces protons In addition, in a recent study α-galactosidase, polarimetry was applied to continuous monitoring of the reaction

Immobilized-enzyme reactors.

Abstract: Publisher Summary The development the methods for the immobilization has produced novel forms of biochemical catalyst, which can be used in reactors to carry out conversions of industrial interest. Many different types of reactor have been employed with immobilized enzymes. These may be classified according to mode of operation and the flow pattern in the reactor. The most common system is the stirred tank normally operated as a batch system, but also semi continuously by repeatedly drawing off part of the reaction liquor at intervals and refilling with fresh substrate solution. The chapter focuses on the problems that may arise in designing and operating an immobilized enzyme reactor. Although these problems may seem daunting, it should be emphasized that many immobilized enzyme reactors are being operated successfully both in the laboratory and in industry. This type of catalyst system can be expected to take its place among the important types available for practical application.

A Split-Flow Enzyme Thermistor

Abstract: The split-flow system is comprised of two identical micro-columns, one of which contains an immobilized enzyme preparation, the other an inert support material. The heat produced in each column on introduction of a sample is measured with thermistors placed in these columns. The use of a reference column virtually eliminates the influence on the measurements of artifactual signals as unspecific heat, i.e., heat not produced by the enzymic reaction. The performance of the split-flow enzyme thermistor at a variety of pH's, ionic strengths or viscosities associated with the sample has been investigated and compared with previously described alternative enzyme thermistor arrangements. In this comparative study glucose at a concentration of 5 · 10−4 M was used throughout. On passage through the imnobilized glucose oxidase preparation this solution gave rise to a heat change At of about 0.01°C. The insensitivity of the system described herein towards such variations makes it particularly suitable for t...

The Chemistry of Enzyme Immobilization

Abstract: Publisher Summary Enzymes immobilized on or within a solid matrix by conjugation with synthetic water-insoluble polymeric supports can serve as reusable and removable highly specific reagents, which possess improved storage and operational stability. Continuous large-scale processes can be carried out in immobilized-enzyme reactors. Immobilized enzymes in conjunction with a detector have led to the development of highly specific electrode systems and similar analytical and monitoring devices. Immobilized enzymes are being explored for clinical application in the form of extracorporeal shunts or microcapsules. The clarification of some of the principles underlying the kinetic behavior of immobilized enzyme systems, that is, effects of the microenvironment imposed by the chemical nature of the support material and the effects of diffusional restrictions on the translocation of substrate and product, make possible the modulation of the properties of a bound enzyme by its conjugation to a support of predetermined chemical and physical characteristics.

Studies on immobilized trypsin in high concentrations of organic solvents

Abstract: It is well known that the rate of enzymic reactions involving ions or dipolar molecules can be modified or effected by the ionic strength of the reaction medium. It has also been shown that the addition of organic solvents to the reaction medium can also change the reaction rates of enzymes (1,2). Recent studies by Royer (3) have indicated that if trypsin is immobilized on control pore glass in the presence of BAEE, several changes occur in the kinetic parameters of the immobilized enzyme. We have attempted in this study to extend the observations of Royer in aqueous systems to systems containing high concentrations of organic solvents of differing dielectric constant and to determine the effect of these solvents on the activity of the immobilized trypsin.

Characterization of sand as a support for immobilized enzymes.

Abstract: The potential of sand as a support for immobilized enzymes was investigated by preparing alkylamine sand and devising methods to measure the total number of amine groups present and the fraction available for immobilization of enzymes. Alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1.) and lactate dehydrogenase (L-lactate:NAD oxidoreductase, EC 1.1.1.27) were immobilized on alkylamine sand, and the stability of the immobilized protein and dehydrogenase activity was measured. Urease (urea amidohydrolase, EC 3.5.1.5) was also immobilized on sand to test the applicability of these methods to larger scale immobilizations. Results suggest that sand shows promise as a support for immobilized enzymes.

Immobilization of enzymes based on hydrophobic interaction. II. Preparation and properties of an amyloglucosidase adsorbate

Abstract: Amyloglucosidase from Aspergillus niger (α-1,4 and 1,6 glucan glucohydrolase, EC 3.2.1.3) was immobilized through adsorption onto a hexyl–Sepharose, containing 0.51 mol hexyl-group per mole of galactose. The adsorption limit of the carrier with respect to this enzyme was about 17 mg per gram wet conjugate. The retention of activity upon immobilization was high, varying from essentially full activity at low enzyme content down to 68% at the adsorption limit. The immobilized preparation, as well as the soluble enzyme, showed apparent zero order kinetics within 60% of the substrate's conversion limit. Product inhibition of the soluble enzyme showed a k1 of 5 · 10−2M. In the presence of 3M NaCl, adsorbates were formed more rapidly and with a higher yield of immobilized protein, but with lower specific activity. Conjugates resulting from adsorption of amyloglucosidase in identical concentrations, but at different salt contents, showed comparable activities and operational stabilities. Continuous operation for three months reduced conjugate activity to 40%. The thermal stability of the adsorbate was inferior to that of the soluble enzyme, but was noticeably enhanced in the presence of substrate.

Immobilized glucuronosyltransferase for the synthesis of conjugates.

Abstract: Partially purified rabbit liver UDPglucuronosyltransferase is immobilized on agarose by the cyanogen bromide activation method. Both soluble and matrix-bound enzyme preparations display very similar Km and pH optimum. The storage stability of the immobilized enzyme at 4 degrees is 5-10 times improved over the soluble preparations. The agarose-bound UDPglucuronosyltransferase is successfully used in the synthesis of p-nitrophenyl glucuronide in an overall yield of 50-70%. The matrix-bound enzyme is reusable over an extended period of time and offers an easy and convenient synthetic tool for various drug glucuronides.

Solid-phase synthesis of drug glucuronides by immobilized glucuronosyltransferase.

Abstract: Rabbit liver glucuronosyltransferase immobilized on beaded agarose has been used to synthesize glucuronic acid conjugates of meprobamate, diethylstibestrol, bilirubin, borneol, benzioc acid, and p-nitrothiophenol. The immobilized enzyme exhibited a high degree of specificity for UDPGA as cofactor when p-nitrophenol is used as substrate. Other cofactors tested were less effective, all producing less than 10% conjugation relative to UDPGA. The effects on agarose-bound enzyme activity of a variety of cosolvents and emulsifiers have been studied. Ethanol, dimethyl sulfoxide, propylene glycol, and bovine serum albumin are among the cosolvents and emulsifiers which can be used within limited concentration ranges to sulubilize lipophilic substrates for conjugation. Concentrations of calcium and magnesium cations between 1.5 and 10.0mM were found to enhance glucuronosyltransferase activity of the immobilized enzyme.

An immobilized-enzyme flow-enthalpimetric analyzer: application to glucose determination by direct phosphorylation catalyzed by hexokinase.

Abstract: A novel flow-enthalpimetric analyzer is described and its use demonstrated by an analysis in which glucose is determined by its hexokinase-catalyzed phosphorylation reaction. The method depends on measurement of the temperature differential across a column packed with glass-supported immoblized enzyme. Sample volumes of 120 mul can be used to obtain a calibration curve that is linear up to 25 mmol of glucose per liter. A precision (within-day) of 5% is generally observed in the optimum concentration range where glucose is quantitatively phosphorylated. Results by the technique correlate reasonably with those by the o-toluidine and the hexokinase/glucose-6-phosphate dehydrogenase methods: Other sugars--including fructose, glucosamine, and mannose--will interfere; galactose does not. The technique is amenable to both routine and emergency analyses.

[41] Enzyme electrodes and solid surface fluorescence methods

Abstract: Publisher Summary This chapter discusses two modern aspects of the analytical uses of immobilized enzymes. Two major techniques used to immobilize an enzyme are (1) the chemical modification of the molecule by the introduction of insolubilizing groups; this technique, resulting in a chemical “tying down” of the enzyme, is in practice sometimes difficult to achieve because the insolubilizing groups can attack across the active site, destroying the activity of the enzyme. (2) The physical entrapment of the enzyme in an inert matrix, such as starch or polyacrylamide gels. Physical entrapment techniques offer advantages of speed and ease of preparation over many chemical methods, except some such as the glutaraldehyde technique. The enzyme electrode and fluorometric analysis by a reagentless solid surface method is also discussed.

Effects of subunit interactions on the activity of lactate dehydrogenase studied in immobilized enzyme systems

Abstract: Rabbit muscle lactate dehydrogenase (LDH) was coupled to Sepharose in such a way that each molecule is expected to be attached via only one subunit. Dissociation of the bound active enzyme by several methods all yielded immobilized subunit derivatives which were inactive. These derivatives were capable of regenerating activity by interacting specifically with subunits in solution formed transiently during renaturation. This ability to peck up soluble subunits is lost fairly rapidly upon storage of the immobilized subunits. Similarly, LDH subunits attached to Sepharose via disulfide bonds were found to be inactive. When these subunits were detached from the matrix by mild reduction with mercaptoethanol, activity was regenerated. The kinetics of this reactivation process suggests that reassociation is required for appearance of activity. All these results can be interpreted as showing that subunit interactions are essential for LDH activity.