Showing papers on "Immobilized enzyme published in 1977"

Transformations of organic compounds by immobilized microbial cells.

Abstract: Publisher Summary This chapter discusses the transformations of organic compounds by immobilized microbial cells. Microbial cells can be immobilized by ionic binding to water-insoluble ion exchangers. However, it is considered that this method is not advantageous, because an enzyme may easily leak out from cells owing to autolysis of cells during continuous enzyme reaction. Microbial cells can be immobilized by cross-linking each other with bifunctional reagents. These immobilized cells showed aspartase activity corresponding to 34.2% of that of intact cells. Further, this chapter attempts the immobilization of E. coli cells by cross-linking with toluene diisocyanate, but active immobilized cells could not be obtained. Microbial cells can be immobilized by entrapping them in a polymer matrix where they are physically restrained. In this method, the following matrixes are employed: polyacrylamide, collagen, cellulose triacetate, agar, alginate, and polystyrene. Further, this chapter compares the stability of aspartase activity of immobilized cells and intact cells.

The immobilization of whole cells

Abstract: The literature of the last three years has seen a renaissance in the use of immobilized enzyme systems made practical by the immobilization of the entire cell. Such systems have proven to have a diversity of application unattainable with isolated pure enzymes and the operation of such systems in continuous reactors has proven to be less costly than more conventional processes based on free intact cells. Thus, whole cell immobilization as a technique should be of expanding practical and academic significance in the future.

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 2. Specific activity and thermal stability of enzymes bound weakly and strongly to Sepharose CL 4B.

Abstract: Ribonuclease A (EC 3.1.4.22) and alpha-chymotrypsin (EC 3.4.21.1) have been covalently coupled, by a varying number of bonds, to Sepharose CL 4B which was activated with different amounts of CNBr. Upon increasing the number (1-8) of points of attachment between the enzyme and the matrix, the specific activities of immobilized ribonuclease A relative to its soluble counterpart decreased from 60 to 15% while the amount of protein coupled increased from 5 to 37 mg per g of sucked gel. Differential scanning calorimetry was used to determine whether the immobilization caused any changes in the physicochemical properties of the enzyme. Ribonuclease A, weakly bound to the matrix, showed almost the same behavior as the soluble enzyme. By contrast strongly immobilized enzyme exhibited a higher transition temperature (by about 5 degrees C) and a broader endotherm. Similar results were found for alpha-chymotrypsin.

Immobilization of enzymes on slowly soluble carriers.

Abstract: The preparation and some properties of microspheres composed of oxidized polysaccharides and some vinyl polymers are described. The microspheres contain immobilized enzyme and can be slowly solubilized in water solutions, thereby releasing active a enzyme into the surrounding medium. The kinetic characteristics of the immobilized enzyme bound with a fragment of matrix after complete solubilization are unchanged, but the enzyme exhibits high thermostability. These preparations could have a wide range of medical applications, e.g., to form a drug "depot" directly in an affected organ.

Nonporous magnetic materials as enzyme supports: studies with immobilized chymotrypsin.

Abstract: Chymotrypsin has been immobilized to several nonporous magnetic materials. Nickel particles were considered to be most suitable as immobilized enzyme supports. Chymotrypsin immobilized to nonporous magnetic supports was not fouled significantly by either whole milk or clarified yeast homogenate. AE-cellulose-chymotrypsin was rapidly fouled by both these materials and chymotrypsin immobilized to acrylic-based ion exchangers was slowly fouled. Immobilized enzyme activity was found to be inversely proportional to particle diameter for nonporous rock magnetic particles. Immobilization by adsorption and then glutaraldehyde crosslinking was used to produce controlled amounts of chymotrypsin on the particles. Esterolytic activity increased with enzyme loading but caseinolytic activity did not increase. Chymotrypsin is inhibited by metal ions from the magnetic supports. It is partially protected by use of a preliminary protein coating and may be reactivated by incubation with EDTA or BSA.

Preparation and properties of immobilized papain and lipase.

Abstract: Papain and lipase were immobilized on derivatized Sepharose 4-B. The activated agarose had a binding capacity of 1.2 micronmol amino groups/ml packed agarose or 17 mg proteins/g dry agarose. The immobilized enzyme preparations were tested for the effects of pH of assay, temperature of assay, and substrate concentrations. The effect of 6M urea on the activity of papain was also determined. Soluble forms of the enzymes were used for comparison. Immobilization of the enzymes resulted in slightly different pH and temperature optima for activities. For immobilized papain Km(app) was similar to the one observed with soluble papain. Immobilization of lipase, however, cause a decrease in Km values. The immobilized enzyme preparations were stable when stored at 4 degrees C and pH 7.5 for periods up to eight months. The soluble enzymes lost their activity within 96 hr under similar storage conditions. Immobilized papain did not lose any activity after treatment with 6M urea for 270 min, whereas soluble papain lost 81% of its activity after the urea treatment, indicating that the immobilization of papain imparted structural and conformational stability to this enzyme.

Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.

Abstract: The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.

Immobilized catalytically active substance and method of preparing the same

Abstract: An enzyme or microorganism is entrapped within the gel matrix of a sulfated polysaccharide (said polysaccharide containing more than 10 w/w% of a sulfate moiety in its molecule) in the presence of ammonium ion, a metal ion, a water-soluble amine or a water-miscible organic solvent. The immobilized enzyme or microorganism thus obtained shows a high level of catalytic activity for a long period of time and can be used for continuous enzymatic reactions with substrates.

Characteristics of yeast invertase immobilized on porous cellulose beads.

Abstract: Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55°C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.

Amperometric determination of total cholesterol in serum, with use of immobilized cholesterol ester hydrolase and cholesterol oxidase.

Abstract: We describe an electrochemical method for simple, rapid, and economical assay of total serum cholesterol with use of immobilized cholesterol esterase (EC 3.1.1.13) and cholesterol oxidase (EC 1.1.3.6). A rotating porous cell was specially designed to hold the immobilized enzymes firmly and to allow the reaction mixture to pass through the enzyme layer easily, thus catalyzing the enzymatic transformation quickly. Hydrogen peroxide resulting from a catalytic reactions was measured amperometrically at +0.60 V cs. a standard calomel electrode. The calibration curve for total serum cholesterol was linear from 0 to 5.00 g/liter. The method is specific, precise, and inexpensive. Our results correlate well with those obtained by the method of Abell et al. [Stand. Methods Clin. Chem. 2, 26 (1958)], the correlation coefficient being 0.992. Ascorbic acid or bilirubin in concentrations up to 700 mg/liter do not interfere. The immobilized enzymes are stable, and the same immobilized-enzyme stirrer can be used for at least 200 accurate, reproducible assays.

Continuous-flow analysis for glucose, triglycerides, and ATP with immobilized enzymes in tubular form.

Abstract: Small-bore ("Autozyme") tubes with immobilized enzymes at the inner wall have been developed and studied for application in the Technicon "SMAC" high-speed continuous-flow biochemical analyzer. Tubes coated with glucose oxidase (D-glucose:oxygen oxidoreductase, EC 1.1.3.4) have been prepared for the assay of glucose, with colorimetric assay of the hydrogen peroxide produced; tubes coated with glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) for the enzymatic assay of triglycerides; tubes coated with hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) and glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NAD+ oxidoreductase EC 1.1.1.49) for the measurement of ATP, an intermediate product in assays for creatine kinase. With use of 10-15 cm lengths of Autozyme tube and SMAC hydraulics (150 samples per hour), assay sensitivity and carryover were similar to values for the corresponding free-enzyme methods. These immobilized enzymes were sufficiently stable for one to eight weeks of continuous use before replacemnt. We conclude that suitable bound-enzyme tubes can replace either single or multiple free-enzyme reagents in many continuous-flow assays at high sampling rates.

Process for immobilizing proteins

Abstract: A protein which can be an enzyme is immobilized by: (a) admixing the protein and an isocyanate-capped liquid polyurethane prepolymer to form an intermediate product; and (b) foaming the intermediate product by reacting it with water to form a polyurethane foam comprising the immobilized enzyme.

Chemical procedures for enzyme immobilization on porous cellulose beads

Abstract: In a previous article, the method of preparation and the physical properties of porous (75 to 80% porosity) cellulose beads were described (Biotechnol. Bioeng., 18, 1057 (1976). The present article reports that the chemical procedures employed for immobilizing enzymes on ordinary cellulose can be applied to the porous cellulose beads. The results showed more enzyme loading on the beads than ordinary cellulose. The choice of the procedures might also affect the mechanical strength of the cellulose beads.

Properties of an Immobilized Pesticide-Hydrolyzing Enzyme

Abstract: A bacterial enzyme(s) capable of hydrolyzing nine organophosphate insecticides was covalently bound to glass. The efficiency of this binding reaction ranged from 4 to 17%. Under continuous column operation, the immobilized enzyme(s) had an extrapolated half-life of 280 days. The specific activity of this glass-covalently bound hydrolase activity for parathion varied from 0.035 to 0.15 μmol/min per g of glass. The bound activity increased with decreasing glass particle size; however, the flow resistance also increased. Immobilized enzyme(s) kinetics were approximately 50% slower than those of the free enzyme(s), but there was no significant difference in the effect pH and temperature had on the activity of immobilized and free enzyme(s).

The use of α‐galactosidase and invertase in hollow fiber reactors

Abstract: Invertase and alpha-galactosidase have been immobilized in hollow fiber cartridges with no detectable enzyme leakage and used for the hydrolysis of sucrose and raffinose, respectively. For both hollow fiber immobilized enzymes nearly complete substrate conversion is possible. Enzyme stabilities in polysulfonate hollow fibers which have been preconditioned with bovine albumin approach the stabilities of the free enzymes.

Affinity Labelling of Alcohol Dehydrogenases

Abstract: 1 dl-α-Bromo-β(5-imidazolyl)-propionic acid is a potential affinity labelling reagent for metallo enzymes. It has been used with the alcohol dehydrogenases from liver and yeast. The liver enzyme is chemically modified and inactivated in a Michaelis-Menten-type reaction, where one molecule of the reagent is bound per subunit. The enzyme is protected from the inhibitor in a competitive manner by imidazole, 2,2′-dipyridyl, 1,10-phenanthroline and cyclohexanone, which all combine with the active-site zinc. The protection by chloride, acetate and NADH, which are considered to bind at the general anion binding site, is not strictly competitive. Inactivation has an optimum at pH 8.5. For the liver enzyme, the reagent was found to decrease the initial rate of ethanol oxidation. Prior to the irreversible alkylation of Cys-46, reversible binding is shown to occur at the active-site zinc atom. The yeast enzyme was extremely resistant to the reagent and no specific modification was found. 2 The potential affinity labelling and crosslinking reagent, symmetrical 1,3-dibromoacetone although unstable, has also been used for chemical modification. With the liver enzyme, concentrations below 5 mM gave a reaction of the Michaelis-Menten-type at pH 7.0. Several ligands known to complex with the active-site region protect the enzyme against the reagent. Dibromoacetone gave rapid inactivation of the yeast enzyme. Despite the fact that a pseudo-first-order reaction was observed with respect to enzyme as well as inhibitor, no saturating effect was found. In this work, dibromoacetone reacted like a monofunctional reagent.

Preparation and Some Properties of Chitosan Bound Enzymes

Abstract: An immobilization method using chitosan prepared from chitin as an insoluble carrier was investigated. Glucose isomerase, urease, glucamylase, trypsin and glucose oxidase were attached to chitosan by the aid of water soluble carbodiimide. Their activity yields were as follows; glucose isomerase 32%, urease 44%, glucamylase 8%, trypsin 10%, glucose oxidase 37%. Immobilized glucose isomerase showed no significant changes in optimal temperature and heat stability. But pH optimum of reaction and pH stability range were somewhat lowered. The inhibitory effects of bivalent metal ions were considerably reduced by immobilization and similar tendency was observed for buffer reagents such as Tris or veronal. Immobilized glucose isomerase was inhibited by 8M urea or 6M guanidine hydrochloride in nearly the same way as free enzyme. With SDS, cysteine or mercaptoethanol free glucose isomerase was scarcely affected by these reagents, while immobilized enzyme considerably suffered to a loss of its activity.

Application of cyanide‐metabolizing enzymes to environmental control; enzyme thermistor assay of cyanide using immobilized rhodanese and injectase

Abstract: The application of the enzyme thermistor in the analysis of cyanide in standard solutions as well as in blast furnace waste water is described. The heat signal is generated in the conversion of cyanide, catalyzed by the immobilized enzymes rhodanese (E.C. 2.8.1.1) and injectase (E.C. 4.4.19). Using the combination of cyanide-metabolizing enzymes and the enzyme thermistor unit, assays down to 20 microM cyanide can be carried out. Linear relationships were obtained at 20-600 microM cyanide for injectase and 20-1000 microM for rhodanese. The stability at 27 degrees C of the heat response was initially decreased, but soon stabilized at about 80% of the initial value and remained so for at least 200 hr. The technique was easily adapted to continuous analysis, applicable to environmental control (e.g., a "cyanide guard") with a response time at present within 2-3 min after a sudden change in cyanide concentration has appeared.

Process for producing dextrose using mixed immobilized enzymes

Abstract: Process for converting starch to dextrose wherein a partially hydrolyzed starch solution containing at least 10 percent hydrolyzed starch is contacted with an enzyme system under conditions whereby substantially complete conversion of the starch to dextrose is achieved. The enzyme system comprises immobilized glucoamylase and alpha-amylase selected from the group consisting of soluble alpha-amylase, immobilized alpha-amylase and mixtures thereof.

High loading of immobilized enzymes on activated carbon supports

Abstract: Enzymes are immobilized on activated carbon supports at high load levels and with high stability by a procedure whereby a carbon support is first activated with a water-soluble carbodiimide derivative which forms a highly reactive intermediate with carboxyl and other active organic radicals on the surface of the carbon support; thereafter the complex of the carbon and carbodiimide is treated with an enzyme solution whereby the enzyme displaces the carbodiimide and forms a carbon-enzyme complex wherein the enzyme is immobilized and yet the carbon retains its surface activity. Preferred immobilizing agents are quaternary ammonium forms of aminocarbodiimides or hydrochloric acid salts of water-soluble carbodiimides. The immobilization of the enzymes on activated carbon provides materials which are easily handled and which are stabilized against denaturation by hydrogen peroxide.

Enzyme immobilization with a protein carrier

Abstract: Enzymes are immobilized by mixing an enzyme solution with a dry insoluble crosslinked protein having a water-absorption capacity of 2 to 8 times its dry weight, allowing the protein to swell and suck up the total amount of enzyme solution and mixing the enzyme-containing swollen protein with a solution of glutardialdehyde to bond the enzymes to the protein. Enzymes immobilized by this method are especially well suited for repeated and continuous use.