Showing papers on "Immobilized enzyme published in 1970"

Chemical fixation of chymotrypsin to water-insoluble crosslinked dextran (sephadex) and solubilization of the enzyme derivatives by means of dextranase.

Abstract: Catalytically active chymotrypsin derivatives can be synthesized from cyanogen bromide-activated Sephadex G200. In most cases the apparent catalytic activity of the covalently fixed enzyme appears to be considerably decreased in comparison to the activity of the free enzyme. However, by proper choice of the reaction conditions for the activation, enzyme conjugates with high activity, even toward a high molecular substrate, can be synthesized. These latter derivatives may be of practical value for the digestion of proteins. Crosslinked dextran as carrier was chosen because of the possbility, of digesting it enzymatically by dextranase. Sephadex G200, if activated at or below pH 10.3, will combine with chymotrypsin to yield digestable products. Changes of apparent kinetic properties of the fixed enzyme can accordingly be studied during the degradation process. On the solubilization of the insoluble conjugate, a total recovery of activity of the fixed enzyme can be obtained in cases the carrier has been activated by a sufficiently mild procedure. The high apparent Michaelis constant Km of insoluble chymotrypsin–Sephadex toward N-acetyl-L-tyrosine ethyl ester shifts back on solubilization to the value of free chymotrypsin. We therefore propose that the decreased activity of an insoluble chymotrypsin–Sephadex is due to diffusional effects shown by the gel matrix toward the substrate. Similarly observed shifts in optimum pH are explained by accumulation of hydrogen ions in the gel. The organic chemical reaction used for coupling the enzyme to the polymer can therefore be performed without decreasing the inherent catalytic activity of the enzyme. The route described for fixing chymotrypsin to Sephadex followed by solubilization of the products may be useful as a synthetic method for binding proteins, peptides, and other amino group-containing substances to soluble carriers, e.g., for the modification of pharmaceuticals.

Application of ultrafiltration for enzyme retention during continuous enzymatic reaction.

Abstract: In most enzymatic reactions, batch or continuous, separation of the enzyme for reuse is difficult if not impossible. A process will be presented in which an Ultrafiltration membrane serves to separate the reaction products from the enzyme and the substrate. In this manner the enzyme may be retained and re-used. Furthermore, under these conditions, the enzyme need only be present in catalytic amounts regardless of the amount of product produced. Under proper operating conditions and proper ultrafiltration membrane selection, a pure solution of α-amylase from Bacillus subtilis may be retained with no loss in enzyme activity over a test period of 30 hr after steadystate has been achieved. In the presence of substrate, the membrane support and ultrafiltration cell serve as the reaction vessel for the hydrolysis of starch. The substrate is continuously pumped into the cell under constant ultrafiltration pressure. The di-, oligo-, and polysaccharides formed from the enzyme reaction then either pass through the membrane as products or are retained. The molecular weight distribution of the products is dependent on the nominal molecular weight cut-off of the membrane, absolute ultrafiltration pressure, enzyme-to-substrate ratio, temperature, and residence time of the substrate in the reactor. In addition to the partial hydrolysis of starch by α-amylase, some preliminary findings on the complete hydrolysis of starch by glucoamylase will also be presented. In these latter studies, the substrate may be completely hydrolyzed to glucose units.

Process and apparatus for obtaining a differential white blood cell count

Abstract: A PROCESS AND APPARATUS FOR OBTAINING A DIFFERENTIAL WHITE BLOOD CELL COUNT IS DISCLOSED. A CYTOLOGICAL FIXING AGENT IS ADDED TO A SAMPLE OF BODY FLUID, SUCH AS BLOOD, CONTAINING WHITE BLOOD CELLS TO KILL THE BLOOD CELLS CONTAINED IN THE SOLUTION AND TO IMMOBILIZE THE CATALYTIC ENZYMES IN THE CELLS. THE ACTIVITY OF THE ENZYMES IS NOT SERIOUSLY IMPARRED, AND SOLUBLE COMPONENTS IN THE EXTRACELLULAR SOLUTION ARE NOT PRECIPITATED. WHEN THE BODY FLUID ALSO CONTAINS RED BLOOD CELLS A HEMOLYZING REAGENT IS ADDED AFTER THE FIXATION STEP TO CAUSE THE RED BLOOD CELLS TO RELEASE THEIR HEMOGLOBIN CONTENT INTO SOLUTION. ADDITION OF A SPECIFIC CYTOCHEMICAL SUBSTRATE, CHROMOGENIC PRECIPITATING COUPLING REAGENT, AND PH BUFFER CAUSES DEPOSITION OF AN INSOLUBLE DYE IN A SPECIFIC TYPE OF CELL CONTAINING THE IMMOBILIZED ENZYME. THE SOLUTION CONTAINING THE DYE BLOOD CELLS IS THEN PASSED THROUGH A PHOTOMETRIC COUNTER WHICH RAPIDLY AND ACCURATELY GIVES A COUNT OF THE DYED CELLS, USING DIFFRENT SPECIFIC SUBSTRATES FOR DIFFERENT ENZYMES CONTAINED IN SPECIFIC KINDS OF CELLS, ABSOLUTE AND RELATIVE COUNTS OF THE DIFFERENT CELLS CAN BE OBTAINED.

Enzyme gel and use therefor

Abstract: A SUBSTANTIALLY RIGID ENZYME GEL, USEFUL IN PROCESS INCORPORATING ENZYME CATALYSTS, SUCH AS SUBSTRATE ANALYSIS, COMPRISING A POLYMER MATRIX HAVING WATER COLLOIDALLY DISPERSED THEREIN AND ACTIVE ENZYME MOLECULES DISTRIBUTED HOMOGENEOUSLY THROUGHOUT AND SUBSTANTIALLY UNREACTED WITH THE POLYMER MATRIX THE POLYMER MATRIX HAVING A SUFFICIENTLY SMALL PORE SIZE TO RETAIN INDIVIDUAL ENZYME MOLECULES, I.E. THE ENZYME IN AQUEOUS SOLUTION. THE GEL IS FORMED BY POLYMERIZING AN AQUEOUS SOLUTION OF A MONOMER, FOR EXAMPLE AN ACRYLAMIDE, CONTAINING AN ENZYME UNDER CONDIDTIONS NOT DETRIMENTAL TO THE ACTIVITY OF THE ENZYME.

A Synthetic Approach to the Study of Microenvironmental Effects on Enzyme Action: The Second Kaj Linderstrøm-Lang Gold Medal Lecture

Abstract: Publisher Summary This chapter presents a synthetic approach to the study of microenvironmental effects on enzyme action The chapter discusses water-insoluble enzyme derivatives Enzyme derivatives in which the biologically active protein is covalently bound to a water-insoluble polymeric carrier may serve as easily removable reagents of considerably improved stability; they are well suited for repeated or continuous use and provide means for a more adequate control of enzyme reactions Immobilized enzyme derivatives can be used as specific adsorbents for the isolation and purification of enzyme inhibitors Moreover, new properties may sometimes be imposed on the immobilized enzyme by the chemical nature of the polymeric carrier In the case of acidic proteins, containing large excess of carboxyl groups, immobilization could be achieved by coupling the protein to aminoethyl cellulose through soluble carbodiimide activation of the protein carboxyls The kinetic behavior of immobilized enzyme systems is dominated by several factors not encountered in the kinetics of free enzymes: (1) effects of the chemical nature of the carrier, stemming from the modified environment within which the immobilized enzyme is located, (2) steric restrictions imposed by the carrier, and (3) diffusion control on the rate of substrate penetration

Purification of low molecular weight enzymes

Abstract: For preparation of an enzyme, the enzyme containing solution is passed through an ultrafilter passing only material having a molecular weight lower than 10,000; a soluble form of a bivalent non-toxic cation is added to it, and this cation is precipitated as a carbonate in the enzyme solution; the enzyme is selectively adsorbed to the said carbonate when so precipitated. The carbonate with the adhered enzyme is recovered for example on a filter or by centrifugation and may be washed and/or dried as such, or treated with acid for recovery of the pure enzyme. The metal carbonate complex of the enzyme is particularly suitable for therapeutic purposes because of its high ability and the ready availability of the enzyme upon contact with stomach acid.

Selección de soportes magnéticos para la inmovilización de Ureasa

Abstract: Fe3O4, Fe3O4-NH2, Fe3O4@SiO2 y Fe3O4@SiO2-NH2 as magnetic supports were studied for the urease immobilization. The solids were characterized by N2-physical adsorption at 77 K, magnetization curves and FTIR. Activity assays were evaluated by urea decomposition. The magnetic properties of solids allow an easy separation. The immobilized enzyme improved the enzymatic activity compared to the free enzyme. In Fe3O4-NH2 and Fe3O4@SiO2-NH2 was observed allows a greater amount of enzyme coupled.