Insolubilized enzymes

The modification of cellulose with alkaline carbon disulfide to introduce xanthate groups has been extensively exploited in the industrial production of viscose. Early work on the preparation and properties of starch xanthate has been discussed. Xanthate derivatives of cellulose and starch have been discussed with respect to general xanthate chemistry, and the xanthation of cellulose in homogeneous medium is known to be a second-order reaction. Cellulose xanthate shows some potential as a matrix for enzyme insolubilization, " and stable derivatives of this xanthate may be prepared by transesterification. Thermal decomposition of cellulose allyl- and benzyl-xanthates gives 5,6-cellulosene. Some thiocarbonyl derivatives of polysaccharides have been prepared. "... 

The structure of Blue Dextran 2000, a water-soluble commercial derivative, involves a triazine type of dye covalently linked to dextran, and, in a way analogous to that used for other dyed polysaccharides, has been employed for the assay of dextranase. The ability of Blue Dextran 2000 to bind proteins cannot be attributed to formation of a covalent link, because no chloro groups remain on the triazinyl rings. The binding must involve an ionic bond between the protein and the sulfonic groups of the dye residue, and, in one case, the association could be reversed by using 0-(2-diethylaminoethyl) cellulose to abstract the dyed polysaccharide. Other chlorotriazinyl dyes have been used in the preparation of dyed derivatives of amylopectin, laminaran, dextrans, pectin, pelvetian, zosterine, and cellulose. As already mentioned, triazine-dyed polysaccharides are useful in enzyme insolubilization. 

Enzymes best retain their activity on insolubilization when hydrophilic rather than hydrophobic matrices are used. Although many non-carbohydrate matrices have been employed, the hydrophilic nature of polysaccharides must be a major feature contributing to their widespread use for enzyme insolubilization. Presumably, the hydroxyl groups of the polysaccharide resemble water molecules sufficiently to provide the enzyme to be attached with an environment suited to its stability. 

Introduction of an arylamino group into a polysaccharide provides diazotizable products of great utility for enzyme insolubilization, on account of the ease with which diazo groups react with a variety of amino acid side-chains, particularly phenolic ones. Many enzyme derivatives have, therefore, been prepared by use of 0-(4-aminobenzyl) cellulose (38), 0-(3-aminobenzyl)oxymethylcelluIose, and cellulose p-aminoben-zoate. However, although these polysaccharide derivatives could be expected to be widely applicable, the lower enzymic activities often ob-... 

A novel means of enzyme insolubilization involves chelation. " " Treatment of cellulose with titanium or other transition-metal ion activates the polysaccharide, which then reacts directly with enzymes. Presumably, in the activation, one of the water molecules in the octahedral, hexahydrated titanium (IV) ion becomes replaced by a polysaccharide hydroxyl group, and, in the second stage, another water molecule is displaced by an amino, carboxyl, or hydroxyl group of the enzyme. 

D-Fructofuranosidase in the cell wall preparation of hypocotyls and roots of germinating sugar beets was investigated with reference to /3-D-fructofurano-sidase isolated from the aged tissue slices of the mature roots.Effect of sodium chloride concentration on release of the enzymes, insolubilization by decreasing... 

C. Oligo- and Poly-nucleotides.—The stepwise enzymatic synthesis of internucleotide bonds has been reviewed. A number of polynucleotides containing modified bases have been synthesised " in the past year from nucleoside triphosphates with the aid of a polymerase enzyme, and the enzymatic synthesis of oligodeoxyribonucleotides using terminal deoxynucleotidyl transferase has been studied. Primer-independent polynucleotide phosphorylase from Micrococcus luteus has been attached to cellulose after the latter has been activated with cyanogen bromide. The preparation of insolubilized enzyme has enabled large quantities of synthetic polynucleotides to be made. The soluble enzyme has been used to prepare various modified polycytidylic acids. ... 

Lowe, C.R., and Dean, P.D.G. (1971) Affinity chromatography of enzymes on insolubilized cofactors. FEBS Lett. 14, 313-316. 

Enzymes. The specificity of an enzyme for its substrate, coenzyme or competitive inhibitor provides the basis for many affinity chromatographic separations. Enzymes may be extracted and purified using insolubilized substrates, coenzyme or inhibitors. Less frequently, enzymes are used as the ligands. 

Antibodies. The reaction between an antibody and its antigen does not result in the chemical modification of the antigen compared with the action of an enzyme and provides the basis for producing chromatographic media capable of selecting the complementary molecules. Either the antigen is insolubilized and used to isolate and purify the appropriate antibodies or with the increased availability of monoclonal antibodies, the reverse procedure is used. 

The classic potentiometric enzyme electrode is a combination of an ion-selective electrode-based sensor and an immobilized (insolubilized) enzyme. Few of the many enzyme electrodes based on potentiometric ion- and gas-selective membrane electrode transducers have been included in commercially available instruments for routine measurements of biomolecules in complex samples such as blood, urine or bioreactor media. The main practical limitation of potentiometric enzyme electrodes for this purpose is their poor selectivity, which does not arise from the biocatalytic reaction, but from the response of the base ion or gas transducer to endogenous ionic and gaseous species in the sample. 

Melrose, G.J.H. (1971) Insolubilized Enzymes— Biochemical applications of synthetic polymers. Rev. PureAppl. Chem., 21, 83-119. 

Salmona M, Saronio C, Garattini S (eds) (1974) Insolubilized enzymes. Raven, New York... 

M. L. Schuler, R. Aris, and H. M. Tsuchiya, Diffusive and electrostatic effects with insolubilized enzymes, /. Theor. Biol. 1972, 35, 67-76. 

Other methods of stabilization include chemical or carbohydrate modification of enzymes. Modifications of reactive groups on proteins without insolubilization has been used to enhance stability in solution. Grafting of polysaccharides or synthetic polymers, alkalation, acetylation and amino acid modification have all been reported (5)... 

The insolubility of enzymes in monophasic organic systems has a controlling influence on the kinetics of enzymatic catalysis in organic media. Insolubilized enzymes are subject to intraparticle and external diffusional limitations which can mask the true, intrinsic kinetics of catalysis. These limitations are particularly severe for highly active and purified enzymes such as horseradish peroxidase. One way to overcome this problem is to increase the surface area of the enzyme in contact with the organic solvent. 

During isolation by conventional methods yeast proteins frequently become denatured, insolubilized and display poor functional properties. These proteins can be rendered more soluble by limited hydrolysis with acid, alkali or proteolytic enzymes. Protein hydrolyzates are most commonly prepared by partial acid hydrolysis and yeast hydrolyzates are popular as food flavorings and ingredients (66). Acid hydrolyzates have flavors resembling cooked meats and are widely used by earners to impart brothy, meaty flavors to soups, gravies, sauces, canned meats. 

In the field of biosensor technology, immobilized enzyme electrode development occupies a place of prominence due to the attractive performance of this hybrid device. Coupling an immobilized enzyme layer with an electrochemical sensor combines the advantages of using an insolubilized enzyme system (see below) with the sensitivity of readily available potentiometric and amperometric electrodes. The resulting biosensor enables direct, reliable, and reproducible... 

We faced the problem of the poor solubility of most N-protected amino aldehydes in water, which might account for the low reactivity observed with D-fructose-1,6-diphosphate aldolase from rabbit muscle (RAMA) (14, 15, 19-21). Increasing the percentage of organic co-solvent (e.g. dimethylformamide) in the medium to make the aldehyde soluble may lead to either a dramatic enzyme deactivation [22] or an insolubilization of the donor (e.g. dihydroxyacetone (DHA) and DHAP sodium salt). As a result, no reaction or insufficient product yields are often obtained. 

Insolubilized Enzymes And Other Enzyme Engineering Sources. 

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