Enzyme preparations, insolubilization

The second example, which is also in daily use, is the specific hydrolysis of natural penicillin G by penicillin amidase in preparative scale, again through a chromatographic process (129). It produces a clean hydrolytic product (6-aminopenicillanic acid) free of possible contaminant and it can then be used to prepare chemically all kinds of semisynthetic penicillin derivatives. Unlike the rather unstable soluble enzyme, the insolubilized preparation shows no loss activity after up to 11 weeks of continuous operation at 37°C. Furthermore, the procedure is free from potentially allergy-inducing contaminants. 

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. ... 

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. 

Another commonly used reaction for insolubilization of proteins and ligands through amino groups to polysaccharides is with 2,4,6-trichlorotriazine [18,19]. This procedure is extensively used in our laboratories in the preparation of agarose immobilized enzymes. 

The formation of cyclic carbonates of polysaccharides for biopolymer insolubilization [20] has been described and has found application in the preparation of immunosorbents. Coupling of enzymes to polysaccharides, glass and nylon structures using titanium halides has been described [21]. In this case coupling is through hydroxyl groupings. 

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. 

Water-insoluble enzymes have now been developed very few references bear dates prior to 1960, and yet the overall principle of attachment of an enzyme to an insoluble matrix is simple and simulates the natural mode of action and environment of enzymes embedded in biological membranes. The insolubilization of enzymes with retention of activity has already made an impact on the chemistry of biological molecules and systems, and, for an essentially new-born subject, a> large number of reviews dealing with their preparation have been published. " ... 

The advantages of insoluble enzymes over their soluble counterparts are numerous, deriving principally from the fact that they can effectively perform the same catalytic action as soluble enzymes, yet without contaminating the substrate solution. The stability of an enzyme is often increased on insolubilization, and, as the derivatives can also be re-used without loss of activity, insoluble enzymes, once prepared, are more economical than soluble enzymes. Many are commercially available, and their numerous applications have been reviewed. - " ... 

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-... 

Preparation of active, insoluble enzymes by physical adsorption to matrices has already been discussed (see p. 362), and, in the case of polysaccharide matrices, most of the enzyme derivatives formed in this way depend upon ionic interaction between charged groups on the polysaccharide and enzyme (see Table II). The instability of these enzyme derivatives may be lessened by cross-linking the coupled enzyme molecules.A few enzymes have been insolubilized by inclusion in microcapsules of collodion, or by entrapment in a polysaccharide gel. - A partition eflFect, in which an enzyme is held in the aqueous phase of a cellulose column while the substrate flows through in an organic phase, has also been used. 

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... 

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