Insolubilization of enzymes

Bromoacetyl) cellulose, formed by the action of bromoacetyl bromide on cellulose, has proved to be a very useful material for the insolubilization of enzymes, etc., because of the ease with which the bromine atom... 

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. " ... 

Uses include simplification of reactors, industrial processes and clinical analyses, and employment in analytical chemistry - and biochemistry, sequence analysis, syntheses, separation techniques, isolation of compounds related to enzymes, and in membrane and column forms. The insolubilization of enzymes may modify their characteristics, particularly if the matrix contains ionic groups, and the kinetics of insolubilized enzymes have been studied. ... 

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

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. 

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. 

Affinity chromatography can be applied as a purification technique to any pair of biologically complementary substances, for example, enzymes and inhibitors, steroids and binding proteins, antigens and antibodies. Insolubilization of one member of the pair yields an affinity column specific for the second member of the pair. Affinity chromatography can also be used to concentrate dilute solutions of purified substances and removal of soluble substances which are present as contaminants in low concentration. 

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 insolubilization of NAD and AMP and the uses of these supports have already been described, as has the use of hydrazide-agarose for immobilization. Other insolubilized nucleotide affinity columns have also been described. For example, Olsen [101] isolated galactosyltransferase from whey using a UDP-Sepharose affinity column. GTP coupled to a hydrazide Sepharose derivative was used to isolate D-erythro-dihydroneopterin triphosphate synthetase, the first enzyme for folate biosynthesis in Lactobacillus plantarum [102]. ATP-agarose has been used in the purification of heavy meromysin, elution being effected with ATP [103]. 

Insolubilization of stem bromelain with carboxymethylcellulose as carrier changes the catalytic behavior of the enzyme against substrates of low and high molecular weight [69-71]. The tyrosyl residues in stem bromelain, which are in an exposed state and hence readily accessible to the solvent, can be acetylated with iV-acetylimidazole at pH 7.5 or nitrated with tetranitromethane at pH 8.0 without change in catalytic activities [72],... 

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. 

The principles of attachment of molecules to polysaccharides with concomitant insolubilization, discussed in the preceding two Sections, also apply to nucleic acids. The insolubilization of nucleic acids and polynucleotides provides materials useful (a) for fractionation and purification of other nucleic acids and related compounds,(b) for multiplication and isolation of single nucleic acid strands by base-pairing, " (c) for base-sequence determination, (d) as afiR-nants, " templates, and substrates for nucleic acid-related enzymes, and (e) as aflBnants for nucleic acid-binding proteins. ... 

Based on the crystallographic data, detailed mechanisms for the carboxypeptidase A enzymic reaction have been proposed. These mechanisms and recent work relating to them have been reviewed.Although probably correct in general, these mechanistic conclusions are based on the assumption that the kinetic and chemical properties are conserved on crystallization. In general coordination chemistry examples abound where the structures of species in the crystd and in solution are markedly different and indeed it has been shown that the detailed kinetics of carboxypeptidase A solutions differ from those of the enzyme crystals. It has been suggested that different conformations of the active site exist in the two physical states,Detailed kinetic studies on crystals over a range of enzyme concentrations, substrate concentrations and crystal sizes have been carried out and the results interpreted in terms of a recent theory for insolubilized enzymes. The marked differences... 

The solubilization of j8-D-fructofuranosidase bound to cell walls of sugar beet Beta vulgaris) seedlings has been studied.Gel filtration etc. studies confirmed that substances which were released from the cell wall together with bound jS-D-fructofuranosidase acted on the enzyme in dilute salt solutions and caused insolubilization of the enzyme. The characteristics of two different fractions of the substances were analogous to those of nucleic acids and nucleoproteins. 

The effect of Blue Dextran on protection from insolubilization of NaCl-released 3-D-fructofuranosidase from sugar beet cell-wall was studied and discussed.The enzyme was insoluble in water or salt solutions of low concentrations but was solubilized by the addition of Blue Dextran. Upon chromatography of the enzyme on a Sepharose 6B column in the presence of Blue Dextran, the enzyme and Blue Dextran were eluted with the same elution volume. However, they were eluted separately by using 0.5 M-NaCl as an eluting solvent. Solubility of enzyme increased with concentration of Blue Dextran added. It was found that the enzyme was absorbed to Sephadex G-lOO, but not to dextran T40 or T500. Some additional experiments revealed that the dependence of pH on solubility of the released enzyme disappeared in the presence of Blue Dextran. 

Other Matrices. A large number of other matrices have been employed, including starch (33), cross-linked dextrins (34,35), a vinyl maleimide polymer (36), chitin (37), mannan (38), and insolubilized proteins (39). The number of potential matrices is almost limitless, and most of the matrices used for immobilization of enzymes have potential application in bioselective adsorption. Among these are nylon (40), metal oxides (41), maleic anhydride-ethylene co-polymers (42), and polystyrene derivatives (43). Few of these have been used because of their potential for nonspecific adsorption either by charge, as with the metal oxides, or by hydrophobic interactions (as would be the case with polystyrene). The derivatized matrix must be free of nonspecific adsorption. Proper derivatization can eliminate nonspecific adsorption or, as with cyanogen bromide activation, create nonspecific adsorption in matrices having no prior nonspecific adsorption properties. 

In nature, they occur often with different degrees of esterification (DE) around 70%. Because of enzymic degradation, they can have a lower DE in that case (if DE < 50%), they are insolubilized by calcium cross-linkage. Extraction requires the use of chelating agent. 

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