Attachment of enzyme

Covalent attachment of enzymes to surfaces is often intuitively perceived as being more reliable than direct adsorption, but multisite physical interactions can in fact yield a comparably strong and stable union, as demonstrated by several biological examples. The biotin/streptavidin interaction requires a force of about 0.3 nN to be severed [Lee et al., 2007], and protein/protein interactions typically require 0.1 nN to break, but values over 1 nN have also been reported [Weisel et al., 2003]. These forces are comparable to those required to mpture weaker chemical bonds such as the gold-thiolate bond (1 nN for an alkanethiol, and even only 0.3 nN for a 1,3-aUcanedithiol [Langry et al., 2005]) and the poly(His)-Ni(NTA) bond (0.24 nN, [Levy and Maaloum, 2005]). 

The supports employed for covalent attachment of enzymes can be classified into two groups a) natural (agarose, dextran, cellulose, porous glass, silica, the optical fiber itself or alumina) and b) synthetic (acrylamide-... 

Ruth, J.L. (1993) Direct attachment of enzymes to DNA probes. In Methods in Nonradioactive Detection (G.C. Howard, ed.), pp. 153-177. Appleton and Lange, Norwalk, Connecticut. 

The covalent attachment of enzymes to a polyurethane is not the only method in which enzymes are used. In many cases, it is more convenient to immobilize the cells that produce the enzymes. The exuacellular enzymes produced by the cells are then used as we will illustrate below. Immobilization is also important for the production and harvesting of enzymes and other proteins with the objective of increasing the useful lives of organisms. This was shown by Bucke, who immobilized Erwinia... 

Mutarotation of 0.3% solutions of the freshly dissolved sugars in 12 ml of 5 mM EDTA, pH 7.4 was followed. Significant differences in mutarotation rates (AK) in the presence and absence of 100 units of bovine kidney enzyme were expressed as the ratio AK/Ksp. Differences of less than 5% in these rate constants were not considered significant. Of the 18 sugars listed, nine have been tested previously as substrates for other mammalian mutarotases with essentially the same pattern as described here. The pattern of specificity indicates that a 3-point attachment of enzyme and substrate is necessary for catalysis of mutarotation. b Data from 72). 

Covalent Attachment The covalent attachment of enzyme molecules via nonessential amino acid residues (that is, amino acids minus water) to water-insoluble, functionalized supports are the most widely used method for immobilizing enzymes. Functional groups of the nonessential amino acid residues that are suitable for the immobilization process are free a-, /3-, or y-carboxyl groups, a- or /3-amino groups, and phenyl, hydroxyl, sulfhydryl, or imidazole groups.2... 

Commonly employed water-insoluble supports for the covalent attachment of enzymes include synthetic supports such as acrylamide-based polymers, maleic anhydride-based polymers, methacrylic acid-based polymers, styrene-based polymers, and polypeptides, and natural supports such as agarose (Sepharose), cellulose, dextran (Sephadex), glass, and starch (Zaborsky, 1973). 

Chemical methods that involve the formation of at least one covalent bond (attachment of enzyme to water-insoluble functionalized polymers, intermolecular cross-linking of enzyme molecules using multifunctional... 

The covalent attachment of enzymes to water-insoluble carriers is usually the preferred immobilization method for sensor manufacturing. Obviously, the selected procedure should avoid the loss of enzymatic activity and keep the accessibility of the binding site to the substrate molecules. Unfortunately, this is usually not the case and due to the severe conditions of many of these procedures, major activity losses and/or changes on the substrate selectivity are produced during immobilization. Some authors have pointed out that the enzyme activity decreases approximately one fifth per formed bond [66]. 

Fig. 31.8. Covalent attachment of enzymes to oxirane-functional polymeric beads. (Courtesy of M. Elizabeth Miller and Rohm and Haas.)...

Attachment of enzymes to activated insoluble polysaccharides is routinely carried out at pH 9.0, and this pH has been found to be suitable for synthesis of most soluble dextran-enzyme... 

Fig. 4 General approach of the bioreactive MALDI mass spectrometer probe tips. Goid piated probe tips are activated through the covalent attachment of enzymes (the general terminology of Au/enzyme is used to indicate the nature of the activated surfaces). The probe tips are then used for protein characterization by direct appiication of the analyte and time given for digestion. The digestions are stopped with the addition of a MALDI matrix, the reaction product-matrix mixture aiiowed to dry, and the probe tips are inserted into the mass spectrometer for MALDI-TOF analysis.

This shift in the pH optimum can be explained by an uneven distribution of H+ and OH between the bulk of the external solution and the polyelectrolyte (carrier) phase. Polyornithine, shown in Figure 4.5, possesses side chains with primary amino groups that are used for the covalent attachment of enzyme. Not all of these groups will react, and those that remain underivatized do so either for steric reasons or due to incomplete coupling reaction steps. 

The reuse of an enzyme can be economically favorable when a high-cost enzyme is used. It can be difficult to separate and reuse an enzyme because enzymes are typically globular proteins that are highly soluble in water. A common technique to facilitate the reuse of a high-value enzyme is to immobilize the enzyme onto a surface, inside of an insoluble matrix or within a semipermeable membrane. Both chemical and physical means can be employed to immobilize enzymes. The former method involves the covalent attachment of enzymes to water-insoluble supports and is the most widely used method for enzyme immobilization. ... 

Many reviews and books on the immobilization of enzymes have been published during the last two decades [5, 11-20]. The intent of this part of the review is to explain the basic principles and to show recent developments of enzyme immobilization for the purpose of preparative biotransformation. The attachment of enzymes onto prefabricated artificial or natural carriers will be given special emphasis (see Sect. 3.2). 

Chitosan is of importance because of its primary amino groups that are susceptible for coupling reactions. Furthermore, porous spherical chitosan particles are commercially available (Chitopearl, Fuji Spinning) allowing noncovalent or covalent attachment of enzymes [55]. This support matrix can be easily prepared [56] and activation methods have been summarized [57]. Treatment with polyethyleneimine or with hexamethylenediamine and glut-ardialdehyde can improve the mechanical characteristics [53,58] of the biocatalyst, which is poor otherwise. However, this is often accompanied by some activity loss or increase of diffusional limitations. 

Figure 13.1 Covalent attachment of an enzyme to derivatized polypyrrole (a) polypyrrole polymer (b) nitration of polypyrrole (c) electrochemical reduction of nitro groups to amine groups (d) attachment of enzyme to amine groups with carbodiimide [32],...

Covalent attachment of enzymes to solid supports exhibit highly stable enzyme immobilizations, with reduced activity however. The sensitivity of such sensors is limited because only monolayers of enzyme molecules can be produced. 

Attachment of proteins to solid supports has been a development primarily of the attachment of enzymes to solid supports to make immobilized enzymes (37). The attachments can be made by many different biochemical reactions. The selection of the appropriate reaction depends... 

Immobilization of whole microbial cells for industrial purposes eliminates the need for the isolation, purification and attachment of enzymes and provides the enzymes with a microenvironment maintained at optimal conditions by cellular metabolic and transport activities. Adhesion of microbial cells to inert substrata often occurs in nature and greater understanding of these natural processes may lead to advances in the technology of whole cell immobilization. Mechanisms of attachment in natural systems involve adhesive microexudates produced by the cells, electrostatic attraction, and anatomical projections which cling to the support surface. The chemical methods which have been used for whole cell immobilization have recently been reviewed by Jack and Zajic (1 ). 

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