Linkage methods, immobilized enzymes

It was reported that PEGylated lipase entrapped in PVA cryogel could be conveniently used in organic solvent biocatalysis [279], This method for enzyme immobilization is more convenient in comparison to other types of immobilization that take advantage of enzyme covalent linkage to insoluble matrix, since the chemical step which is time consuming and harmful to enzyme activity is avoided. The application of this catalytic system to the hydrolysis of acetoxycoumarins demonstrated the feasibility of proposed method in the hydrolysis products of pharmaceutical interest and to obtain regioselective enrichment of one of the two monodeacetylated derivatives. 

Physical adsorption relies on non-specific physical interaction between the enzyme protein and the surface of the matrix. In this method only weak bonds are involved, e.g. hydrogen bonds, multiple salt linkages, van der Waals forces, which on one hand is less desruptive towards enzyme, while on the other hand leads to desorption from the surfaces once there are changes in temperature, pH, ionic strength etc. Physical forces are non-specific and further adsorption of other proteins or other substances can occur as the immobilized enzyme is used which may alter the properties of the immobilized enzyme. 

The adsorption of an enzyme onto a support or film material is the simplest method of obtaining an immobilized enzyme. Basically, the enzyme is attached to the support material by noncovalent linkages and does not require any preactivation step of the support. The interactions formed between the enzyme and the support material will be dependent on the existing surface chemistry of the support and on the type of amino acids exposed at the surface of the enzyme molecule. Enzyme immobilization by adsorption involves, normally, weak interactions between the support and the enzyme such as ionic or hydrophobic interactions, hydrogen bonding, and van der Waals forces (see Figure 44.1). ... 

Creation of covalent bonds between surface amino acids of the enzyme and an insoluble matrix is perhaps the most frequendy exploited method of immobilization. Polar amino acids, which are likely to be present on the protein surface, have struaures that lend themselves to the chemical manipulation necessary for immobilization. e-Amino groups of lysine residues are the most frequendy employed points of linkages, though cysteine (via -SH), tyrosine, histidine, aspartic and glutamic acids, tryptophan, and arginine can also be used. 

Alkaline phosphatase has been reversibly immobilized on cross-linked agarose by two methods, both based on disulphide linkages.Prior to coupling with 5,5 -dithiobis-(2-nitro-benzoic acid)-activated thiol-agarose (57), the enzyme was modified by thiolation with 4-mercaptobutyrimidate. The unmodified enzyme could be coupled directly to dithiobis(succinimidyl propionate)-agarose (58). 

Conditions for achieving efficient DET via enzyme immobilization are dictated partly by materials architecture. Enzyme immobilization techniques may include nonspecific adsorption, covalent linkage, entrapment in conductive polymeric films, association with metal colloids, and encapsulation within porous matrices (see Chapter 11). The simplest method is nonspecific adsorption, but control is limited various noncovalent interactions will yield different orientations of the redox center with respect to the electrode interface and, as a result, inefficient DET. 

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