The most intensely studied of the immobilization techniques is the formation of covalent bonds between the enzyme and the support matrix. When trying to select the type of reaction by which a given protein should be immobilized, the choice is limited by two characteristics: (1) the binding reaction must be performed under conditions that do not cause loss of enzymatic activity, and (2) the active site of the enzyme must be unaffected by the reagents used.
The covalent binding method is based on the binding of enzymes and water-insoluble carriers by covalent bonds. The functional groups that may take part in this binding are listed below:
Hydroxyl group Imidazole group Phenolic group
Thiol group Threonine group Indole group
This method can be further classified into diazo, peptide and alkylation methods according to the mode of linkage. The conditions for immobilization by covalent binding are much more complicated and less mild than in the cases of physical adsorption and ionic binding. Therefore, covalent binding may alter the conformational structure and active center of the enzyme, resulting in major loss of activity and/or changes of the substrate. However, the binding force between enzyme and carrier is so strong that no leakage of the enzymes occurs, even in the presence of substrate or solution of high ionic strength.
Covalent attachment to a support matrix must involve only functional groups of the enzyme that are not essential for catalytic action. Higher activities result from prevention of inactivation reactions with amino acid residues of the active sites. A number of protective methods have been devised:
Hence, covalent binding can be brought about by the following:
The active site of the enzyme must not be hindered. There must be ample space between the enzyme and the backbone.
It is possible in some cases to increase the number of reactive residues of an enzyme in order to increase the yield of the immobilized enzyme. This provides alternative reaction sites to those essential for enzymatic activity. As with cross-linking, covalent bonding should provide stable, immobilized enzyme derivatives that do not leach enzyme into the surrounding solution. The wide variety of binding reactions and insoluble carriers (with functional groups capable of covalent coupling or being activated to give such groups) makes this a generally applicable method of immobilization. This is true even if very little is known about the protein structure or active site of the enzyme to be coupled.