Catalytic Activity

Immobilisation almost invariably changes the catalytic activity of an enzyme, and this is clearly reflected in alterations in the characteristic kinetic constants of the enzyme-catalysed reaction. In particular, the maximum reaction velocity (Vmax) obtained with an immobilised
enzyme is usually lower than that obtained with the corresponding soluble enzyme under the same reaction conditions. The Michaelis constant (Km), which reflects the affinity that the enzyme has for its substrate, is usually changed upon immobilisation, indicating that binding of substrate to the active site has been altered. Four principal factors influence the catalytic activity of immobilised enzymes: (a) conformation, (b) steric, (c) micro-environment and (d) diffusion.

The conformation of an enzyme refers to the particular shape adopted by the polypeptide chain, which is essential for maintaining the active site structure . Immobilisation procedures that involve modification or interaction with amino acid residues on the polypeptide chain can sometimes disturb protein structure and thereby affect the enzyme activity. Covalent immobilisation is most likely to cause an alteration in the protein conformation of an enzyme. A steric problem arises if the enzyme is immobilised in a position that causes the active site to be less accessible to the substrate molecules. In solution, a free enzyme molecule is surrounded by a homogeneous micro-environment in which the enzyme is fully integrated with all components of the solution.

Immobilisation creates a heterogeneous micro-environment consisting of two phases, i.e. the immobilised enzyme and the bulk of the solution from which the immobilised enzyme is separated . Therefore, all components of the reaction, substrate, products, activators, ions, etc., are partitioned between the immobilised enzyme phase and the bulk solution phase. This feature can significantly alter the characteristics of an enzyme reaction even if the enzyme molecule itself is not changed by immobilisation. The support material may influence the partitioning effect. If the support material attracts substrate then this can improve catalytic activity. Reaction rate is also reduced by diffusion restriction. As the substrate is consumed, more substrate must diffuse into the enzyme from the bulk solution and product must diffuse away from the active site. This is normally a problem for all forms of immobilised enzymes, but particularly so for encapsulated enzymes .
Diffusional limitations may be divided into two types, external diffusion restriction and internal diffusion restriction. The external type refers to a zone or barrier that surrounds the support material, called the Nernst layer. Substrate molecules can diffuse into this layer by normal convection and by a passive molecular diffusion. If substrate molecules pass through this layer slowly, then this may limit the rate of enzyme reaction. External diffusion restriction can be improved by speeding up the flow of solvent over and through the immobilised enzyme by increasing the stirring rate.
Internal diffusion restrictions are due to a diffusion limitation inside the immobilised enzyme preparation . In this case, diffusion of substrate molecules occurs by a passive molecular mechanism only, which may be more difficult to overcome if it is a seriously limiting factor. The overall rate of diffusion is markedly influenced by the method of immobilisation. Covalent and adsorption procedures cause less diffusion limitation than do entrapment and encapsulation
procedures.