Entrapment Method of Immobilisation

by entrapment differs from adsorption and covalent binding in that enzyme molecules are free in solution, but restricted in movement by the lattice structure of a gel . The porosity of a gel lattice is controlled to ensure that the structure is tight enough to prevent leakage of enzyme or cells and at the same time allow free movement of substrate and product. Inevitably the support will act as a barrier to mass transfer and, although this can have serious
implications for reaction kinetics, it can have useful advantages as harmful cells, proteins and enzymes are prevented from interaction with the immobilised biocatalyst. There are several methods of entrapment:
- ionotropic gelation with multivalent cations (e.g. alginate);
- temperature-induced gelation (e.g. agarose, gelatin);
- organic polymerisation by chemical/photochemical reaction (e.g. polyacrylamide);
- precipitation from an immiscible solvent (e.g. polystyrene).
Entrapment can be achieved by mixing an enzyme with a polyionic polymer material and then cross-linking the polymer with multivalent cations in an ion-exchange reaction to form a lattice structure that traps the biocatalyst (ionotropic gelation).Temperature change is a simple method of gelation by phase transition using 1–4% solutions of agarose or gelatin. However, the gels formed are soft and unstable. A significant development in this area has been an introduction of k-carrageenan polymers, which can form gels by ionotropic gelation and by temperature-induced phase transition, and this has introduced a greater degree of flexibility in gelation systems for immobilisation. Alternatively, it is possible to mix an enzyme with chemical monomers that are then polymerised to form a cross-linked polymeric network, trapping the enzyme in the interstitial spaces of a lattice . The latter method is more widely used and a number of acrylic monomers are available for the formation of hydrophilic copolymers.
For example, acrylamide monomer is polymerised to form polyacrylamide and methyl acrylate is polymerised to form polymethacrylate.
In addition to a monomer, a cross-linking agent is added during polymerisation to form cross-linkages between polymer chains and help to create a three-dimensional network lattice. The pore size of a gel and its mechanical properties are determined by the relative amounts of monomer and cross-linking agent. It is possible to vary these concentrations to influence the porosity of a lattice structure. The polymer formed may be broken up into particles of a desired size or polymerisation can be arranged to form beads of defined size to suit particular bioreactor requirements. Precipitation occurs by phase separation rather than by chemical reaction, but does bring a biocatalyst into contact with a water-miscible organic solvent and most biocatalysts are not tolerant of such solvents. Hence this method is limited to highly stable/previously stabilised enzymes or non-living cells.