Adsorption Method of  Immobilisation

Immobilisation by adsorption is the simplest method and involves reversible non-covalent interactions between biocatalyst and support material (Figure 1).The forces involved are mostly weak electrostatic such as van der Waals forces and ionic and hydrogen bonding interactions, although hydrophobic bonding can be significant. These forces are individually very weak, but sufficiently large in number to provide good binding. For example, it is known that yeast cells have a surface chemistry that is substantially negatively charged so that use of a positively charged support material will allow immobilisation. Existing surface chemistry on a biocatalyst and on a support is utilised for adsorption, so no chemical activation or modification of support is required and little damage is inflicted on biocatalysts in this method of immobilisation.
 

Figure 1. Principal methods of biocatalyst immobilisation.

The procedure consists of mixing together biocatalyst and support under suitable conditions of pH, ionic strength, etc., for a period of incubation, followed by collection of immobilised biocatalyst and extensive washing to remove non-bound biocatalyst.
Advantages:
- little or no damage to a biocatalyst;
- simple, cheap and quick to obtain immobilisation;
- no chemical changes required for support or biocatalyst;
- easily reversed to allow regeneration with fresh biocatalyst.
Disadvantages:
- leakage of biocatalyst from support/contamination of product;
- non-specific binding of other material to the support;
- overloading of a support with biocatalyst;
- steric hindrance of a biocatalyst by a support.
The most significant disadvantage is leakage or desorption of a biocatalyst from support material. This can occur under circumstances such as environmental changes in pH, temperature and ionic strength. Sometimes a firmly adsorbed enzyme is readily desorbed during reaction as a result of substrate binding, binding of contaminants present in the substrate, product production or other condition leading to change in conformation (molecular shape) of the adsorbed enzyme.
Physical characteristics of bioreactors such as flow rate, bubble agitation, particle–particle abrasion and scouring effect of particulate materials on reactor vessel walls can lead to desorption.Desorption can be turned to advantage if regeneration of support is built into the operational regime to allow rapid expulsion of ‘exhausted’ biocatalyst and replacement with fresh biocatalyst. In some commercial biotransformations, the economics of a process require the use of crude substrates containing contaminants that ‘poison’ the biocatalyst and so regular regeneration of biocatalyst is an essential part of the operation (Figure 2).
Non-specific binding can become a problem if substrate, product andor residual contaminants possess charges that allow interaction with a support material. This can lead to diffusion limitations and reaction kinetics problems, with consequent alteration in enzyme kinetic parameters . Further, binding of hydrogen ions to a support material can result in an altered pH microenvironment around a support with a consequent shift in pH optimum (1–2pH units) of a biocatalyst, and this may be detrimental to enzymes with precise pH requirements. Unless carefully controlled, overloading a support with excess biocatalyst can lead to low catalytic activity and in some circumstances a spacer molecule situated between an enzyme molecule and a support may be required to overcome problems of steric hindrance .

Figure 2. Graph showing the steady loss of immobilised enzyme with time and regeneration of activity at monthly intervals by addition of fresh enzyme.