3.3 Immobilization
In practice, three significant drawbacks are often encountered in enzyme-catalyzed
reactions.
• Many enzymes are not sufficiently stable under the operational conditions and
they may lose catalytic activity due to auto-oxidation, self-digestion and/or
denaturation by the solvent, the solutes or due to mechanical shear forces.
• Since enzymes are water-soluble molecules, their repeated use, which is important to ensure their economic application [439], is problematic because their
separation from substrates and products and their recovery from aqueous systems is difficult.
• The productivity of industrial processes, measured as the space-time yield is
often low due to the limited tolerance of enzymes to high concentrations of
substrate(s) and product(s).
These problems may be overcome by ‘immobilization’ of the enzyme [440–
452]; two main strategies exist: (i) The enzyme is attached onto a solid support via
coupling onto a carrier or the enzyme molecules are linked onto each other via
cross-linking. (ii) Alternatively, the biocatalyst may be spatially confined to a
restricted area from which it cannot leave but where it remains catalytically active
by entrapment into a solid matrix or a membrane-restricted compartment. Thus,
homogeneous catalysis using a native enzyme turns into heterogeneous catalysis
when the biocatalyst is immobilized. Depending on the immobilization technique,
enzyme properties, such as stability, selectivity [453, 454], k cat and K M value, pH
and temperature characteristics, may be significantly altered [455] – sometimes for
the better, sometimes for the worse. At present, predictions about the effects of
immobilization on the catalytic performance of proteins are very difficult to make.
The following immobilization strategies are most widely employed (Fig. 3.5)
[456, 457].
Adsorption
Adsorption of a biocatalyst onto a water-insoluble macroscopic carrier is the easiest
and oldest method of immobilization. It may be equally well applied to isolated
enzymes as well as to whole viable cells. For example, adsorption of whole cells of
Acetobacter onto wood chips for the fermentation of vinegar from ethanol was first
used in 1815! Adsorbing forces are of different types, such as van der Waals
(London) forces, ionic interactions, and hydrogen bonding, and are all relatively
weak. The appealing feature of immobilization by adsorption is the simplicity of the
procedure. As a result of the weak binding forces, losses in enzyme activity are
usually low, but desorption (leakage) from the carrier may be caused by even minor
changes in the reaction parameters, such as a variation of substrate concentration,
the solvent, temperature, or pH.
3.3 Immobilization
369
In practice, three significant drawbacks are often encountered in enzyme-catalyzed
reactions.
• Many enzymes are not sufficiently stable under the operational conditions and
they may lose catalytic activity due to auto-oxidation, self-digestion and/or
denaturation by the solvent, the solutes or due to mechanical shear forces.
• Since enzymes are water-soluble molecules, their repeated use, which is important to ensure their economic application [439], is problematic because their
separation from substrates and products and their recovery from aqueous systems is difficult.
• The productivity of industrial processes, measured as the space-time yield is
often low due to the limited tolerance of enzymes to high concentrations of
substrate(s) and product(s).
These problems may be overcome by ‘immobilization’ of the enzyme [440–
452]; two main strategies exist: (i) The enzyme is attached onto a solid support via
coupling onto a carrier or the enzyme molecules are linked onto each other via
cross-linking. (ii) Alternatively, the biocatalyst may be spatially confined to a
restricted area from which it cannot leave but where it remains catalytically active
by entrapment into a solid matrix or a membrane-restricted compartment. Thus,
homogeneous catalysis using a native enzyme turns into heterogeneous catalysis
when the biocatalyst is immobilized. Depending on the immobilization technique,
enzyme properties, such as stability, selectivity [453, 454], k cat and K M value, pH
and temperature characteristics, may be significantly altered [455] – sometimes for
the better, sometimes for the worse. At present, predictions about the effects of
immobilization on the catalytic performance of proteins are very difficult to make.
The following immobilization strategies are most widely employed (Fig. 3.5)
[456, 457].
Adsorption
Adsorption of a biocatalyst onto a water-insoluble macroscopic carrier is the easiest
and oldest method of immobilization. It may be equally well applied to isolated
enzymes as well as to whole viable cells. For example, adsorption of whole cells of
Acetobacter onto wood chips for the fermentation of vinegar from ethanol was first
used in 1815! Adsorbing forces are of different types, such as van der Waals
(London) forces, ionic interactions, and hydrogen bonding, and are all relatively
weak. The appealing feature of immobilization by adsorption is the simplicity of the
procedure. As a result of the weak binding forces, losses in enzyme activity are
usually low, but desorption (leakage) from the carrier may be caused by even minor
changes in the reaction parameters, such as a variation of substrate concentration,
the solvent, temperature, or pH.
3.3 Immobilization
369