Biomedical Engineering Reference
In-Depth Information
Figure 8.1
Enzyme immobilization strategies: (a) Entrapment, (b) encapsulation, (c) solid support, and (d) self-immobilization.
Enzymes are represented by circles. (From Brady, D. and Jordaan, J. 2009. Biotechnol Lett 31: 1639-1650. With
permission.)
Enzyme entrapment (Figure 8.1a) and encapsulation (Figure 8.1b) protects enzymes by
preventing direct contact with the environment, thereby minimizing the effects of gas
bubbles, mechanical shear, and hydrophobic solvents, but has the drawback of mass
transfer limitations. For support-based immobilization (Figure 8.1c), adsorption is a rela-
tively simple and inexpensive method of immobilization, and does not chemically modify
the enzyme, but it has limitations as the enzyme tends to leach out, especially in aqueous
solvents. This can result in difficulties in process design and downstream processing.
Hence the method is best suited to immobilize lipases for use in organic solvents. Ionic
binding is another simple noncovalent immobilization technique. Enzymes can be bound
to polysaccharide biopolymers such as dextran, agarose, and chitosan. The most interest-
ing recent developments are in the area of immobilization through covalent binding.
Improvements in current strategies for carrier-based immobilization have been developed
using heterofunctionalized supports that enhance binding efficacy and stability through
multipoint attachment (MPA) [5].
Multipoint covalent attachment of enzymes on highly activated preexisting supports via
short spacer arms and involving many residues placed on the enzyme surface promotes a
rigidification of the enzyme structure of the immobilized enzyme (Figure 8.2). The relative
distances among all residues involved in the multipoint immobilization have to be main-
tained unaltered during any conformational change induced by any distorting agent (heat,
organic solvents, and extreme pH values). This should reduce any conformational change
involved in enzyme inactivation and greatly increase the enzyme stability. The character-
istics of the support, reactive groups, and immobilization conditions need to be carefully
selected to be able to involve the maximum number of enzyme groups in the immobiliza-
tion. A support suitable for protein multipoint immobilization requires fulfilling some
characteristics, for example, large internal surface, high superficial density of reactive
groups, minimal satiric hindrances of reactive groups between the protein and the sup-
port, and enough stable reactive groups placed in the enzyme surface [6].
Moreover, novel methods of enzyme self-immobilization (Figure 8.1d) have been devel-
oped (cross-linked enzyme crystals (CLECs) and cross-linked enzyme aggregates (CLEAs)).
As mentioned above, the use of solid supports for enzyme immobilization can reduce the
specific and volumetric activity of the biocatalyst by a factor of 10 or more. Carrier-free
enzyme immobilization is possible using bifunctional cross-linkers, such as glutaralde-
hyde (GA), to bind enzymes to each other without resorting to a support. CLEC formation
requires extensive protein purification and method development and, although broadly
applicable, it only works for crystallizable enzymes. Then, a less-expensive method of
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