Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 341

or entrapped); (II-iii) on the agent-activated support; (II-iv) cross-linking by
covalent binding of the enzyme to an agent-activated support following cross-
linking with other agents.
III. Entrapment: (III-i) entrapment of the enzyme within a polymer network; (III-ii)
covering the surface of the enzyme with a film.
IV. Electrochemically conjugated protein.
V. Enzyme-chitosan conjugate.
Methods such as adsorption, entrapment, and cross-linking involve some immobiliza-
tion processes described in Section 8.2.1. However, the operations in detail take on diver-
sity due to special properties of chitosan/chitin. In addition, some peculiar enzyme
immobilization methods (IV and V) are also presented here.
Immobilization of the enzyme on chitosan/chitin powder can be easily fulfilled by phys-
ical adsorption. Furthermore, chitosan has been widely used as a sorbent for transition
metal ions and organic species because the amino (-NH 2 ) and hydroxyl (-OH) groups on
chitosan chains can serve as the coordination and reaction sites. Thus chitosan particle-
adsorbed metal ions or dye can be used as an affinity support for enzyme immobilization
in chromatography or biosensors (see Section 8.3.4.3).
In addition, a protein engineered to carry affinity tags from heterologous sources is able
to bind specifically to its unnatural cognate ligands. This approach has become widely
acceptable for enzyme immobilization based on the following merits: (1) strong binding of
enzymes to the support, (2) proper exposure of the enzyme active site, (3) mild conditions
for immobilization, and (4) the lack of substrate diffusion barriers. Chao et al. have explored
the utilization of the ChBD as an affinity tag to retain d-hydantoinase or Z. mobilis levansu-
crae (encoded by levU ) on chitin beads. ChBD of chitinase A1 from Bacillus circulans WL-12
comprises 45 amino acids and exhibits remarkably high specificity to chitin. To investigate
the feasibility of exploiting ChBD as affinity tags to confine enzymes of interest on chitin,
ChBD fused to the C-terminus of the gene encoding enzyme was constructed. After direct
absorption of the protein mixture from Escherichia coli onto chitin beads, the enzyme tagged
with ChBD was found to specifically attach to the affinity matrix. Subsequent analysis indi-
cated that the linkage between the enzyme and chitin beads was substantially stable.
In comparison with its unbound counterpart, d-hydantoinase immobilized in this way
exhibits higher tolerance to heat and can be reused 15 times to achieve conversion yields
exceeding 90% [18]. With 20% sucrose, the production of levan was enhanced by 60% to
reach 83 g/L using the immobilized levansucrase as compared with that by the free coun-
terpart [19]. As illustrated in the above studies, this approach is marked by high stability as
well as facile operation and provides a promising way for enzyme immobilization.
Protocols for covalent enzyme immobilization often begin with a surface modification
or activation step. Chitosan has reactive amino and hydroxyl groups that, after further
chemical modifications, can make covalent bonds with reactive groups of the enzyme.
Mostly, immobilization of enzymes on such prepared gels does not require chemical
activation, as the cross-linker, normally a bifunctional agent, fulfills two functions: cross-
linking and activation. GA, as the most commonly used cross-linking agent, was applied for
activating the -NH 2 group. Other bifunctional agents, such as carbodiimide and epoxide
reactants (glycidol (Gly) and epichlorohydrin (ECO)), were used to activate -OH groups.
Spagna et al. presented various enzyme immobilization trials by covalent binding.
α-l-Arabinofuranosidase (Ara) from Aspergillus niger was immobilized on chitin or chito-
san (particle sizes between 75 and 125 μm) by means of the activation of the supports with