Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 361

metal ions-chelated matrices could be suitable for immobilization of enzyme due to their
intrinsically high specific surfaces, providing the quantity and accessibility of the binding
sites necessary for a high immobilization capacity and a large surface area for enzymatic
reaction.
Bayramoglu et al. prepared a chitosan membrane by using the phase-inversion tech-
nique and then cross-linking with ECO under alkaline conditions. Itaconic acid was
grafted on the cross-linked chitosan membrane via ammonium persulfate initiation under
a nitrogen atmosphere. Poly(itaconic acid)-grafted and/or Fe(III) ions-incorporated chito-
san membranes were used for reversible immobilization of CAT (from bovine liver) via
adsorption. In this method, grafted poly(itaconic acid) acted as a metal-chelating ligand,
and there is no need of any reaction step to activate the matrix for the chelating-ligand
immobilization. In addition, grafted poly(itaconic acid) brush provides a hydrophilic
microenvironment for the guest enzyme. It was observed that the same support enzyme
can be repeatedly used for immobilization of CAT after regeneration without significant
loss of adsorption capacity or enzyme activity [76].
8.4.2.4 Chitosan-Grafted-Polyethyl Acrylate
A method has been developed to immobilize HRP on modified chitosan beads by means
of graft copolymerization of polyethyl acrylate (PEA) in the presence of potassium persul-
fate and Mohr's salt redox initiator. The immobilization of HRP on modified chitosan
beads was carried out in three steps. The first step involves the graft copolymerization of
EA onto chitosan by potassium persulfate and Mohr's salt as the combined redox initiator.
In the second step, the majority of the grafted polyethylacrylate chains were converted into
polyacrylic hydrazide by the reaction with hydrazine hydrate, followed by beads activa-
tion through acyl azide formation and then coupling of the enzyme by the immersion of
the activated beads in the HRP enzyme solution in the third step. The activity of immobi-
lized HRP decreased slowly with time as compared with that of free HRP and could retain
65.8% residual activity after six consecutive operations. This immobilization remarkably
improved the temperature and operational stability, which made it more attractive in
application aspects [77].
8.4.3 enzyme-Chitosan Conjugate
8.4.3.1 Laccase Conjugation to Chitosan
Different from the conventional immobilization of laccase on preformed solid supports
or by physically trapping in gels, conjugation of laccase and chitosan occurs in a solution
by forming covalent bonds between the protein and polysaccharide molecules. Once
bound, the molecules are strongly coupled and distributed uniformly in the conjugate
aggregates or gels. Since the protonation of amino groups on the chitosan backbone is
dependent on the solution pH, the laccase conjugate becomes a pH-sensitive biomaterial
and can adjust its aggregation state in response to pH variation, as indicated in the upper
part of Figure 8.9. The conjugates, therefore, can undergo repeated phase change with
little loss of enzyme proteins, a useful feature for novel applications in bioremediation,
organic synthesis, biosensing, and immunoenzyme assays. Additionally, chitosan's
amino groups are nucelophilic and reactive at higher pH values. The reactivity of these
amino groups allows chitosan to be cross-linked under mild conditions to create gel