Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 342

GA before bringing them into contact with the enzyme (functionalization, the method of
II-iii) or, alternatively, the adsorption of the enzyme on chitosan and its cross-linking,
always with GA, which is added either in the absence (cross-linking, the method of II-i) or
in the presence (conjugation, the method of II-i) of the enzyme solution. Conjugation
proved to be a better method as it ensured good biocatalyst activity and stability, better
than that reported for the free enzyme. It may be assumed in this case that the enzyme
was at least partially cross-linked already in the solution itself before forming any bonds
with the support, thus being able to benefit from the protective effects previously described
for the free enzyme [20]. However, functionalization, the method of II-iii, was most used in
enzyme immobilization by covalent binding (see Section 8.3.4.1).
An MPA mode would occur when the enzyme is immobilized via epoxy activation of
the support. Chitosan hydroxyl groups can be activated by using epoxide reactants such as
Gly and ECO, for instance, followed by oxidation with sodium periodate to produce reac-
tive aldehyde-glyoxyl groups. These aldehyde groups are less reactive than the ones from
GA. While the latter can immobilize the enzyme after linking with only one amine group,
the former will only be able to keep the enzyme linked if at least two bonds are formed.
Thus the higher the concentration of amine groups in the enzyme and of glyoxyl groups
in the support, more bonds can occur [21]. The method of immobilization involving the
amino and hydroxyl groups of chitosan is named “binary immobilization,” which is also
MPA [22] (see Section 8.3.4.2). In addition, changing the gel structure and immobilization
conditions led to significant improvement in the covalent MPA of chymotrypsin on chito-
san [23]. Adequate geometrical congruence between the enzyme and the support is very
important: the greater the enzyme-support congruence, the higher the possibility of
achieving an intense MPA. Therefore, the internal area of the support is one important
variable to obtain active and stable enzyme derivatives. The use of sodium alginate, gela-
tin, or κ-carrageenan, activation with GA, Gly, or ECO, and the addition of microorgan-
isms followed by cellular lysis allowed modification of the chitosan gel structure.
Entrapment usually refers to an immobilization enzyme within a polymer network (II-i).
Covering the surface of the enzyme with a film can also be considered as a kind of entrap-
ment. For example, Miao and Tan developed a silica sol-gel/organic hybrid material for
the fabrication of an amperometric H 2 O 2 biosensor by using TMOS as the silylating agent.
The homogeneous stock sol-gel/chitosan solution was pipetted to cover the HRP-modified
carbon paste electrode (CPE) and finally it was dried for ca. 10 min at room temperature.
The developed HRP electrode exhibits high sensitivity and fast response. It also shows
very good reproducibility and stability [16].
Chitosan films can be activated for protein assembly by anodic oxidation of the underly-
ing electrode in the presence of NaCl (method IV, electrochemically conjugate protein) [24].
Although the mechanism of anodic activation of chitosan has not been definitively estab-
lished, the working hypothesis is that a reactive mediator (possibly hypochlorite OCl-) is
electrochemically generated at the anode, and this mediator reacts with the chitosan film
to generate reactive substituents (possibly aldehydes) along chitosan's backbone. The elec-
trodeposited and electroactivated chitosan films react with proteins to assemble them
with spatial selectivity and quantitative control. The evidence presented indicates that the
assembled proteins retain their native structure and biological functions. This method for
on-demand biofunctionalization of individual electrode addresses should offer a generic
approach to assemble proteins for multiplexed analysis.
Polymer conjugation has been reported to reduce the autolysis of proteolytic enzymes
and to allow enzymes to dissolve in nonaqueous solvents. Advantages of these biocatalytic
composites are that the matrix can be selected to enhance performance and the composites