Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 345

of electrodes. For example, the chitosan-modified electrode was fabricated by casting the
chitosan solution on the surface of a glassy carbon electrode (GCE) and evaporating the sol-
vent under room temperature in air. A cross-linking agent may also be mixed with the
initial chitosan solution before drying. For composites of chitosan and polymer or inor-
ganic materials, such as PECs, metal oxide-chitosan nanocomposite, chitosan-silica hybrid
material, and so on, this technique involves depositing/casting a complex fluid on a sub-
strate by dip, spin coating, or spray drying, followed by allowing the film to evaporate and
form self-assembled structures. Sometimes, repeated cross-linking is necessary. Enzymes
may be immobilized on such prepared membranes by adsorption or entrapment or chemi-
cal cross-linking.
8.3.1.1 Spin Coating
Spin coating is a procedure used to apply uniform thin films to flat substrates. In short, an
excess amount of a solution is placed on the substrate, which is then rotated at high speed
in order to spread the fluid by centrifugal force. A machine used for spin coating is called
a spin coater, or simply a spinner. Azmi et al. described an optical biosensor based on glu-
tamate dehydrogenase (GLDH) immobilized in a chitosan film for the determination of
ammonium in water samples. The GLDH-chitosan mixture was deposited onto a glass
slide. Then it was spun at 2000 rpm for 3 s. The biosensor was kept at 4°C for drying. The
reproducibility of the biosensor was good [25].
8.3.1.2 Spray Drying
The spray drying technique is widely used in the food and pharmaceutical industries. The
method is based on drying atomized droplets in a stream of hot air. González Siso et al.
prepared microbeads by spray drying of a 0.3% chitosan solution with different concentra-
tions of an enzyme and a reticulating agent (GA). The average size of the microbeads was
2.5-4 μm. Microencapsulation on chitosan beads has been shown to be an effective immo-
bilization method for α-amylase and invertase and the observed differences in their behav-
ior are explained mainly by the molecular weight of their substrates [26].
8.3.2 Phase-inversion Technique
The phase-inversion technique can produce chitosan gel via precipitation under an alkali
solution or an organic medium or their mixture solution. This method is exploited to pro-
duce chitosan precipitates, membranes, and fibers, but most importantly spherical beads of
different sizes and porosities. However, these supports cannot endure low pH and their
intensity is low; therefore, chemical cross-linking is often used. Enzyme immobilization is
similar to the solvent evaporation method. It is a simple, easy, and inexpensive method for
using the deposition of chitosan as an enzyme immobilization support.
8.3.2.1 Chitosan Powders
Briante et al. described a methodology to produce in high yield hydroxytyrosol from a
commercially available oleuropein by using the immobilized recombinant β-glycosidase
from the hyperthermophilic archaeon Sulfolobus solfataricus on a chitosan support.
Chitosan precipitates were prepared by adding NaOH (1.5 M final concentration) into a
low-molecular-weight chitosan acetic acid solution. Neutralized chitosan suspension was