Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 347

neutralized by 2.0 wt% NaOH aqueous solution. Hb is immobilized in GA-activated
chitosan film. The enzymatic assay indicates that immobilized Hb showed a higher thermal
stability than free Hb, and catalytic activity in organic solvents was also enhanced [32].
8.3.3 ionization gelation Method
Chitosan can form gel by ionotropic gelation with certain anionic counterions, such as
polyphosphates. Sodium TPP is a multivalent anion that can cross-link by ionic interaction
between positively charged amino (-NH 3 + ) groups of chitosan and multivalent negatively
charged TPP molecules in an acid medium. The method is utilized chiefly for the prepara-
tion of gel beads or nanoparticles, within which enzymes are usually entrapped. Chitosan
complexation with anionic polyelectrolytes will be described in Section 8.5.3.
8.3.3.1 Chitosan Beads
Betigeri and Neau prepared immobilized lipase by entrapment in three polysaccharides.
Chitosan beads are obtained by adding a mixture of acidic chitosan solution and lipase
dropwise into a TPP solution, after which the characteristics of immobilized enzyme are
compared with other hydrophilic polymers, including alginate beads prepared by ionic
gelation using calcium chloride and agarose beads by adding the heated solution drop-
wise into chilled vegetable oil. Agarose beads exhibited undesirable swelling in the leach-
ing and activity medium and the polymer was not used further. The fact that the lipase
activity in chitosan beads is higher than that in alginate beads could be attributed to an
alginate-enzyme interaction [33].
8.3.3.2 Micro/Nanoparticles
Micro- or nanochitosan can be obtained by a reversed process by comparing with the
formation of chitosan beads discussed in ref. [33]. Fernandes et al. developed a novel
biosensor based on laccase immobilized on microspheres of chitosan cross-linked with
tripolyphosphate for rutin determination in pharmaceutical formulations by square wave
voltammetry. Microspheres of chitosan were obtained by adding 2% (m/v) TPP solution
into 1% (m/v) chitosan solution (1% acetic acid) under stirring and subsequently spray dry-
ing this mixture solution. The microspheres had an irregular shape and were 3.0-8.9 μm
in diameter. This proposed bioelectrode exhibited high sensitivity, good reproducibility,
low detection, rapid response and excellent long-term stability. The determination of rutin
in three pharmaceutical formulations was successfully carried out without any separation
step before the electrochemical measurement [34].
In order to investigate the characterization of chitosan nanoparticles prepared by the
ionization gelation method, neutral proteinase and lipase were selected as model enzymes
to assess its potential applicability for enzyme immobilization. Chitosan nanoparticles of
mean particle size smaller than 100 nm were obtained by adding 0.75 mg/mL TPP to chi-
tosan solution (20 mg of chitosan was dissolved in 40 mL of 2.0% (v/v) acetic acid). Enzyme
could be immobilized on the surface of chitosan nanoparticles by adsorption. The thermal,
operational, and storage stabilities of immobilized enzymes were improved after they
were immobilized on chitosan nanoparticles. They could improve 13.17% of neutral protei-
nase or lipase activity than that of free neutral one [35,36].
Biró et al. prepared macro-, micro-, and nanosized chitosan particles suitable as immo-
bilization carriers by precipitation, emulsion cross-linking, and ionic gelation methods,