Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 346

lyophilized overnight. Enzyme immobilization was carried out by absorption and subse-
quently cross-linking by GA [27]. Zhang et al. reported that a powdery carrier of chitosan
was obtained by adding chitosan solution to 2 mol/L NaOH and subsequently cross-
linking with GA. Laccase is immobilized in the GA-activated powdery carrier. Immobilized
laccase could be used repeatedly, and its removal efficiency for 2,4-dichlorophenol
(2,4-DCP) remained above 50% for up to six usages [28].
8.3.2.2 Spherical Beads
Juang et al. prepared highly swollen beads by spraying a chitosan acetic acid 0.1 L solution
into 125 mL of deionized water containing 15 g NaOH and 25 ml of 95% ethanol through
a nozzle (diameter 1.2 mm). The diameter of the wet beads was approximately 2.3 mm.
The activities of both acid phosphatase and β-glucosidase immobilized onto GA-activated
chitosan beads remained 76% and 94% of the original ones, respectively, up to 42 days [29].
Chitosan beads were also prepared by the emulsion method and used for the immobili-
zation of ω-transaminase of Vibrio fluvialis. . The water-in-oil emulsion was poured into a
12% NaOH solution with stirring and was left for 3 h to allow the formation of chitosan
beads. The average sizes of the prepared wet and dried chitosan beads were 1 mm and
150 μm, respectively. The specific surface area and the average pore size of the dried chito-
san beads were measured to be 90 m 2 /g and 15 nm, respectively. Enzyme was immobi-
lized in GA-activated chitosan beads. The yield of enzyme immobilization (54.3%) and its
residual activity (17.8%) were higher than those obtained with other commercial beads
(Eupergit ® C and Tentagel SCOOSu) [30].
8.3.2.3 Nanoparticles
In recent years, chitosan nanoparticles have been synthesized through different approaches,
such as the chemical coprecipitation process, sol-gel self-propagation and using a water-
in-oil microemulsion. The use of water-in-oil microemulsion is a potentially very useful
technique for the preparation of nanoparticles. The nanosized water droplets offer a unique
microenvironment for the formation of highly monodispersed nanoparticles. The growth
of particles is controlled by the size of the microemulsion droplets, particularly in anionic
microemulsion systems; therefore, chitosan nanoparticles could be prepared by micro-
emulsion. Commonly, hydrochloric acid (HCl) or acetic acid (HAc) was used to dissolve
chitosan in the water phase, and NaOH or organic solution was used as precipitant.
Wu et al. prepared chitosan nanoparticles in a water-in-oil microemulsion by using 2%
(wt) HAc and 30% (wt) tri- n -octylamine (TOA) as solvent and precipitant, respectively. It
was found that particle diameters were about 7 nm and the particles formed ovoid-shaped
aggregates. Using 0.05% HCl and 5.0 M NaOH as solvent and precipitant resulted in nano-
particles 10 nm in size that aggregated in the form of snowflakes. Enzyme immobilization
was carried out by absorption and subsequently cross-linking by GA. These particles
showed good loading ability for lipase immobilization and little loss of enzyme activity
was observed. The stability of the catalyst was very good; only 9% of enzyme activity was
lost after five cycles [31].
8.3.2.4 Chitosan Films
Zhang et al. reported that hemoglobin (Hb) can be effectively immobilized on chitosan
films as mimetic peroxidase. The dried film obtained by solvent evaporation is further