Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 364

material that can be further removed. The preparation of macroporous beads and membranes
in the presence of silica during gel formation, followed by the removal of the silica after
dissolution at alkaline pH, has already been reported. When a similar approach is used,
the gel could be formed in the presence of cells of microorganisms, followed by cell lysis.
After removing the cellular material, the resulting polymer may have a higher internal
surface area, favoring enzyme immobilization. It was shown that changing gel structure
and immobilization conditions led to a significant improvement of the covalent MPA of
chymotrypsin on chitosan. Enzyme immobilization for 72 h at pH 10.05 and 25°C and
reduction with NaBH 4 in chitosan 2.5%-carrageenan 2.5%, with the addition of S. cerevisiae
5% and activation with ECO, led to the best derivative, which was 9900-fold more stable
than the soluble enzyme [23].
8.5.2 Chitosan-Tethered Membrane
8.5.2.1 Chitosan-Tethered Poly(Acrylonitrile) Membrane
The chemically modified polyacrylonitrile (PAN) membrane, which possesses excellent
properties, such as good thermal and mechanical stability, has been successfully applied
as a membrane matrix for enzyme immobilization.
8.5.2.1.1 Chitosan-PANCMA Membrane
Ye et al. proposed a protocol to prepare a dual-layer biomimetic membrane as a support for
enzyme immobilization by tethering chitosan on the surface of a poly(acrylonitrile- co -maleic
acid) (PANCMA) ultrafiltration hollow fiber membrane in the presence of EDC and NHS.
Lipase from C. rugosa was immobilized on this dual-layer biomimetic membrane using GA
and on the nascent PANCMA membrane using EDC/NHS as a coupling agent. It was found
that both the activity retention of the immobilized lipase and the amount of bound protein
on the dual-layer biomimetic membrane were higher than those on the nascent PANCMA
membrane. After immobilization, the pH, thermal, and reuse stabilities of the immobilized
enzyme increased [83]. In order to further raise enzyme loading on the support and to reduce
the diffusion resistance for the immobilized enzyme, PANCMA was fabricated into nanofi-
brous membranes by the electrospinning process. It was found that there is an increase of
activity retention of the immobilized lipase on the chitosan-modified nanofibrous mem-
brane (45.6 ± 1.8%) and on the gelatin-modified one (49.7 ± 1.8%) as compared with that on
the nascent one (37.6 ± 1.8%). The kinetic parameter K m of the immobilized lipase on the
nanofiber membranes was lower than that of the hollow fiber membrane [17].
Immobilization of lipase on this dual-layer biomimetic support by adsorption is also
investigated. The activity retention of the immobilized lipase on the chitosan-tethered
membrane by adsorption (54.1%) is higher than that by chemical bonding (44.5%).
Additionally, the experimental results on thermal stabilities indicate that the residual
activity of the immobilized lipase at 50°C is 38% by adsorption and 65% by chemical bond-
ing [84]. These results demonstrated that the dual-layer biomimetic membrane was a
potential support in the enzyme immobilization technology for industrial applications
such as the manufacture of acid by the hydrolysis of triglyceride.
8.5.2.1.2 Chitosan-Poly(Acrylonitrile-MethylMethacrylate-Sodium Vinylsulfonate)
(PANMV) Membrane
Godjevargova and coworkers report the formation and characterization of ternary copoly-
mer poly(acrylonitrile-methylmethacrylate-sodium vinylsulfonate) (PANMV)/chitosan