Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 357

8.4.1 Chitosan Derivatives
8.4.1.1 Hydrophobically Modified Chitosan
8.4.1.1.1 Modified by Linolenic Acid
Chitosan (CS) was modified by linolenic acid (LA), which can form nanoparticles on LACS
in pH 7.4 phosphate-buffered saline buffer after sonication and can also be used as an
enzyme carrier. Trypsin could be immobilized on linolenic acid chitosan using GA as a
cross-linking agent. The immobilized trypsin at certain GA concentrations (from 0.03% to
0.07% v/v) can form nanoparticles after sonication, which still have catalytic activities. The
kinetic constant ( K m ) of trypsin immobilized on nanoparticles (71.9 mg/mL) was higher
than that of pure trypsin (50.2 mg/mL), indicating that the immobilized process slightly
decreased the affinity of trypsin on nanoparticles to the substrate (casein). On the other
hand, this formation can improve the thermal stability and optimum temperature of
trypsin, which makes it more attractive in the application aspect [65].
8.4.1.1.2 Modified by LA
Modified by aldehydes with long alkyl chain lengths. Chitosan also can be easily hydro-
phobically modified by reductive amination using aldehydes with long alkyl chain lengths
(butanal, hexanal, octanal, or decanal). Although chitosan modified with small alkyl
chains (i.e., <4 carbon chain length) is soluble in dilute aqueous acetic acid, the introduc-
tion of longer chain side groups renders the modified polymer relatively increasingly less
soluble in dilute acidic aqueous solution.
Klotzbach et al. examines how hydrophobic modification of both Nafion and chitosan
alters the selectivity, ion-exchange capacity, morphology, and mass transport of redox spe-
cies through the membrane. It was shown that hydrophobically modified micellar poly-
mers alter the transport properties of redox species to the electrode surface as a function of
the size and charge of the redox species. GOx was immobilized within the hydrophobically
modified chitosan. It was shown that the increase in hydrophobicity increases the enzyme
activity, resulting in a more optimal membrane for enzyme immobilization [66].
As in the above descriptions, over the last decade, researchers have employed reductive
amination to hydrophobically modify chitosan to induce a micellar structure. However,
commercial sources of chitosan vary in their degree of deacetylation and there remains a
paucity of information regarding how this can impact the modified polymer's functional-
ity for enzyme immobilization. Sjoholm et al. evaluate the effect that the degree of deacety-
lation has on the hydrophobic modification of medium-molecular-weight chitosan via
reductive amination with long-chain aldehydes (butyraldehyde) and the resulting changes
in enzyme activity after the immobilization of GOx in the micellar polymeric structure.
The chitosan was deacetylated to differing degrees via autoclaving in 40-45% NaOH solu-
tions. The results suggest that a high degree of deacetylation provides optimal enzyme
immobilization properties (i.e., high activity), but that the deacetylation method begins to
significantly decrease the polymer molecular weight after a 20 min autoclave treatment,
which negatively affects immobilized enzyme activity [67].
8.4.1.2 2-diethylaminochloroethane (DE)-Chitosan
α-l-Rhamnopyranosidase (Rha; EC 3.2.1.40) is an enzyme of considerable importance to
food technology in increasing the aroma of wines, musts, fruit juices, and other beverages.
Chitosan only adsorbs the Rha if activated with GA; nevertheless, the enzyme immobilized