Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 350

complex, acylisourea, which then complexed with the amino groups of the enzyme to
form the immobilized enzyme (product I). On the other hand, it could be rearranged to
form another product, acylurea (product II). The process appreciably increased the activity
of lipase immobilized on wet chitosan beads [43].
8.3.4.1.3 Epoxide Reactants as a Cross-Linking Agent
Chitosan hydroxyl groups can also be activated by using epoxide reactants such as Gly
and ECO. An MPA mode appeared when the enzyme was immobilized via epoxy activa-
tion of the support [21].
Spagna et al. studied the coimmobilization of two glycosidases, Ara and β-d-glucopy-
ranosidase (βG), on chitosan or glyceryl chitosan (GCh), a derivative especially prepared
by the reaction of chitosan with Gly. The glycosidases adsorbed on this latter support
were then cross-linked with GA to prevent them from being released into the wine.
Finally, the biocatalyst was reduced with sodium borohydride to increase its stability
over time. GCh further increased the adsorption of Ara while allowing significant
increases in activity. When cross-linking the enzymes adsorbed on GCh with GA, the
activity of βG was not affected even though that of Ara was reduced [44].
Giordano and coworkers reported that trypsin was immobilized on chitosan gels coagu-
lated with 0.1 or 1 M NaOH and activated with GA or Gly. Activation with Gly led to lower
immobilization yields than the ones obtained with GA, but allowed obtaining the most
stable derivative chitosan-glyoxyl (ChGly), which was 660-fold more stable than the soluble
enzyme at 55°C and 70°C. The ChGly derivative also presented the highest stability during
incubation at pH 11. Analyses of lysine residue contents in soluble and immobilized trypsin
indicated the formation of multipoint bonds between the enzyme and the support for
glyoxyl derivatives [45].
Supports activated with ECO displayed a higher degree of activation than the ones acti-
vated with Gly. However, chitosan-EDC was only 1.6-fold more thermally stable than the
soluble enzyme CALB, which indicates that immobilization of CALB on chitosan-ECO is
not the result of a multipoint covalent attachment. It was observed that chitosan activated
by ECO lost a great amount of water, which may decrease the porous diameter of the sup-
port and increase its hydrophobicity. Then, despite the high aldehyde concentration, the
enzyme would not reach most of these groups. Therefore, physical changes in the support
surface may not favor immobilization of CALB [21].
8.3.4.1.4 Tris(Hydroxymethyl)Phosphine as Cross-Linking Agent
The most commonly used coupling reagent is GA, although its chemistry creates some
inherent difficulties because of the continuing polymerization of GA upon storage and
the reversibility of the Schiff base linkage. To overcome these difficulties of coupling
with GA, Cochrane et al. reported that a coupling agent, P(CH 2 OH) 3 , which contains
>P-CH 2 -OH groups, is well known to undergo Mannich-type condensation reactions at
room temperature with N-H group-containing compounds. The potential advantages
of using P(CH 2 OH) 3 as a coupling agent include an increase in the number of immobi-
lizing groups, together with an improved hydrolytic stability of the resulting P-CH 2 -N-
enzyme linkages. The use of P(CH 2 OH) 3 as a coupling reagent for the immobilization of
alcohol dehydrogenase onto chitosan films and for the attachment of the chitosan film
to a glass support resulted in enzyme activities far above those obtained by adsorption
of enzyme and greater than those observed when using the more conventional
GA-coupling protocol [46].