Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 362

NH 2

HO

O

Enzyme +

nH +

O

OH

n

Insoluble precipitate

NH +

HO

Enzyme O

O

OH

n

Soluble

Enzyme

Hydrogel matrix

Figure 8.9
Enzyme-chitosan conjugate for pH-responsive solubility (upper path) and biocatalytic hydrogels (lower path).
(From Vazquez-Duhalt, R. et al. 2001. Bioconjug Chem 12: 301-306. With permission.)

matrixes of various shapes and sizes including beads, membranes, and fibers (the lower
path in Figure 8.9) [78].
One major problem of enzyme conjugation or immobilization is the loss of enzyme activ-
ity, which is attributed to many factors involving enzyme, polymer, reagents, and process
conditions. Delanoy et al. investigated the effect of the molecular size of chitosan on lac-
case conjugation and the activity loss in the conjugation process. It was demonstrated that
the chitosan molecular size has little effect on the first moderate activity loss in the conju-
gation reaction, but has a visible effect on the substantial activity loss associated with
phase change. Small chitosan molecules gave high residual activity. The conjugated lac-
case exhibited a high stability in the following repeated phase changes and had the same
temperature and pH profile as those of free laccase. Compared with free laccase, the con-
jugated laccase had a similar affinity ( K m ) but reduced turnover ( k cat ) that was adversely
affected by an increase of the molecular mass of chitosan [79].

8.4.3.2 Invertase-Chitosan Conjugate

Adsorption of enzymes on charged supports through electrostatic interactions has been
widely used for immobilizing enzymes because of the easiness of preparation and the pos-
sibility of reuse of the supports. However, immobilization of native enzymes on conven-
tional ionic exchanger resins is frequently not very strong, and most proteins are fully
desorbed from such matrices at moderate ionic strength or pH changes.
Gómez et al. developed a new procedure for immobilizing enzymes on charged solid sup-
ports via electrostatic interactions and it received special attention in enzyme technology.
PEC formation mediated immobilization of an invertase-chitosan conjugate on sodium