Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 349

able to rapidly immobilize proteins via a direct covalent attachment, the monomer yields
a very low immobilization rate. The activity/stability properties of enzymes immobilized
on GA-activated supports depend on the exact immobilization protocol employed.
Moreover, due to the existence of one or two ionic groups (amino groups) under GA, which
provide a certain anionic exchanger nature to the support, altering the ionic strength dur-
ing immobilization can modify the immobilization rate and also the region of the protein
that is implied in the interaction with the support. Since in this contribution immobiliza-
tion was performed in neutral medium, the reaction should have involved the most reac-
tive amino groups in the protein [40,41].
It was possible that enzyme conformation was not changed by covalent binding to
GA-pretreated chitosan beads. Desai et al. reported that the Michaelis constant ( K m ) and
the maximum reaction velocity ( V max ) of the free and immobilized porcine pancreatic
lipase were almost the same, indicating no conformational change taking place during
immobilization [42]. Moreover, the different structures of the lipases chosen for catalysis
led to very different activity levels. Lipases from Candida rugosa , Pseudomonas fluorescens, ,
and C. antarctica B were immobilized onto chitosan and GA-pretreated chitosan powders,
the latter derivatives being the most active [41].

8.3.4.1.2 Carbodiimide as a Cross-Linking Agent
Carbodiimide is generally utilized as a carboxyl-activating agent for amide bonding with
primary amines. It has been used in peptide synthesis, in cross-linking of proteins to
nucleic acids, and in the preparation of immunoconjugates, for example.
Chiou and Wu have demonstrated that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) can be used for activating the hydroxyl groups of chitosan. Chitosan beads were
produced by the deposition of chitosan solution under a mixture of ethanol and NaOH
solution. Figure 8.4 shows the hypothetical illustration of lipase immobilized on chitosan
beads using the activation method with EDC in the pH range of 4.0-6.0. It was assumed
that the hydroxyl groups of chitosan upon activation using EDC formed an unstable

R

N

pH 4-6

Chitosan

CH 2 OH + R N C N ′

CH 2 O

Chitosan C

Reaction of the same as with CH 2 OH

OH

NH

Acylisourea

R ′

E

NH 2

R

E

Chitosan CH 2 NH

I

N

Chitosan CH 2 O C Immobilized enzyme

NH

II

Chitosan CH 2 NH R ′ CONHR

R ′

Acylurea

Figure 8.4
Scheme of lipase immobilized on chitosan by EDC activation. (From Chiou, S. H. and Wu, W. T. 2004. Biomaterials
25: 197-204. With permission.)