Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 338

Pure immobilization of enzymes Multipoint immobilization

OH

OH

OH

No rigidification Enzyme rigidification

Figure 8.2
Effect of immobilization on enzyme stability. (From Mateo, C. et al. 2007. Enzyme Microb Technol 40: 1451-1463.
With permission.)

enhancing enzyme proximity for cross-linking is by simply precipitating the protein and
cross-linking the aggregates to form particles of about 50-100 μm diameter. These CLEAs
were developed in Sheldon's laboratory and commercialized by CLEA Technologies (the
Netherlands) [5].
Several enzymes have been successfully immobilized using CLEA techniques, includ-
ing penicillin G acylase, lipases, and nitrilases [7-9]. Precipitants were found to have a
profound influence on both specific activities and total activity recovery of CLEAs, as
exemplified by Candida antarctica lipase B (CALB) [9]. Among the CLEAs of CALB studied,
those obtained using PEG600, ammonium sulfate, PEG200, and acetone as precipitants
were observed to attain over 200% total activity recovery in comparison with acetone pow-
der directly precipitated from the liquid solution by acetone. PEG200-precipitated CLEA
gave the best specific activity (139% relative to acetone powder). The results of kinetic stud-
ies showed that V max / K m does not significantly change upon CLEA formation. The diver-
sity of enzymes able to form active CLEA, however, might be restricted by their ability to
resist chemical cross-linking. Extensive enzyme aggregation in CLEAs must also be
avoided to prevent mass-transfer limitations during catalysis.

8.2.2 Several Supports of enzyme immobilization

The results of immobilization, including the performance of immobilized enzymes,
strongly depend on the properties of supports, which are usually referred to as material
type, composition, structure, and so on. So far, different nanostructured materials have
been used as supports, such as nanoparticles, nanofibers, nanotubes and mesoporous sil-
ica, and single enzyme nanoparticles [10,11]. They stand out from other supports because
of their extremely high surface area-to-volume ratios, which can provide large specific
surface areas for highly efficient immobilization as well as stabilize enzymes. Several
involved materials in this chapter are briefly described below.
Nanoparticles provide an ideal remedy for the usually contradictory issues encountered in
the optimization of immobilized enzymes: minimum diffusional limitation, maximum sur-
face area per unit mass, and high enzyme loading. Moreover, the MPA of enzyme molecules