Thanks to the latest breakthroughs in the research for improving the enzymes, nowadays most enzymes are produced for a commercially acceptable price. Nonetheless, the industrial

utilization of cellulases could be even more convenient by improving their stability in long­term operations and by developing methods/processes for the downstream recovery and reuse. These objectives can be achieved by the immobilization of the enzymes (Cao, 2005). The main advantages of the enzyme immobilization are:

1. more convenient handling of enzymes

2. easy separation from the product

3. minimal or no protein contamination of the product

4. possible recovery and reuse of enzymes

5. enhanced stability under storage and operational conditions (e. g. towards denaturation by heat or organic solvents or by autolysis) (Sheldon, 2007).

The main methods of enzyme immobilization can be classified into four classes: support binding (carrier), entrapment, encapsulation and cross-linking.

Support binding is based on fixing the enzyme to the external or internal surface of a substrate, by physical (adsorption), ionic or covalent bonding. Adsorption is a simple and inexpensive method of immobilization, and does not modify the enzyme chemical structure. However, it does not produce strong bonds between enzyme and substrate and this could cause a progressive lost of the enzyme from the support. Ionic-binding determines a strong bond between enzyme and support. The supports may be functionalized with a variety of chemical groups to achieve the ionic interaction, including quaternary ammonium, diethylaminoethyl and carboxymethyl derivates (Brady & Joordan, 2009). Covalent binding is the most widely used method of immobilization. Here the amino group of lysine is typically used as point of covalent attachment (Brady & Joordan, 2009). Lysine is a very common amino-acid in proteins, often localized on the surface of proteins. It has a good reactivity and provides acceptable bonds stability (Krenkova & Forest, 2004). Supports containing epoxy groups are widely used in the immobilization by covalent binding. These can react with lysine and with many other nucleophilic groups on the protein surface (e. g. Cys, Hys, and Tyr). Epoxy groups also react, in a slower way, with carboxylic groups (Mateo et al., 2007). The support used in this immobilization method is typically a prefabricated carrier, such as synthetic resins, biopolymers, inorganic polymers such as silica or zeolites.

Entrapment is based on inclusion of the enzyme in a polymer network (i. e. organic polymer, silica sol-gel). Unlike the previous methods, entrapment requires the synthesis of the polymeric network in the presence of the enzyme (Sheldon, 2007). This method has the advantage of protecting the enzyme from direct contact with the environment, reducing the effects of mechanical sheer and hydrophobic solvents. However, low amount of enzymes can be immobilized (Lalonde & Margolin, 2002).

Encapsulation is a method similar to entrapment, but, in this case, the enzyme is enclosed in a membrane that acts as a physical barrier around it (Cao L., 2005). The disadvantage is that entrapping or encapsulating matrix offer a certain resistance to the substrates diffusion.

Cross-linking results in the formation of enzyme aggregates by using bifunctional reagent, like glutaraldehyde, able to bind enzymes to each other without resorting to any support. In 1996, cross-linked enzyme crystals (CLEC; St. Clair and Navia 1992) were commercialized by Altus Biologics (Margolin, 1996). However the CLEC formation requires laborious and expensive processes of protein purification and it is applicable only to crystallisable enzymes. In addition, only one kind of enzyme can be used in the CLEC formation (Brady & Joordan, 2009). In 2001 a less-expensive method, known as CLEA (cross linked enzyme aggregates) was developed in Sheldon’s Laboratory and commercialized by CLEA Technologies (Netherlands), (Sheldon et al., 2005). Recently a new method has been developed, especially suitable for lipase immobilization. It is defined Spherezymes and it is based on the formation of a water-in-oil emulsion, in which lipases and surfactant are dissolved. Following the addition of a bifunctional cross-linker, permanent spherical particles of enzyme are generated (Brady & Joordan, 2009).

The most interesting immobilization procedures are in the area of covalent binding. Supports containing epoxy groups are widely used in the immobilization by covalent binding because these generate intense multipoint covalent attachment with different nucleophiles present on the surface of the enzyme molecules (Mateo et al., 2007). One limitation of the epoxy supports is the slow reaction of immobilization. To overcome this problem, Mateo and coworkers have designed epoxy supports able to ensure a mild physical adsorption of the enzymes followed by a very fast intramolecular covalent binding with the material epoxy groups. These supports were used to immobilize and stabilize enzymes such as glutaryl acylase (Mateo et al., 2001), |3- galactosidase from Thermus sp. (Pessela et al., 2003), and peroxidase (Abad et al., 2002). Epoxy supports, known as Sepabeads® are marketed by Resindion s. r.l. and quickly have begun to supersede another commercial support, known as Eupergit. This last is a microporous, epoxy­activated acrylic beads with a diameter of 100-250p, used for a wide variety of different enzymes (Boller et al., 2002).