To minimize substrate diffusion limitations, enzymes are usually attached on non­porous materials. However, the non-porous supports exhibit low enzyme loading capabilities (Chen and Su, 2001). On the other hand, porous materials have high enzyme loading capabilities, but suffer from a high limitation of substrate (Hayashi et al., 1993). In order to minimize the substrate diffusion limitation and enhance the enzyme loading at the same time, nano-size particles have been receiving great attention in recent years due to their large interfacial area and unique physical properties. Nanoparticle materials have been used in various bioprocesses including enzyme immobilization. For example, Tang et al. (2007) immobilized lipase onto nano-sized biopolymer Chitosan particles.

On the other hand, due to their high mechanical strength and thermal resistance, polyacrylonitrile (PAN) were used to generate electrospun nanofibrous membranes, which were used as the support for immobilizing C. rugosa lipase (Li and Wu, 2009). Lipase was bound covalently to PAN nanofibers ranged from 150 to 300 nm by amidination and used in a membrane reactor. The reactor was used for hydrolysis of oil and could also be used for biodiesel production.

Nano-sized magnetite (NSM) particles have been used as support for immobilization of enzymes. In addition to the larger surface area, due to the nano­size used, immobilization on magnetite materials allows easy enzyme recovery from the medium under the magnetic force, due to the magnetic response of the support material. Hence, there is no need for expensive liquid chromatography systems, centrifuges or filters. However, efficient loading of enzymes onto nano-sized magnetite (NSM) particles requires the surface functionalization by polymerization or sol-gel entrapment, which reduces the magnetic response of NSM particles (Lee et al., 2009). To avoid this limitation, Huang et al. (2003) immobilized lipase covalently to NSM particles. However, the covalent binding results in structural changes that can greatly reduce the activity of the enzyme. Therefore, coordinating NSM particles with a low molecular weight ligand has been proposed to overcome the abovementioned problem as the attachment would be via physical adsorption in this case, rather than by covalent bonding (Lee et al., 2009). At the same time, the particle sizes do not increase, as when the NSM particles are wrapped with polymers (Ma et al., 2003). In addition, the ligand acts as a spacer between NSM and the immobilized enzyme to prevent direct contact of lipase to the surface of the magnetites that may hinder the flexible enzyme structure. The immobilized lipase on the NSM particles showed higher specific activity and thermal stability than the free one and the activity of the immobilized lipase remained almost constant over five uses and recoveries (Bastida et al., 1998). The stable reuse as well as the convenience in the recovery offered by magnetic separation ensures that a surface-modified NSM particle is a good support material for lipase immobilization.