Beside the adsorption technique, lipase can be immobilized on support surfaces by covalent anchorage, electrostatic binding and entrapment within inorganic or organic
inert matrices. Adsorption techniques are simple, but the binding forces between the enzyme and the support are weak and enzyme leaching often occurs. A higher degree of stability can be achieved by covalent bonding between the enzyme and the solid surface (Shamel et al., 2005; Shamel et al., 2007); however this requires several chemical steps that are accompanied by loss in enzyme activity. High stability can also be achieved by electrostatic interaction, but this technique is limited to be used at pH values compatible with the electrostatic point, which also may affect the activity of the enzyme, since the enzyme conformation changes as function of pH (Macario et al., 2007). On the other hand, the immobilized lipase by entrapment within a polymer matrix is much more stable than physically adsorbed lipase (Hartmeier, 1985), and unlike the covalent bonding this method uses a relatively simple procedure. Enzyme entrapment in a silica matrix by sol-gel offers a good compromise between stability of the heterogeneous biocatalyst and activity loss, and hence this technique has received considerable attention in recent years (Frings et al., 1999). Entrapment of lipase in an inorganic polymer matrix, which is based on sol-gel process, is well documented (Reetz, 1997). The method involves an aqueous solution of the enzyme, an acid or base (NaOH, NaF or HCl) as catalyst and an alkoxysilanes as inorganic — organic matrix precursor. The sol-gel material is then obtained by hydrolysis and condensation of the precursor to result in an amorphous silica matrix that entraps the enzyme. The lipase entrapped in sol-gel has been used for biodiesel production (Orcaire et al., 2006; Al-Zuhair et al., 2008) and was easily recovered from reaction media. However, under the same operating conditions, it was found that immobilized lipases, from P. cepacia, on ceramic beads were more capable of transesterifying WO of high water contents to biodiesel than lipase, from the same course, entrapped in sol-gel matrix (Al-Zuhair et al., 2008), which is mainly due to diffusional limitations.

Covalently immobilized lipases are usually prepared in almost anhydrous media. This usually results in a problem, especially in porous structures, which is mainly used to enhance the interfacial area. At the oil water interface, lipases are in open active form, where a flap (or lid) that would seclude the active cites is moved to allow substrate accessibility to the active sites (Verger et al., 1973; Brady et al., 1990). When inside a porous structure, lipase molecules become inaccessible to external surfaces, which prevent their activation. Therefore, it has been proposed to use hydrophobic support that resembles the surface of drops of the natural substrates to immobilize lipase on. In this case, the adsorbed lipases are in open form, with the active sites accessible for substrate and the immobilized enzyme in this case exhibit significantly enhanced activity (Bastida et al., 1998). Based on that Palomo et al. (2002) used an epoxy acrylic matrix, Sepabeads, with the surface covered by octadecyl groups, yielding a very hydrophobic surface that has large pores to allow intensive protein interaction. The support permits in one step immobilization, purification, hyper-activation and stabilization of surface in a very simple protocol: the mere addition of support to the lipase solution at very low ionic strength. In addition, the support is rigid enough to be used in packed — bed reactor and does not swell in any reaction media. The stability and activity of lipases from C. antarctica, C. rugusa and M. miehei immobilized on this support were found to be superior to other covalently attached derivatives.