In cross-linking, intermolecular linkages are formed between enzyme molecules by means of bifunctional or multifunctional reagents such as glutaraldehyde, bisdiazo — benzidine, and hexamethylene diisocyanate. In cross-linking, the immobilized lipase is stable due to the strong interactions among enzyme and cross-linking agent. The application of this biocatalyst in biodiesel gives considerable good yield, but the small size of these aggregates creates problem during reuse (Cao et al. 2003; Andrade and Hlady 1986).

12.3.1.2 Entrapment

Entrapment of lipase entails capture of the lipase within a matrix of a polymer. Benefits of entrapment include cheap, fast, and easy procedure with mild operating conditions. Entrapped lipase is more stable than the physically adsorbed ones.

Application of entrapped lipase in biodiesel production has given average yields. This is due to the mass transfer limitations of entrapped lipase and erosion of enzyme from the surface of the support (Xavier et al. 1990; Trevan 1988).

12.3.1.3 Encapsulation

In this method of immobilization, the lipase is retained inside within a porous mem­brane forming a bilayered system. Direct contact between the enzyme and the sub­strates can be avoided in encapsulation. Enzyme leakage can be reduced due to the less leaching out of enzyme molecules from the matrix. When applied for produc­tion of biodiesel, encapsulated lipase has shown to give very low yields. This is due to the strong mass transfer limitations of encapsulated enzyme and clogging of pores with the substrate and product molecules (Yadav and Jadhav 2005).

12.3.1.4 Hybrid Immobilization Techniques

A combination of two or more of the above-mentioned techniques results in “hybrid immobilization.” This technique has given good results on industrial scale to food and pharmaceutical industry. Now this approach is being applied for transesterifica­tion reactions, and the results are quite promising (Bonrath et al. 2002). So this approach looks better in designing an efficient biocatalyst for biofuel industry.