Protocols for simple immobilization of unstable enzymes are plenty, but the vast majority of them, unfortunately, have not reached their massive implementation for the preparation of improved heterogeneous biocatalyst. In this context, the science of enzyme immobilization demands new protocols capable of fabricating heterogeneous biocatalysts with better properties than the soluble enzymes. The preparation of very stable immobilized biocatalysts enables the following: (1) higher operational times of enzyme, increasing their total turnover numbers; (2) the use of enzymes under non-conventional media (temperatures, solvents, etc.) in order to increase the concentrations of substrates for intensification of processes or in order to shift reaction equilibria; (3) the design of solvent-free reaction systems; and (4) the prevention of microbial contaminations. These benefits gained with the immobilization are critical to scale up chemical processes like the synthesis of biodiesel, synthesis of food additives or soil decontamination, where the cost of the catalysts has an enormous impact on their economic feasibility. The science of enzyme immobilization requires a multidisciplinary focus that involves several areas of knowledge such as, material science, surface chemistry, protein chemistry, biophysics, molecular biology, biocatalysis, and chemical engineering. In this chapter, we will discuss the most relevant aspects to do "the science of enzyme immobilization." We will emphasize the immobilization techniques that promote multivalent attachments between the surface of the enzymes and the porous carriers. Finally, we will discuss the effect that the structural rigidification promotes at different protein regions on the functional properties of the immobilized enzymes.
Keywords: Biocatalysis; Colocalization; Directed immobilization; Enzyme immobilization; Enzyme stabilization; Multipoint attachment.