The primary enzymes of importance in the nutritional and functional food applications are the hydrolases, which work by adding a water molecule to the substance to be catalyzed. The main types of hydrolytic enzymes are protease, amylase, lipase and cellulase, targeting protein, starch, lipids and cellulose, respectively. Enzymes only act upon a specific substrate, lowering the active energy needed for conversion without any direct impacts on unrelated substrates. Within each class, individual enzymes are also highly specific with respect to the substrate they affect. Enzymes can improve the digestion of food, reduce stress in the gastrointestinal (GI) tract, help maintain normal pH levels and promote the growth of healthy intestinal flora. Many foods contain the enzymes necessary for their biotransformation. However, enzymes are highly susceptible to changing environmental conditions, such as pH, moisture and temperature, and are destroyed by the high temperatures used in cooking and processing. They can be sourced from animal tissue, plants or microorganisms, such as fungi or bacteria. Fungal or bacterial enzymes offer the broadest range of beneficial properties in the biotransformation process. To produce commercial quantities of enzymes, many manufacturers isolate microbial strains that produce the desired enzyme or develop strains to produce the desired enzyme by genetic engineering . Through fermentation carried out under optimal conditions, manufacturers can produce sufficient quantities of the enzyme product needed. Enzymes can be used to provide biotransformed nutrients through functional and fortified foods, rather than simply in a pill. Enzymes are available in a variety of physical forms (liquids, slurries, granules and powders), which can be used under appropriate conditions as in-vivo digestive aids and in processed foods to reduce the negative effective of food components like lactose, tannins, phytates, proteins, carbohydrates and lipids. It could also be targeted for improving pharmaceutical products. This chapter analyses and describes scientific and technological information about the production and recovery of enzymes for the processing of functional foods, emphasizing the novel catalytic requirements associated for production of the functional foods.