Basics Considerations on Enzymes

Enzymes have been used for millennia in food processing, such as bread baking, brewing, cheese and wine making, although only in the later decades of the 20th century, processes were developed that allowed the production in well-characterized formulations, even at large scale (Kirk et al., 2002; Mishra et al., 2017). Enzymes are globular proteins that act as catalysts, thus they speed up the rate of a reaction by lowering the energy of activation. Some enzymes require cofactors, small organic molecules or metal ions, for catalytic activity. Unlike chemical catalysts, enzymes are natural in origin, operate under mild temperature and pressure, display high specificity and are biodegradable (van Oort, 2010; Subin and Bhat, 2015). Biologically active enzymes can be obtained from animals, microorganisms and plants, but microbial sources are favored. Microbial enzymes can be produced in high yield, in relatively low-cost and short time processes, and are typically more stable than enzymes from the remaining sources. Particularly preferred are microbial produced enzymes that are secreted to the fermentation medium, as this eases separation and purification. Genetically modified microorganisms expressing exogenous enzymes (from plant or animal sources and from pathogenic or difficult to grow microbial strains) are also used in commercial enzyme production (Chandrasekaran et al., 2015; Subin and Bhat, 2015). Enzyme activity and stability are influenced by operational conditions, e.g., pH, temperature, substrate concentration, presence of metal ions and enzyme concentration. Enzymes have optimal pH and temperature conditions for activity and stability that may not fully match in an industrial process. An increase in substrate concentration increases activity up to a given point, henceforth the rate of reaction stabilizes or may even decrease, in case of substrate inhibition. Also depending on the enzyme, given metal ions may be required for activity (e.g., Ca2þ for most a-amylases), or may inhibit enzyme activity (Subin and Bhat, 2015). Thus, careful selection of operating conditions is critical for high enzyme performance. This is relatively easy to implement in laboratory condition with model systems, but may prove difficult to reproduce with real systems, due to the complexity of the matrix to be processed, e.g., hydrolysis of lactose in buffer system or industrial scale hydrolysis of lactose in milk. Enzymes can be used in free form or immobilized, e.g., attached to/entrapped in an inert support, to allow the repeated/continuous use of the enzyme, and to also increase its stability. Still, the implementation of an immobilized enzyme based system at industrial scale requires a careful evaluation as in addition to technical issues, e.g., loss of activity during immobilization, mass transfer limitations, the economics of immobilized enzymes, e.g., cost of immobilization, cost of immobilization carrier and chemicals for immobilization, must be considered (DiCosimo et al., 2013; Sheldon and van Pelt, 2013).