Measuring and reporting enzyme’s immobilization efficiency

NICOLÁS, PAULA; JOSE CARLA; LLERENA SUSTER CARLOS; LASSALLE, VERÓNICA; LAURA BRIAND; MARIA LUJAN FERREIRA

Biocatalyst Immobilization Foundations and Applications

Elsevier

Año: 2022; p. 115 - 147

When in a laboratory around the world, a scientist, a researcher, or a student wants to prepare a biocatalyst by immobilizing one or several enzymes onto a support, one key parameter is the amount ofenzyme immobilized in the support. This amount gives the percentage of the mass of the biocatalystthat is truly the core of the biocatalyst: the active catalyst. Generally, this amount is reported not only asmilligrams or grams of protein per gram of biocatalyst (or in % w/w) but also as milligrams of proteinper gram of support, being the whole mass of biocatalyst protein plus support. However, due to manyreasons, it is very difficult to know exactly the amount of enzyme immobilized. First, what is quantifiedis the protein using different methods. Only if the solution includes one protein, the enzymatic one, thequantification of this protein would give the amount of immobilized enzyme. Besides, only if the enzyme is purified enough, the solution includes a main unique isoenzyme. Therefore, we have to takeinto account that the enzyme solution includes many times different proteins besides the enzymaticone, and the enzymatic proteins may be present as several isoenzymes. Second, the enzyme—providedas a solid or in an aqueous solution—may include other compounds (unknown to the scientist or researcher or student) that may be reactive with the compounds used for the protein quantification. Third,if the enzyme has to be diluted in an aqueous solution, the possibility of the presence of solids as in asuspension has to be taken into account to avoid mistakes in the quantification methods. Filtration andother operations such as centrifugation may be needed to avoid interferences in the quantificationmethods. Fourth, the differences between the quantification methods and the reaction media haveto be taken into account. If a method is valid for an aqueous solution, it should not be applied to otherimmobilization media directly. Fifth, the calibrating protein used to calibrate the equipment to use iskey to avoid mistakes.With these ideas in mind in the following section, several definitions will be presented.