Importance of the analytical method choice for enzyme loading determination in immobilization

PAULA NICOLÁS; VERONICA LASSALLE; MARÍA LUJÁN FERREIRA

Buzios

Simposio; 1 Simposio Latinoamericano de Biocatálisis y Biotecnología; 2014

When immobilizing enzymes, to know the protein content in the solution used as enzyme source and the final protein loading in the biocatalyst is of the outmost importance. This is needed for comparison of specific activity per milligram of total protein between the free and immobilized enzyme. Traditionally, the Bradford method (1) is employed for this purpose, using bovine serum albumin (BSA) as standard (2). It relies on the interaction of Coomasie Brillant Blue G-250 (CBB) with protein side-chains. Protein is determined in the solution before and after performing the immobilization, to calculate the enzyme loading (EL) by difference. The object of this contribution is to warn about the use of this method as tool to quantify any protein, but especially lipases during immobilizations. Quantification data from CALB immobilization onto magnetic nanoparticles are presented. Sulphur determination by Atomic Emission (AE-ICP) is presented as a novel approach attempting to avoid or at least minimize erroneous results derived from the dye-binding method flaws: aggregation of protein molecules, the presence of chemical interferences and the nature of the standard.