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

PAULA NICOLÁS; VERONICA LASSALLE; MARÍA LUJÁN FERREIRA

Simposio; I Simposio Latinoamericano de Biocatálisis y Biotransformaciones; 2014

Whenimmobilizing enzymes, to know the protein content in the solution used asenzyme source and the final protein loading in the biocatalyst is of theoutmost importance. This is needed for comparison of specific activity permilligram of total protein between the free and immobilized enzyme.Traditionally, the Bradford method (1) is employed for this purpose, usingbovine serum albumin (BSA) as standard (2). It relies on the interaction of Coomasie Brillant Blue G-250 (CBB) withprotein side-chains. Protein isdetermined in the solution before and after performing the immobilization, tocalculate the enzyme loading (EL) by difference. The object of thiscontribution is to warn about the use of this method as tool to quantify anyprotein, but especially lipases during immobilizations. Quantification data fromCALB immobilization onto magnetic nanoparticles are presented. Sulphurdetermination by Atomic Emission (AE-ICP) is presented as a novel approach attemptingto avoid or at least minimize erroneous results derived from the dye-bindingmethod flaws: aggregation of protein molecules, the presence of chemicalinterferences and the nature of the standard.