Native Enzyme Mobility Shift Assay (NEMSA): a new method for monitoring carboxyl group modification of carboxymethylcellulase from Aspergillus niger

A simple, sensitive, accurate and more informative assay for determining the number of modified groups during the course of carboxyl group modification is described. Monomeric carboxymethylcellulase (CMCase) from Aspergillus niger was modified by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) in the presence of glycinamide. The different time-course aliquots were subjected to non-denaturing PAGE and the gel stained for CMCase activity. The number of carboxyl groups modified are directly read from the ladder of the enzyme bands developed at given time. This method showed that after 75 min of modification reaction there were five major species of modified CMCases in which 6 to 10 carboxyls were modified.