DETECTION OF ENZYME-ACTIVITY IN FRACTIONS COLLECTED FROM FREE SOLUTION CAPILLARY ELECTROPHORESIS OF COMPLEX SAMPLES

Crude fermentation broth from a fermentation of Aspergillus oryzae was analyzed using free solution capillary electrophoresis (FSCE) in a alkaline running buffer. Fractions as large as possible were collected after FSCE separation and analyzed for alkaline protease activity with Suc-Ala-Ala-Pro-Phe-p-nitroanilide as substrate. Two peaks were isolated; one of them was unknown and therefore was further investigated. After amplification of the activity by incubation with Suc-Ala-Ala-Pro-Phe-p-nitroanilide or casein as substrate, the reaction mixtures were analyzed by FSCE. In this way as little as 3 ng of enzyme were identified as an alkaline protease of the subtilisin family.