Purification and immunochemical quantitation of Penicillium roqueforti acid aspartyl proteinase

Abstract

Acid aspartyl proteinase of Penicillium roqueforti was purified from culture filtrates using FPLC ion-exchange chromatography on Mono Q and size exclusion chromatography on Superdex 75. The purity obtained allowed us to produce, using rabbits, a specific antiserum which was then used to develop a sandwich ELISA. The test was sensitive (detection limit, 0·25 ng/ml), reproducible (CV, 3·1–6·9%) and closely correlated with enzyme activity measurement (r=0·995, P<0·001). This ELISA should be a valuable tool for monitoring acid aspartyl proteinase level in culture filtrates and for determining the enzyme activity[ratio ]enzyme protein ratio of acid aspartyl proteinase obtained after genetic recombinations or site-directed mutagenesis.