Purification and some properties of an alkaline protease inhibitor-inactivating-enzyme from Aspergillus oryzae W-1 [Japanese]

摘要

An alkaline protease inhibitor-inactivating enzyme (AIE) of Aspergillus oryzae W-1 was purified to homogeneity by the combination of various column chromatographies. The molecular mass of the enzyme was estimated to be 42 kDa by SDS-PAGE; the isoelectric point was 4.7. AIE was optimally active at around 55degreesC and pH 6. The enzyme was stable up to 40degreesC and in the pH range of 6-10. AIE was inactivated by HgCl2 or p-chloromercuribenzoate (p-CMB) but not by trypsin inhibitor, leupeptin, and phenylmethylsulfonylfluoride (PMSF). It was also inactivated by chelating agents, like ethylenediaminetetraacetate (EDTA) and o-phenanthroline. The enzyme treated with HgCl2, however, was reactivated by the addition of 2-mercaptoethanol (2-ME). On the other hand, EDTA- inactivated AIE was reactivated in the presence of 2-ME and CaCl2. When the protease-inhibitor complex was incubated with AIE at 37degreesC and pH 5, the activation of AP was detected. From these results, it was supposed that AlE is responsible for the activation of alkaline protease (AP) in the cells of mold.