Abstract
The autolysis of semi-alkaline proteinase (SAP) purified from Aspergillus melleus was studied from the viewpoint of enzyme stability. We also prepared inactive phenylmethylsulfonyl semialkaline proteinase (PMS-SAP) in order to avoid the influence of autolysis and used it to study the denaturation profile or structural change in urea solution. Experiments with native SAP and PMS-SAP were performed in parallel under the same conditions. It was found that the rate of inactivation of this enzyme, as determined from the decrease in enzyme activity, followed firstorder kinetics and that there was a good relationship between the degree of inactivation and the amount of autolyzed products during urea treatment. The rate of urea denaturation of PMS-SAP was followed by high-performance liquid chromatography (HPLC) and circular dichroism (CD) spectral measurement ; the denatured enzyme could be completely separated from the intact PMS-SAP by HPLC. The results suggested that the inactivation of the enzyme was a result of the denaturation, which was accompanied by conformational change. Thus, it seems likely that the cause of the inactivation of SAP is denaturation rather than autolysis, because during autolysis, SAP was proteolyzed through the denatured form produced in the process of inactivation.
