Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2092843 | Microbiological Research | 2010 | 10 Pages |
SummaryThe SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6× His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS–PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54 kDa. The optimal temperature and pH of the purified rSAP6 were 40 °C and 3.4, respectively. The enzyme was stable below 45 °C and between pH 2.6 and 5.0. The results show that Mn2+ had an activating effect on the enzyme, while Cu2+, Mg2+, Zn2+ and Ag+ acted as inhibitors of the enzyme. However, Ca2+ had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.