Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli

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.