RESUMO
The production and purification of a calcium-dependent protease by Bacillus cereus BG1 were studied. The production of the protease was found to depend specifically on the calcium concentration in the culture medium. This suggests that this metal ion is essential for the induction of protease production and/or stabilisation of the enzyme after synthesis. The calcium requirement is highly specific since other metal ions (such as Mg(2+) and Ba(2+), which both activate the enzyme) are not able to induce protease production. The most appropriate medium for growth and protease production comprises (g L(-1)) starch 5, CaCl(2) 2, yeast extract 2, K(2)HPO(4) 0.2 and KH(2)PO(4) 0.2. The protease of BG1 strain was purified to homogeneity by ultrafiltration, heat treatment, gel filtration on Sephacryl S-200, ion exchange chromatography on DEAE-cellulose and, finally, a second gel filtration on Sephacryl S-200, with a 39-fold increase in specific activity and 23% recovery. The molecular weight was estimated to be 34 kDa on SDS-PAGE. The optimum temperature and pH of the purified enzyme were determined to be 60 degrees C and 8.0, respectively, in 100 mM Tris-HCl buffer + 2 mM CaCl(2).
