中国生物工程杂志

Bacillus subtilis ZC-7 was obtained by implantation with N+ ions beam to B. subtilis AS1.398, and compared with the AS1.398 neutral protease, the enzyme activity of ZC-7 neutral protease was about 1 times higher in previous research. A neutral protease was purified from the culture of B. Subtilis ZC-7 by the procedures including amoninium sulfate precipitation, ultrafiltration, DEAE-Sepharose Fast Flow chromatography and Sephadex G-75 chromatography. By multi-step purification, the ZC-7 neutral protease was purified to 78.5 folds and its yield was 27.7%, at last, the specific activity of ZC-7 neutral protease was up to 4.1×105U/mg. Analysed by SDS-PAGE, the purified protease has shown a molecular mass of about 42kDa. The Km for casein hydrolysis was 3.67×10-3 ug/mL and the Vmax was 12.21ug/min. The optimum pH and temperature for hydrolysis of casein were 7.0 and 55℃, respectively. This protease was stable up to 40℃ within the pH range of 6.5 and 8.0. EDTA, isopropanol and alcohol nearly inhibited its activity while some ions such as Ca2+，Mg2+，Fe3+ can improve its activity. In addition, it could resist 1 mol/L H2O2.