Abstract: Milk-clotting enzyme has an important role in the process of cheese procution and maturation.
In this study, a bacterium producing extracellular milk-clotting enzyme was isolated from the soil in Tibetan
Plateau and identified as Bacillus licheniformis using 16S rDNA. Then the milk-clotting enzyme from B.
licheniformis HN1 was purified to 26.25-fold with 18.39 % recovery by precipitation in ammonium sulfate and
ion-exchange chromatography. The molecular mass of the enzyme was 51.9 kDa as determined by SDS-PAGE,
and the result of pepstatin A inhibition treatment showed that this enzyme was an aspartic proteas. The enzyme
was active in the pH range 5.5-9.5 and was inactivated completely by heating at 60°C for 20 min. The highest
level of enzyme activity was obtained at 55°C, pH 5.5, and in the presence of 20 mM CaCl2. The high level of milkclotting
activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from
licheniformis HN1 was a potential substitute for calf rennet.