Purification and characterization of Bacillus subtilis milk-clotting enzyme from Tibet Plateau and its potential use in yak dairy industry

A milk-clotting enzyme named YS-1 was purified from a Bacillus subtilis (B. subtilis) YB-3, which we have isolated from Tibetan Plateau, Gansu, China. The enzyme YS-1 was identified as a metalloproteinase. SDS-PAGE and MALDI-TOF-MS analysis of the purified enzyme gave a molecular weight of 42 kDa. YS-1 was stable over a wide range of temperature from 20 to 60 °C. Purified YS-1 was also active over a wide range of pH from 5.0 to 9.0. It can be activated by Ca²⁺ and Al³⁺ but inhibited by Zn²⁺, Fe²⁺ and Cu²⁺. The milk-clotting enzyme YS-1 exhibited high specificity to substrate β-casein and yak milk casein and led to a 75% more rapid coagulation of yak milk than cow milk, due to high β-casein content in yak milk. Together, our findings confirmed that the enzyme YS-1 has a potential to be used in yak cheese industry.