Discloses a protease with milk-clotting activity which comprises an amino acid sequence at least 75% identical to SEQ ID NO: 3, said protease has at least one mutation selected from the group consisting of: (A) replacement of glutamine corresponding to glutamine at position 265 in SEQ ID NO: 3 with an acidic amino acid； and (B) replacement of glutamine at position 266 in SEQ ID NO: 3 with an acidic amino acid. Further discloses the said protease with other selected mutations. Further discloses a method for producing a protease with milk-clotting activity comprising the steps of culturing a transformed cell with the disclosed protease sequence in a culture medium and collecting the protease in the culture medium. The disclosed protease finds application in production of cheese and dairy products.
