Purification and properties of milk‐clotting enzyme from Bacillus subtilis K‐26

A milk‐clotting enzyme from Bacillus subtilis K‐26 was purified by gel filtration and ion‐exchange chromatography resulting in a 24‐fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a Km of 2.77mg/ml for κ‐casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk‐clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy‐metal ions (Hg2+, Pb2+) inactivated the enzyme.