Production and Immobilization of a Bacterial Milk-Clotting Enzyme

An extracellular milk-clotting enzyme was obtained from a strain of Bacillus licbeniformis isolated from raw milk. Production studies improved yield of the enzyme. The enzyme was purified partially by fractionation with 70% ammonium sulfate. Milk-clotting properties of the partially purified enzyme were investigated and compared to the corresponding properties of Mucor miebei protease, a chymosin-like enzyme. Maximum activity of both enzymes was in milk at 65C with added calcium chloride dihydrate concentration of 50 mM. The bacterial enzyme had maximum activity at pH 5.0 to 5.5 and the Mucor protease at pH 5.0. Both enzymes showed high inactivation after heating for 3 min above 65C. The enzymes were immobilized by embedding in paraffin wax. This immobilization technique had no appreciable effect on factors affecting the enzymes, such as pH, temperature, and calcium 2+ concentration. Both the enzymes were used after immobilization in a continuous milkclotting system.