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To isolate and enhance the yield of a bacterial milk clotting protease (MCP) through process optimization and scale up.Bacillus subtilis was isolated as MCP producer with good milk clotting activity (MCA) per proteolytic activity (PA) index. The enzyme production was inducible with casein and enhanced with fructose and ammonium nitrate resulting in 571·43 U ml−1 of enzyme.Medium containing 4% fructose, 0·75% casein, 0·3% NH4NO3 and 10 mmol l–1 CaCl2, pH 6·0, inoculated with 4% (v/v) inoculum, incubated at 37°C, 200 rev min−1 for 72 h gave maximum production. A 6·67-fold increase in MCP yield with very high MCA per PA index was observed after final optimization indicating similarity to rennets.Mostly fungal MCPs have been reported. The MCA and MCA per PA index of this bacterium is comparable to that of many fungal reports and better than quite a few bacterial MCPs. Thus, this enzyme by B. subtilis has good probability of successful use in cheese production.