Metalloprotease from Bacillus thuringiensis

Bacillus thuringiensis var. kurstaki was shown to produce an extracellular, metal chelator-sensitive protease during the early stages of sporulation. Protease production in nutrient broth was dependent upon supplementation with Mn 2+ or Ca 2+ . The addition of Ca 2+ was required for enzyme stabilization. Protease production occurred in nutrient broth supplemented with 7 � 10 -3 M Ca 2+ , 5 � 10 -4 M Mn 2+ , and 10 -3 M Mg 2+ . The protease had optimum activity in the pH range 6.5 to 7.5. It was inhibited by chelating agents but not by a serine protease inhibitor. The culture supernatant and the partially purified protease lacked esterase activity. Partial purification of the enzyme (92.3 �) by (NH 4 ) 2 SO 4 fractionation and starch adsorption yielded an enzyme whose molecular weight was estimated to be 37,500 by acrylamide gel-sodium dodecyl sulfate electrophoresis or 40,800 by sucrose density gradient centrifugation. In the presence of Ca 2+ , the partially purified enzyme retained 78% of its activity after heating at 70 C for 10 min but only 8% of its activity after heating at 80 C for 10 min.