Directed Evolution of a Subtilisin with Calcium-Independent Stability

Extracellular proteases of the subtilisin-class depend upon calcium for stability. Calcium binding stabilizes these proteins in natural extracellular environments, but is an Achilles' heel in industrial environments which contain high concentrations of metal chelators. Here we direct the evolution of calcium-independent stability hi subtilisin BPN′. By deleting the calcium binding loop from subtilisin, we initially destabilize the protein but create the potential to use new structural solutions for stabilization. Analysis of the structure and stability of the loop-deleted prototype followed by directed mutagenesis and selection for increased stability resulted in a subtilisin mutant with native-like proteolytic activity but 1000-times greater stability in strongly chelating conditions.