title: Directed evolution of a subtilisin with calcium-independent stability.
abstract: 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 in 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.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, MD 20850, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Strausberg
authors.forename: P A
authors.initials: PA
authors.lastname: Alexander
authors.forename: D T
authors.initials: DT
authors.lastname: Gallagher
authors.forename: G L
authors.initials: GL
authors.lastname: Gilliland
authors.forename: B L
authors.initials: BL
authors.lastname: Barnett
authors.forename: P N
authors.initials: PN
authors.lastname: Bryan
mesh_terms: D005075:Biological Evolution; D002118:Calcium / Q000502:physiology*; D002872:Chromosome Deletion; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013381:Subtilisins / Q000235:genetics*
chemical_list: D013381:Subtilisins; D002118:Calcium