Random mutagenesis of the substrate-binding site of a serine protease can generate enzymes with increased activities and altered primary specificities

Several mutated versions of alpha-lytic protease were created and their cleavage sites and proteolytic activity were studied. Random replacements of four residues (Gly191, Arg192, Met213 and Val218) resulted in the formation of the library, with the active mutants showing substitution of Met213 with a His residue. This substitution increases enzyme specificity, with preferred cleaving occurring at His residues in synthetic amide substrates.