A new library of alpha-lytic protease S1 mutants generated by combinatorial random substitution.

Four positions in the S1 site of alpha-lytic protease (positions 190, 192, 213 and 218) were subjected to targeted random mutagenesis. In the resulting library we found active mutant proteases whose cleavage preferences could be grouped into three distinct families. Some potentially useful enzymes (such as one that prefers to cleave at Asn and Cys residues) were identified. In addition, we discuss instances where it was possible to relate changes in substrate specificity to alterations in the structure of the substrate binding site.