ENGINEERING OF THE SUBSTRATE-BINDING REGION OF THE SUBTILISIN-LIKE, CELL-ENVELOPE PROTEINASE OF LACTOCOCCUS-LACTIS

The substrate-binding region of the cell-envelope proteinase of Lactoc occus lactis strain SK11 was modelled, based on sequence homology of t he catalytic domain with the serine proteinases subtilisin and thermit ase. Substitutions, deletions and insertions were introduced, by site- directed and cassette mutagenesis of the prtP gene encoding this enzym e, based on sequence comparison both with subtilisin and with the homo logous L.lactis strain Wg2 proteinase, which has different proteolytic properties. The engineered enzymes were investigated for thermal stab ility, proteolytic activity and cleavage specificity towards small chr omogenic peptide substrates and the peptide alpha(s1)-casein(1-23). Mu tations in the subtilisin-like substrate-binding region showed that Se r433 is the active site residue, and that residues 138 and 166 at eith er side of the binding cleft play an important role in substrate speci ficity, particularly when these residues and the substrate are opposit ely charged. The K748T mutation in a different domain also affected sp ecificity and stability, suggesting that this residue is in close prox imity to the subtilisin-like domain and may form part of the substrate -binding site. Several mutant SK11 proteinases have novel properties n ot previously encountered in natural variants. Replacements of residue s 137 - 139AKT along one side of the binding cleft produced the 137 - 139GPP mutant proteinase with reduced activity and narrowed specificit y, and the 137-139GLA mutant with increased activity and broader speci ficity. Furthermore, the 137 - 139GDT mutant had a specificity towards alpha(s1)-casein(1 - 23) closely resembling that of L. lactis Wg2 pro teinase. Mutants with an additional negative charge in the binding reg ion were more stable towards autoproteolysis.