1. Homology modelling of cell-envelope proteinase Engineering of the substrate binding region of the subtilisin- like, cell-envelope proteinase of Lactococcus lactis Protein Engineering 6 (1993) 927-937 Roland Siezen, Paul Bruinenberg, Pieter Vos, Ingrid van Alen-Boerrigter, Monique Nijhuis, Arno Alting, Fred Exterkate, and Willem de Vos

2. Flavour formation in cheese Milk Lactose/ citric acid citric acidCaseinFat peptides amino acids flavour compounds fatty acids chymosin protease peptidases amino acid convertases lipases esterases metabolic enzymes pyruvate metabolic enzymes

3. Proteolysis by lactic acid bacteria L.lactis Casein peptides PrtPPepN PepX PepA

4. N C Ca1 Ca2 Ser His Asp +28 +8 +14 +15 substrate binding cleft PrtP protease domain +151 I-domain 137-139 166

5.  s1 -casein(1-23) degradation by PrtP R-P-K-H-P-I-K-H-Q-G-L-P-Q-E-V-L-N-E-N-L-L-R-F wild-type N166D AKT(137-139)GDT AKT(137-139)GPP AKT(137-139)GLA  238-388) 5 10 15 20 Main cleavage sites: pH 6.5, low NaCl

6. Conclusions Homology modelling substrate-binding region of PrtP can be modelled several (large) loops cannot be modeled model suffices for general predictions Protein engineering mutations in substrate-binding region, based on homology modelling mutants show altered proteolytic specificity tested mutants under cheese making conditions