Effect of genetically modified Lactococcus lactis cell-envelope proteinases with altered specificity on the course of casein degradation under cheeseconditions

The specificity of the cell-bound, wild-type proteinase (PrtP) of Lactococc us lactis subsp. cremoris SK11 towards the alpha (s1)-casein(1-23) fragment under simulated cheese conditions was compared with that of several mutant s of PrtP. A unique specificity resulted from substitution of the substrate -binding site residues AKT 137-139 in the wild-type with GLA in the mutant PrtP. This different specificity clearly modifies the breakdown of chymosin -generated primary cheese peptides under cheese conditions. The consequence s for amino acid production were investigated with cells which were optimal ly permeabilized with respect to accessibility and activity of the intracel lular proteolytic system. Unlike untreated cells, these cells showed effici ent peptide uptake and intracellular conversion (involving peptides of up t o 15 residues), similar to that occurring in a normal Gouda cheese. No sign ificant differences were found in the composition of the amino acid pools g enerated by permeabilized cells containing either the wild-type or the muta nt PrtP. (C) 2001 Elsevier Science Ltd. All rights reserved.