DIVERSITY OF CELL-ENVELOPE PROTEINASE SPECIFICITY AMONG STRAINS OF LACTOCOCCUS-LACTIS AND ITS RELATIONSHIP TO CHARGE CHARACTERISTICS OF THESUBSTRATE-BINDING REGION

The biochemical and genetical diversity of the subtilisin-like cell en velope proteinase (CEP) among Lactococcus lactis strains was investiga ted. The specificities of the proteinases of 16 strains toward the imp ortant cheese peptide alpha(s1)-casein fragment 1 to 23 and toward two differently charged chromophoric peptides have been determined. On th e basis of the results, these strains could be classified into seven g roups. The contribution to the specificity of specific residues in the large C-terminal segment, which differentiates this proteinase from m ost other members of the subtilisin family, was established with hybri d proteinases, even in the case of the small substrates. These remote residues and the subtilisin-like substrate-binding region are therefor e assumed to be spatially close to each other and together constitute most of the binding region of CEP. DNA sequence analysis of fragments of the gene (prtP) encoding segments of the proteinase which contain t he relevant residues of the substrate-binding region shows that among the strains studied, this-binding region is the most negatively charge d in the CEP group represented by strain HP and the most positively ch arged in the CEP group represented by strains AM1 and SK11. Consequent ly, these two proteinase groups show the most divergent specificities. Each of the proteinases of the other groups shows a different interme diate specificity which in part is the reflection of an intermediate c harge in the binding region. However, the results suggest that amino a cid residues outside the segments known to be part of the CEP-binding region also contribute to specificity. We propose a new nomenclature o f CEPs which is based on their defined specificities toward the alpha( s1)-casein fragment 1 to 23 and on their origins.