The lactococcal cell envelope proteinases: Differences, calcium-binding effects and role in cheese ripening

Abstract

Abstract The lactococcal cell envelope proteinase (CEP) has been extensively characterized during the last decade, both biochemically and genetically. The enzyme belongs to a family of subtilisin-like serine proteinases, the subtilases, and is characterized by a large extension C-terminal of the proteinase domain. Biochemical characterization has involved the truncated so-called release-CEP obtained by the autoproteolytic release from cells which occurs in the absence of calcium. A comparative study of the specificities of the release-CEP of different strains of Lactococcus lactis revealed a broad diversity. Differences could in part be related to charge characteristics of the subtilisin-like substrate binding region and of other residues outside this region and outside the proteinase domain. The structural organization of the bound CEP at the periphery of the cell wall seems to be the main determinant for specific catalytic properties different from those of the release-CEP. Calcium plays a role in inducing and (or) stabilizing active conformations of the bound CEP. Thermal stabilization is most obvious in type III CEP; it involves a change of conformation induced by binding of one calcium ion to a relatively high affinity binding site and accompanied by an increase of specific activity. A simultaneous anchoring of the actual subtilisin-like proteinase domain to the cell wall surface is assumed to prevent autoproteolytic release. Binding of additional calcium ions increases its stability and specific activity. The action of CEP during the ripening of cheese is essential to secondary proteolysis and flavour development.