PURIFICATION AND CHARACTERIZATION OF THE MATURE, MEMBRANE-ASSOCIATED CELL-ENVELOPE PROTEINASE OF LACTOBACILLUS-HELVETICUS L89

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb- CEP) that is biochemically and genetically related to that of the lact ococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysi s and remains associated with the membrane during lysozyme treatment o f cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that p reserves the complete in-situ proteinase (mature proteinase) assumed t o be the N-terminally processed translation product including the memb rane anchor: its monomer molecular mass is approximately 180 kDa. The purified enzyme appeared to be more stable towards heat than hitherto known related, but C-terminally truncated cell-envelope proteinases of lactobacilli and lactococci, which were released from the cells by au toproteolysis. On the basis of its specificity towards caseins, toward s the alpha(sl)-casein-(1-23)-fragment and towards two differently cha rged chromophoric peptides, the proteinase was recognized as an (Lb-)C EP(I/III) mixed-type variant different from those identified so far am ong the lactococcal proteinases.