Purification and characterization of the mature, membrane-associated cell- envelope proteinase of Lactobacillus helveticus L89

Abstract

Lactobacillus helveticus L89 possesses a cell-envelope proteinase (Lb- CEP) that is biochemically and genetically related to that of the lactococci (Lc-CEP). The in-situ proteinase is resistant to autoproteolysis and remains associated with the membrane during lysozyme treatment of cells and subsequent mechanical disruption of the treated cells. The proteinase was purified from isolated membranes by a procedure that preserves the complete in-situ proteinase (mature proteinase) assumed to be the N-terminally processed translation product including the membrane 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 autoproteolysis. On the basis of its specificity towards caseins, towards the α(s1)-casein-(1-23)-fragment and towards two differently charged chromophoric peptides, the proteinase was recognized as an (Lb-)CEP(1/1H) mixed-type variant different from those identified so far among the lactococcal proteinases.