A study of the cell wall-associated proteinase of lactic streptococci : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in biochemistry at Massey University

Abstract

The cell wall proteinase of Streptococcus lactis 4760 was released by incubation of milk­ grown cells in Ca++- free buffer. The effects of duration of incubation, pH and presence of Ca++ ions on the release of proteinase activity was investigated. The extent of leakage of intracellular enzymes during incubation was monitored by the appearance of lactate dehydrogenase activity in the incubation buffer. The proteinase released from the cells was partially purified by ion- exchange and gel permeation chromatography and then analysed for activity towards various milk- proteins. Only a single proteinase was evident from the purification. This enzyme was active towards - casein but showed no apparent cleavage of a 81- and K- caseins nor the whey proteins, a- lactalbumin and - lactoglobulin. The enzyme cleaved the - casein molecule within the C- terminal 49 residues, generating four main peptides containing residues 167- 175, 176- 182, 183- 193 and 194-209, and smaller amounts of peptides corresponding to the overlapping sequences 161- 166, 164- 169 and 166- 175. The four main peptides are identical to those generated by an S. lactis 763 proteinase described by Monnet et al. (1986) and by an S. cremoris HP proteinase recently described by Visser et al. (1987). No apparent specificity of enzyme action was evident. A preliminary study of the cell wall proteinase from S. cremoris SKl 1, a strain reported to produce a proteinase with a different specificity, suggested that the enzyme may hydrolyse the - casein molecule at the same sites as those cleaved by the S. lactis 4760 enzyme.