PROTEOLYTIC-ENZYME ACTIVITY IN LACTOCOCCI GROWN IN DIFFERENT PRETREATED MILK MEDIA

Lactococcus lactis ssp. lactis MG1363, harbouring plasmid pNZ521, whic h encodes the extracellular serine proteinase (PrtP) from strain SK110 , was used to study the effect of two different treatments of the grow th medium milk on the activity levels of PrtP and the intracellular lo calized aminopeptidase PepN and X-prolyldipeptidyl aminopeptidase PepX P. All three proteolytic enzymes showed lower activity levels in cells grown in high heat-treated milk as compared to cells grown in non-hea t-treated milk. Highest activity levels of the three studied enzymes w ere found in cells grown in milk heat treated for 30 min at 63 degrees C. Using cells of strain L. lactis ssp. lactis MG1363, harbouring pla smid pNZ544, which encodes reporter gene gusA under control of the prt P promoter, it was demonstrated that the regulation of PrtP takes plac e at the transcription initiation level. After separation of the pH 4. 6 soluble fraction of high heat-treated milk with reverse phase HPLC, it was found that the hydrophilic small peptide fraction of the milk w as responsible for this regulation. Amino acid analysis of this fracti on confirmed that this fraction consisted of peptides only. Ultrafiltr ation of milk, which increases the dry matter of the milk specifically through increase of its protein content, only significantly affected the levels of PrtP and PepN in cells of strain MG1363. Highest activit y was found during growth in unconcentrated milk, and the lowest level was found during growth in four times concentrated retentate. Using c ells of strain MG1363(pNZ544), it was demonstrated that also in this c ase regulation of PrtP takes place at the level of transcription initi ation. Approximately 40% of the decrease in activity of PrtP and PepN could be explained by the presence of higher amounts of purified whey proteins and higher amounts of dry mass. This suggests the presence of another factor, concentrated by ultrafiltration, which controls the p roduction of different proteolytic enzymes in concentrated retentate.