Purification and characterization of the free form of the lactococcal extracellular proteinase and its autoproteolytic cleavage products.
In addition to the cell wall proteinase, Lactococcus lactis subsp. cremoris produced significant amounts of a free extracellular proteinase. The free proteinase activity was highest in the late exponential and early stationary phase of growth, whereas the cell wall activity was highest in the last half of the exponential phase. Both proteinase forms had a pH optimum between 4-6 and 5-8, and they behaved similarly upon anion exchange and hydrophobic interaction chromatography, chromatofocusing and gel filtration, indicating that they were related. Purification to homogeneity, as judged by SDS-PAGE, resulted in a 50,000-80,000-fold increase in the specific activity of the free proteinase. It contained two major protein species (termed pro150 and pro115) with proteinase activity. As judged by SDS-PAGE, the Mr values of pro150 and pro115 were 150,000 and 115,000, respectively, and by chromatofocusing the isoelectric points were 4.3 and 4.1, respectively. Upon gel filtration, pro150 and pro115 had Mr values of 300,000 and 125,000, respectively, indicating that pro150 was a dimer and pro115 a monomer. Pro115 was an autodegradation product of pro150. Other distinct autodegradation products had Mr values of 90,000 (p90), 53,000 (p53), 37,000 (p37) and 30,000 (p30). These had little if any proteinase activity. Pro115, p90 and p53 had a common N-terminal sequence with that reported for the cell wall proteinase. Judging from its N-terminal sequence and Mr, p30 was derived from the C-terminal half of p53. Cleavage of pro150 to pro115 generated p37.
Nissen-Meyer
J
J
Laboratory of Microbial Gene Technology, Norway.
Sletten
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002473:Cell Wall / Q000201:enzymology; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000254:growth & development; D046911:Macromolecular Substances; D008969:Molecular Sequence Data
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D010450:Endopeptidases