Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

Abstract

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca2+-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca2+ inhibited, while ethylene glycol-bis(O-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca2+-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.

Characterization of cell envelope-associated proteinases of thermophilic lactobacilli

“…In other words, free a S1 -casein could be less susceptible to the action of proteinase than when it is in a complex with other casein fractions. Release of lactococcal proteinase from the cell envelope was found to be an autoproteolytic event (Laan and Konings 1989;Haandrikman et al 1991). It was also found that Ca 2+ ions play an important role in maintaining the attachment of the enzyme to the cell.…”

Genetics of the proteolytic system of lactic acid bacteria

“…Interestingly, the 80-kDa proteinase fragment was not only active, but also showed the same specificity towards casein as the larger enzyme ( [62][63][64]; see below). Recently, it was shown that an 87-kDa degradation product of the Wg2 proteinase still had the same N-terminal amino acid (Asp.188) as the largest proteinase band of 165 kDa present in this preparation, indicating that extensive C-terminal degradation had taken place [54].…”

“…At the N-terminus a typical signal peptide-like sequence of 33 amino acids is present. The N-terminal amino acid sequences of the isolated proteinases have been determined and indicate that in all three cases the mature enzyme starts with the aspartic acid residue at position 188 of the predicted amino acid sequence [47,52,54].…”

“…The size of the proteinase of strain Wg2 as deduced from the nucleotide sequence of its gene is 200 kDa [51]. The putative signal peptide contains 33 amino acids [51,52], while the pro-region consists of 154 amino acids [47,51,54]. Removal of this N-terminal stretch of amino acids would result in a mature enzyme of 1715 amino acids with a molecular size of 187 kDa.…”

“…Removal of this N-terminal stretch of amino acids would result in a mature enzyme of 1715 amino acids with a molecular size of 187 kDa. Additional proteolytic cleavage was postulated to account for the difference between this calculated size and that of the isolated enzyme (140-165 kDa) [22,51,54]. As the purified enzyme starts with Asp.188 [47,51,54], the cleavage occurs at the Cterminus of the proteinase.…”