Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763

Monnet, Véronique; Ley, Jakob P.; González, Sebastián A.

doi:10.1016/0020-711x(92)90004-k

Cited by 46 publications

(47 citation statements)

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“…Interestingly, the proposed three-dimensional molecular model of ␤-casein A 2 (11) indicates that these preferential cleavage sites are on the outside of the molecule. The positive influence of prolyl residues in the close environment (positions P2, P4, and P6) of these early accessible cleavage sites and that of serine in position PЈ1 has been confirmed (30). These cleavages may result in a modification of the conformation of the substrate and the exposure of new sites for hydrolysis.…”

Section: Discussionmentioning

confidence: 77%

“…The importance of particular amino acids in the flanking regions of the cleavage sites was analyzed as described previously (30). The positions of the amino acids at the N-and C-terminal sides of the cleavage site are indicated as PX and PЈX, respectively, in which X represents the distance from the cleavage site in number of residues (designation according to reference 37).…”

Section: Discussionmentioning

confidence: 99%

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The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides

et al. 1995

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The peptides released from ␤-casein by the action of P I -type proteinase (PrtP) from Lactococcus lactis subsp. cremoris Wg2 have been identified by on-line coupling of liquid chromatography to mass spectrometry. After 24 h of incubation of ␤-casein with purified PrtP, a stable mixture of peptides was obtained. The trifluoroacetic acid-soluble peptides of this ␤-casein hydrolysate were fractionated by high-performance liquid chromatography and introduced into the liquid chromatography-ion spray mass spectrometry interface. Multiply charged ions were generated from trifluoroacetic acid-soluble peptides under low nozzle voltage conditions, yielding the MH ؉ mass of each eluted peptide. All peptides corresponding to each of the MH ؉ calculated masses were determined. In those cases in which different peptides were possible, further identification was achieved by collision-induced dissociation under higher nozzle voltage conditions. Hydrolysis of ␤-casein by PrtP was observed to proceed much further than reported previously. More than 40% of the peptide bonds are cleaved by PrtP, resulting in the formation of more than 100 different oligopeptides. With the exception of Phe, significant release of amino acids or di-and tripeptides could not be observed. Interestingly, one-fifth of the identified oligopeptides are small enough to be taken up by the oligopeptide transport system. Uptake of these peptides could supply L. lactis with all amino acids, including the essential ones, indicating that growth of L. lactis might be possible on peptides released from ␤-casein by proteinase only.Lactococci have very limited capacities of synthesizing amino acids and therefore must utilize exogenous nitrogen sources for optimal growth. The amino acid requirement is strain dependent, but Glu or Gln, Ile, Leu, His, Met, and Val are essential for the growth of most Lactococcus lactis strains (6). The addition of several other amino acids was found to be growth stimulatory (34,25). The concentrations of essential amino acids in milk are very low, especially those of Ile and Leu (less than 1 mg/liter) (27). Moreover, the concentrations of other free amino acids are too low to explain the growth of L. lactis to the cell densities normally reached in coagulated milk (27,44). Thus, for optimal growth in milk, lactococci depend on the utilization of milk proteins, such as caseins. Casein hydrolysis by lactococci is mediated by a complex proteolytic system which includes a cell envelope-located proteinase (P I -or P III -type proteinase [PrtP]) and several peptidases (for recent reviews, see references 31, 32, and 42).According to proposed models, PrtP is involved in the first step of casein degradation (32, 39). The action of purified PrtP on ␤-casein has been studied extensively (28,29,33,46,47). After separation of the proteolytic products by reverse-phase high-performance liquid chromatography (HPLC), the different peptides are collected, purified when needed, and identified by biochemical methods (i.e., amino acid composition, determina...

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Section: Discussionmentioning

confidence: 77%

Section: Discussionmentioning

confidence: 99%

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides

et al. 1995

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“…I). The sequences of most of these peptides did not match with the sequences of peptides released during the incubation of purified cell wall proteinase from the wild-type strain with purified caseins [12,13]. This observation suggests that either the peptides present in the supernatant did not derive from casein hydrolysis by cell wall proteinase alone, or that cell wall proteinase did not exhibit the same specificities in cell-bound and free forms, as has already been demonstrated for other L. lactis strains [4].…”

Section: Peptide Content Of Fermented Milks With Peptidase Mutants Ofmentioning

confidence: 95%

Article

Algaron

Miranda

Bars

et al. 2003

Lait

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-The proteolytic system of lactic acid bacteria has been characterised in detail and numerous modified strains with null or increased specific proteolytic activities have been constructed or identified among natural strains. Based on this knowledge, our objective was to ferment milk with modified strains and produce mixtures of peptides with specific features corresponding to potential bio-activities. We used a collection of Lactococcus lactis negative mutants for peptidase activities available in the laboratory to ferment the milk. In particular, we focused our work on mutants lacking either aminopeptidase N, X-prolyl dipeptidyl aminopeptidase or tripeptidase in order to test their ability to form peptides with immunomodulating or antihypertensive activities. At the end of fermentation, supernatants were fractionated by RP-HPLC. Each fraction collected was analysed by Maldi-Tof MS and sequencing. We observed that mutants accumulated specific peptides consistent with the specificity of the missing peptidases. Some of the peptides identified present similarities with peptides having immunomodulating or anti-hypertensive effects. One of these was quantified. At the same time, we observed that the inhibition of angiotensin converting enzyme was stronger in supernatants obtained with mutant strains than in supernatant obtained using the wild-type strain. In conclusion, we showed that in some cases, modifications to the proteolytic system of Lactococcus lactis gave rise to significant differences in the mixtures of peptides produced during milk fermentation. The differences in bio-activity of these peptide mixtures were only partially determined in vitro and evidently need to be demonstrated in vivo. Exploitation of the biodiversity of the proteolytic system of lactic acid bacteria may enable a direct application of this work and undoubtedly a promising means of directly producing natural bio-active peptides in fermented milk products. Milk fermentation / Lactococcus lactis / bio-active peptide / proteolysis / mutantRésumé -Conséquences de modifications du système protéolytique de Lactococcus lactis sur l'accumulation de peptides potentiellement bio-actifs pendant la fermentation du lait. Le système protéolytique des bactéries lactiques a été caractérisé en détail et de nombreuses souches aux activités protéolytiques modifiées ont été construites ou identifiées parmi des souches sauvages. Notre objectif est de produire des mélanges de peptides ayant des activités biologiques, en utilisant ces souches modifiées pour fermenter le lait. Pour cela, nous avons utilisé une collection de mutants de Lactococcus lactis dont certaines activités peptidasiques ont été supprimées. En particulier, nous nous sommes intéressés à des souches n'ayant plus l'aminopeptidase N, la X-prolyl-dipeptidyl aminopeptidase, ou la tripeptidase pour tester leur aptitude à former des peptides ayant des activités * Corresponding author: algaron@jouy.inra.fr 116 F. Algaron et al.

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“…on April 27, 2019 by guest http://aem.asm.org/ eight amino acids (31). Consequently, the environment of each cleavage site has been studied.…”

Section: Vol 66 2000 Autoproteolysis Of L Lactis Lactocepin Iii 5137mentioning

confidence: 99%

The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

Flambard

Juillard

2000

Appl Environ Microbiol

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The effect of autoproteolysis of Lactococcus lactis lactocepin III on its specificity towards ␤-casein was investigated. ␤-Casein degradation was performed by using either an autolysin-defective derivative of L. lactis MG1363 carrying the proteinase genes of L. lactis SK11, which was unable to transport oligopeptides, or autoproteolyzed enzyme purified from L. lactis SK11. Comparison of the peptide pools by high-performance liquid chromatography analysis revealed significant differences. To analyze these differences in more detail, the peptides released by the cell-anchored proteinase were identified by on-line coupling of liquid chromatography to mass spectrometry. More than 100 oligopeptides were released from ␤-casein by the cell-anchored proteinase. Analysis of the cleavage sites indicated that the specificity of peptide bond cleavage by the cell-anchored proteinase differed significantly from that of the autoproteolyzed enzyme.Due to their limited capacity for synthesizing amino acids (3), lactococci have to utilize exogenous nitrogen sources for optimal growth. The amino acid requirements appear to be strain specific, but most Lactococcus lactis strains need at least Leu, Ile, Val, and His for growth (15,28). In milk, the concentrations of several essential amino acids, especially those of Ile and Leu (less than 1 mg liter Ϫ1 ), are very low (10, 18). In addition, milk peptides are a poor source of Leu and Met (16). Thus, for optimal growth in milk, lactococci depend on utilization of casein (18,29). A complex proteolytic system is needed for casein hydrolysis. According to proposed models, lactocepin (EC 3.4.21.96; previously named cell envelope proteinase PrtP) (34) is involved in the first step of casein degradation. Only some of the oligopeptides released by lactocepin are taken up by the oligopeptide transport system (Opp) and subsequently cleaved into amino acids by intracellular peptidases (for recent reviews, see references 17 and 22).Two different types of lactocepins (lactocepin I and lactocepin III) have been identified in lactococci on the basis of their specificity for caseins (44). Lactocepin I cleaves ␤-casein preferentially and -casein to a lesser extent. In contrast, lactocepin III cleaves ␤-, -, and ␣ s1 -caseins. There are only 44 differences in the amino acid sequences of lactocepins I and III, and 5 of them are responsible for the differences in specificity between the two lactocepins (7, 40).Lactocepin purification requires release of the enzyme from the cell (30), which results from autoproteolysis of the protein in a Ca 2ϩ -free buffer (24, 25). Autoproteolysis takes place in the C-terminal part of the protein, presumably in the B domain of the protein, and results in a 145-kDa enzyme, compared to the 186-kDa cell-anchored lactocepin III (1). The action of purified lactocepins towards caseins has been studied extensively (32,33,37,38). In particular, most, if not all, of the peptides released from ␤-casein by purified lactocepin I have been identified by on-line coupling of liquid c...

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Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCDO 763

Cited by 46 publications

References 18 publications

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides

The extracellular PI-type proteinase of Lactococcus lactis hydrolyzes beta-casein into more than one hundred different oligopeptides

Article

The Autoproteolysis of Lactococcus lactis Lactocepin III Affects Its Specificity towards β-Casein

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