Cell Envelope Proteinase Research Articles

High Pressure Effects on Proteolytic and Glycolytic Enzymes Involved in Cheese Manufacturing

The activity of chymosin, plasmin, and Lactococcus lactis enzymes (cell envelope proteinase, intracellular peptidases, and glycolytic enzymes) were determined after 5-min exposures to pressures up to 800MPa. Plasmin was unaffected by any pressure treatment. Chymosin activity was unaffected up to 400MPa and decreased at 500 to 800MPa. Fifty percent of control chymosin activity remained after the 800MPa treatment. The lactococcal cell envelope proteinase (CEP) and intracellular peptidase activities were monitored in cell extracts of pressure-treated cells. A pressure of 100MPa increased the CEP activity, whereas 200MPa had no effect. At 300MPa, CEP activity was reduced, and 400 to 800MPa inactivated the enzyme. X-Prolyl-dipeptidyl aminopeptidase was insensitive to 5-min pressure treatments of 100 to 300MPa, but was inactivated at 400 to 800MPa. Aminopeptidase N was unaffected by 100 and 200MPa. However, 300MPa significantly reduced its activity, and 400 to 800MPa inactivated it. Aminopeptidase C activity increased with increasing pressures up to 700MPa. High pressure did not affect aminopeptidase A activity at any level. Hydrolysis of Lys-Ala-ρ-NA doubled after 300-MPa exposure, and was eliminated at 400 to 800MPa. Glycolytic enzyme activities of pressure-treated cells were evaluated collectively by determining the titratable acidity as lactic acid produced by cell extracts in the presence of glucose. The titratable acidities produced by the 100 and 200MPa samples were slightly increased compared to the control. At 300 to 800MPa, no significant acid production was observed. These data demonstrate that high pressure causes no effect, activation, or inactivation of proteolytic and glycolytic enzymes depending on the pressure level and enzyme. Pressure treatment of cheese may alter enzymes involved in ripening, and pressure-treating L. lactis may provide a means to generate attenuated starters with altered enzyme profiles.

Exchanging lactocepin plasmids in lactococcal starters to study bitterness development in Gouda cheese: a preliminary investigation

Most fast-milk-coagulating Lactococcus lactis starter strains contain a plasmid-encoded cell envelope proteinase (lactocepin; EC 3.4.21.96) having PI-type (lactocepin I) or PI/III intermediate-type (lactocepin I/III) specificity (those with PIII-type specificity are rare). We assessed the effect of lactocepin I and lactocepin I/III on peptide accumulation and bitterness development in dry-salted Gouda cheese by exchanging lactocepin plasmids between two L. lactis starter strains low in autolysis, thus eliminating the effects of other starter variables. Gouda was made using starter strains, either L. lactis subsp. cremoris HP (lactocepin I) or L. lactis subsp. lactis U (lactocepin I/III), or using constructs of these two strains with exchanged lactocepin plasmids. Bitterness in Gouda was consistently greater when the starter strains contained lactocepin I. Following ripening, cheese peptide profiles (determined by high-performance liquid chromatography) were found to vary more in relation to starter lactocepin specificity than in relation to starter strain background or sub-species (lactis or cremoris).

Peptides identified during Emmental cheese ripening: origin and proteolytic systems involved.

To determine the proteolytic changes occurring during Emmental cheese ripening, peptides released in cheese aqueous phase were analyzed by reversed-phase HPLC and identified by tandem mass spectrometry sequencing, for which different strategies were illustrated by some examples. Among the 91 peptides identified, most of them arose from alpha(s1)- (51) and beta-caseins (28), and a few arose from alpha(s2)- (9) and kappa-caseins (1). An attempt was made to correlate the released peptides with the proteolytic systems potentially involved during Emmental cheese manufacture. Besides the well-known action of plasmin on beta- and alpha(s2)-caseins, and in the absence of residual fungal coagulant from Endothia parasitica, two other proteinases seem to be involved in the hydrolysis of alpha(s1)-casein in Emmental cheese: cathepsin D originated from milk and cell-envelope proteinase from thermophilic starters. Moreover, peptidases from starters were also active throughout ripening, presumably like those from nonstarter lactic acid bacteria, in contrast to those from propionic acid bacteria.

Nucleotide sequence and characterization of the cell envelope proteinase plasmid in Lactococcus lactis subsp. cremoris HP.

The major cell envelope proteinase (lactocepin; EC 3.4.21.96) produced by Lactococcus lactis cheese starter bacteria is required for starter growth and acid production in milk. The aim of this study was to characterize a lactocepin plasmid from a L. lactis subsp. cremoris cheese starter strain. A restriction map of the lactocepin plasmid pHP003 from strain HP was constructed, fragments were cloned in Escherichia coli vectors, and the complete DNA sequence (13,433 bp) was determined. Among 120 industrial L. lactis starter strains screened, five contained the same specificity-type lactocepin as pHP003. The lactocepin gene in these strains was invariably linked with a partially-deleted abiB gene. The lactocepin specificity type of strain HP, conferred by a known configuration of key residues, is relatively uncommon. The gene is invariably linked with a partially deleted abiB gene on each lactocepin plasmid. This is the first complete sequence reported for a lactocepin plasmid, and provides the basis for better understanding, or manipulation, of lactocepin production.

Action of the lactococcal proteinase during Camembert-type curd making

We emphasise the role of the lactococcal cell envelope proteinase (lactocepin) on the proteolysis in a curd of soft cheese. During cheese manufacture, proteolysis occurred as soon as the renneting stage with the action of the chymosin, and the level of proteolysis intensified rapidly. The peptides, present within the whey collected throughout the drainage, were identified using tandem mass spectrometry. From the sixteen peptides identified at the first turn, we showed the early predominant action of the lactocepin type III. From the forty eight peptides identified at the end of the drainage, we demonstrated, in addition to the predominant action of lactocepin, other activities that could result from the action of lactococcal peptidases. In addition, this study of proteolysis confirmed that the action of proteinases on casein in solution differed from their action in situ: some sites cleaved in solution were not cleaved in cheese and vice versa.

Effect of genetically modified Lactococcus lactis cell-envelope proteinases with altered specificity on the course of casein degradation under cheese conditions

Abstract The specificity of the cell-bound, wild-type proteinase (PrtP) of Lactococcus lactis subsp. cremoris SK11 towards the αs1-casein(1–23) fragment under simulated cheese conditions was compared with that of several mutants of PrtP. A unique specificity resulted from substitution of the substrate-binding site residues AKT 137–139 in the wild-type with GLA in the mutant PrtP. This different specificity clearly modifies the breakdown of chymosin-generated primary cheese peptides under cheese conditions. The consequences for amino acid production were investigated with cells which were optimally permeabilized with respect to accessibility and activity of the intracellular proteolytic system. Unlike untreated cells, these cells showed efficient peptide uptake and intracellular conversion (involving peptides of up to 15 residues), similar to that occurring in a normal Gouda cheese. No significant differences were found in the composition of the amino acid pools generated by permeabilized cells containing either the wild-type or the mutant PrtP.

Enzymatic ability of Lactobacillus casei subsp. casei IFPL731 for flavour development in cheese

Abstract Lactobacillus casei subsp. casei IFPL731 is a wild strain isolated from artisanal goats’ milk cheese. The strain shows a multiple enzymatic system that involves esterase, cell-envelope proteinase, aminopeptidases, dipeptidases, specialized peptidases for proline-containing peptides, and amino acid converting enzymes. The broad enzymatic system of Lb. casei IFPL731 is responsible for its hydrolyzing activity towards a number of peptides, including bitter and methionine-containing peptides. Both characteristics are of great interest as regards the use of the strain as a starter culture adjunct to influence the development of cheese flavour, which has been demonstrated in the manufacture of goats’ milk and low fat cheeses. Moreover, Lb. casei IFPL731 shows methionine aminotransferase activity that leads to the production of the typical cheese aroma. The different enzymes from Lb. casei IFPL731 described in this review and its utilization as a starter culture adjunct in cheese manufacture make the strain one of the best characterized lactobacilli in the literature.

Streptococcus thermophilus cell wall-anchored proteinase: release, purification, and biochemical and genetic characterization.

Streptococcus thermophilus CNRZ 385 expresses a cell envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to most classical methods of extraction from the cell envelopes, we developed a three-step process based on loosening of the cell wall by cultivation of the cells in the presence of glycine (20 mM), mechanical disruption (with alumina powder), and enzymatic treatment (lysozyme). The pure enzyme is a serine proteinase highly activated by Ca(2+) ions. Its activity was optimal at 37 degrees C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of the hydrolysis of the chromogenic and casein substrates indicated that PrtS presented an intermediate specificity between the most divergent types of cell envelope proteinases from lactococci, known as the PI and PIII types. This result was confirmed by the sequence determination of the regions involved in substrate specificity, which were a mix between those of PI and PIII types, and also had unique residues. Sequence analysis of the PrtS encoding gene revealed that PrtS is a member of the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an LPXTG motif. PrtS bears similarities to cell envelope proteinases from pyogenic streptococci (C5a peptidase and cell surface proteinase) and lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one domain (the B domain) present in cell envelope proteinases from all other lactic acid bacteria.

Varying influence of the autolysin, N-acetyl muramidase, and the cell envelope proteinase on the rate of autolysis of six commercial Lactococcus lactis cheese starter bacteria grown in milk.

The autolysin, N-acetyl muramidase (AcmA), of six commercial Lactococcus lactis subsp. cremoris starter strains and eight Lc. lactis subsp. cremoris derivatives or plasmid-free strains was shown by renaturing SDS-PAGE (zymogram analysis) to be degraded by the cell envelope proteinase (lactocepin; EC 3.4.21.96) after growth of strains in milk at 30 degrees C for 72 h. Degradation of AcmA was less in starter strains and derivatives producing lactocepin I/III (intermediate specificity) than in strains producing lactocepin I. This supports previous observations on AcmA degradation in derivatives of the laboratory strain Lc. lactis subsp. cremoris MG1363 (Buist et al. Journal of Bacteriology 180 5947-5953 1998). In contrast to the MG1363 derivatives, however, the extent of autolysis in milk of the commercial Lc. lactis subsp. cremoris starter strains in this study did not always correlate with lactocepin specificity and AcmA degradation. The distribution of autolysins within the cell envelope of Lc. lactis subsp. cremoris starter strains and derivatives harvested during growth in milk was compared by zymogram analysis. AcmA was found associated with cell membranes as well as cell walls and some cleavage of AcmA occurred independently of lactocepin activity. An AcmA product intermediate in size between precursor (46 kDa) and mature (41 kDa) forms of AcmA was clearly visible on zymograms, even in the absence of lactocepin I activity. These results show that autolysis of commercial Lc. lactis subsp. cremoris starter strains is not primarily determined by AcmA activity in relation to lactocepin specificity and that proteolytic cleavage of AcmA in vivo is not fully defined.

Deletion of various carboxy-terminal domains of Lactococcus lactis SK11 proteinase: effects on activity, specificity, and stability of the truncated enzyme.

The Lactococcus lactis SK11 cell envelope proteinase is an extracellular, multidomain protein of nearly 2,000 residues consisting of an N-terminal serine protease domain, followed by various other domains of largely unknown function. Using a strategy of deletion mutagenesis, we have analyzed the function of several C-terminal domains of the SK11 proteinase which are absent in cell envelope proteinases of other lactic acid bacteria. The various deletion mutants were functionally expressed in L. lactis and analyzed for enzyme stability, activity, (auto)processing, and specificity toward several substrates. C-terminal deletions of first the cell envelope W (wall) and AN (anchor) domains and then the H (helix) domain leads to fully active, secreted proteinases of unaltered specificity. Gradually increasing the C-terminal deletion into the so-called B domain leads to increasing instability and autoproteolysis and progressively less proteolytic activity. However, the mutant with the largest deletion (838 residues) from the C terminus and lacking the entire B domain still retains proteolytic activity. All truncated enzymes show unaltered proteolytic specificity toward various substrates. This suggests that the main role played by these domains is providing stability or protection from autoproteolysis (B domain), spacing away from the cell (H domain), and anchoring to the cell envelope (W and AN domains). In addition, this study allowed us to more precisely map the main C-terminal autoprocessing site of the SK11 proteinase and the epitope for binding of group IV monoclonal antibodies.

Structural changes and interactions involved in the Ca(2+)-triggered stabilization of the cell-bound cell envelope proteinase in Lactococcus lactis subsp. cremoris SK11.

The cell-bound cell envelope proteinase (CEP) of the mesophilic cheese-starter organism Lactococcus lactis subsp. cremoris SK11 is protected from rapid thermal inactivation at 25 degrees C by calcium bound to weak binding sites. The interactions with calcium are believed to trigger reversible structural rearrangements which are coupled with changes in specific activity (F. A. Exterkate and A. C. Alting, Appl. Env. Microbiol. 65:1390-1396, 1999). In order to determine the significance of the rearrangements for CEP stability and the nature of the interactions involved, the effects of the net charge present on the enzyme and of different neutral salts were studied with the stable Ca-loaded CEP, the unstable so-called "Ca-free" CEP and with the Ca-free CEP which was stabilized nonspecifically and essentially in its native conformation by the nonionic additive sucrose. The results suggest that strengthening of hydrophobic interactions is conducive to stabilization of the Ca-free CEP. On the other hand, a hydrophobic effect contributes significantly to the stability of the Ca-loaded CEP; a phased salting-in effect by a chaotropic salt suggests a complex inactivation process of this enzyme due to weakening of hydrophobic interactions and involving an intermediate enzyme species. Moreover, a Ca-triggered increase of a relatively significant hydrophobic effect in the sucrose-stabilized Ca-free CEP occurs. It is suggested that in the Ca-free CEP the absence of both local calcium-mediated backbone rigidification and neutralization of negative electrostatic potentials in the weak Ca-binding sites, and in addition the lack of significant hydrophobic stabilization, increase the relative effectiveness of electrostatic repulsive forces on the protein to an extent that causes the observed instability. The conditions in cheese seem to confer stability upon the cell-bound enzyme; its possible involvement in proteolysis throughout the ripening period is discussed.

Role of calcium in activity and stability of the Lactococcus lactis cell envelope proteinase.

The mature lactococcal cell envelope proteinase (CEP) consists of an N-terminal subtilisin-like proteinase domain and a large C-terminal extension of unknown function whose far end anchors the molecule in the cell envelope. Different types of CEP can be distinguished on the basis of specificity and amino acid sequence. Removal of weakly bound Ca2+ from the native cell-bound CEP of Lactococcus lactis SK11 (type III specificity) is coupled with a significant reversible decrease in specific activity and a dramatic reversible reduction in thermal stability, as a result of which no activity at 25 degrees C (pH 6.5) can be measured. The consequences of Ca2+ removal are less dramatic for the CEP of strain Wg2 (mixed type I-type III specificity). Autoproteolytic release of CEP from cells concerns this so-called "Ca-free" form only and occurs most efficiently in the case of the Wg2 CEP. The results of a study of the relationship between the Ca2+ concentration and the stability and activity of the cell-bound SK11 CEP at 25 degrees C suggested that binding of at least two Ca2+ ions occurred. Similar studies performed with hybrid CEPs constructed from SK11 and Wg2 wild-type CEPs revealed that the C-terminal extension plays a determinative role with respect to the ultimate distinct Ca2+ dependence of the cell-bound CEP. The results are discussed in terms of predicted Ca2+ binding sites in the subtilisin-like proteinase domain and Ca-triggered structural rearrangements that influence both the conformational stability of the enzyme and the effectiveness of the catalytic site. We argue that distinctive primary folding of the proteinase domain is guided and maintained by the large C-terminal extension.

Multi-domain, cell-envelope proteinases of lactic acid bacteria.

The multi-domain, cell-envelope proteinases encoded by the genes prtB of Lactobacillus delbrueckii subsp. bulgaricus, prtH of Lactobacillus helveticus, prtP of Lactococcus lactis, scpA of Streptococcus pyogenes and csp of Streptococcus agalactiae have been compared using multiple sequence alignment, secondary structure prediction and database homology searching methods. This comparative analysis has led to the prediction of a number of different domains in these cell-envelope proteinases, and their homology, characteristics and putative function are described. These domains include, starting from the N-terminus, a pre-pro-domain for secretion and activation, a serine protease domain (with a smaller inserted domain), two large middle domains A and B of unknown but possibly regulatory function, a helical spacer domain, a hydrophilic cell-wall spacer or attachment domain, and a cell-wall anchor domain. Not all domains are present in each cell-envelope proteinase, suggesting that these multi-domain proteins are the result of gene shuffling and domain swapping during evolution.

Characterisation of a cell-envelope proteinase from Lactobacillus helveticus

The cell-envelope proteinase from Lactobacillus helveticus CRL 1062 was detected in the cell membrane fraction. The enzyme remained associated with the cells even after treatment with lysozyme and was not released from washed cells in absence of calcium. The proteinase was maximally active at pH 6.5–7.0 and 42 °C and hydrolysed α- and β-caseins at different rates. Activity was inhibited (98%) by 1 mM PMSF, suggesting it was a serine-type protease.

Selective Hydrolysis of Milk Proteins To Facilitate the Elimination of the ABBOS Epitope of Bovine Serum Albumin and Other Immunoreactive Epitopes

Milk proteins are hydrolyzed to prevent immunological reactions, but immunoreactive epitopes, including the ABBOS epitope of bovine serum albumin (BSA), can still be detected in commercially available milk protein hydrolysates. We used lactococcal cell-envelope proteinase (CEP) for the hydrolysis of the individual milk proteins and of mixtures thereof, or for the hydrolysis of sodium caseinate (contaminated with whey proteins). CEP exclusively degraded casein, leaving the four major whey proteins intact. This property facilitated the removal of the intact whey proteins from the casein fragments by ultrafiltration. Depending on the molecular mass of the whey protein to be removed, membranes with cutoff values between 3 and 30 kDa were used, resulting in casein hydrolysates free of protein fragments with cross-reactive whey-protein-specific IgE (immunoglobulin E) or ABBOS antibody-binding sites. Even the casein itself was degraded in such a way by CEP that cross-reactive casein-specific IgE antibody-binding sites could be eliminated. The product could find application in infant formulas for therapeutic and preventive treatment of children with cow's milk allergy; in addition, the preventive use of such formulas in children genetically susceptible to the development of insulin-dependent diabetes mellitus (IDDM) should be considered if a relationship between the consumption of BSA and IDDM were to become more apparent. The method is also applicable for preparing casein-free whey protein preparations.

The contribution of caseins to the amino acid supply for Lactococcus lactis depends on the type of cell envelope proteinase.

The ability of caseins to fulfill the amino acid requirements of Lactococcus lactis for growth was studied as a function of the type of cell envelope proteinase (PI versus PIII type). Two genetically engineered strains of L. lactis that differed only in the type of proteinase were grown in chemically defined media containing alphas1-, beta-, and kappa-caseins (alone or in combination) as the sources of amino acids. Casein utilization resulted in limitation of the growth rate, and the extent of this limitation depended on the type of casein and proteinase. Adding different mixtures of essential amino acids to the growth medium made it possible to identify the nature of the limitation. This procedure also made it possible to identify the amino acid deficiency which was growth rate limiting for L. lactis in milk (S. Helinck, J. Richard, and V. Juillard, Appl. Environ. Microbiol. 63:2124-2130, 1997) as a function of the type of proteinase. Our results were compared with results from previous in vitro experiments in which casein degradation by purified proteinases was examined. The results were in agreement only in the case of the PI-type proteinase. Therefore, our results bring into question the validity of the in vitro approach to identification of casein-derived peptides released by a PIII-type proteinase.

Peptide Accumulation and Bitterness in Cheddar Cheese Made Using Single-Strain Lactococcus lactis Starters with Distinct Proteinase Specificities

This study investigated peptide accumulation and bitterness in reduced- and full-fat Cheddar cheeses that were manufactured with single-strain Lactococcus lactis starters that had distinct cell envelope proteinase specificities. Micellar electrokinetic capillary electrophoresis of aqueous cheese extracts detected three large peaks, designated O, P, and Q, that eluted with peptide standards and increased in area during cheese maturation in a pattern that was distinct for each starter. Regression analysis of bitter flavor scores from trained sensory panels and individual OQ peak areas suggested that peaks P and Q had a negative and positive correlation, respectively, to this defect. Then, HPLC, capillary electrophoresis, peptide sequencing, and mass spectrometry were used to identify five peptides from αS1-casein (CN), one from β-CN, and one from αS2-CN that accumulated in 6-mo-old cheeses. Most of the peptides derived from αS1-CN (f 1–23) accumulated in a manner that corresponded with starter proteinase specificity. All of the peptides identified in the study except αS2-CN (f 1–21) eluted in the O-P-Q region of micellar electrokinetic capillary electropherograms. The αS1-CN (f 1–16), αS1-CN (f 1–17) and β-CN (f 193-209) eluted in peak O, αS1-CN (f 1–13) and αS1-CN (f 1–14) eluted in peak P, and αS1-CN (f 1–9) eluted in peak Q.

Specificity of the bound and free forms of the cell-envelope proteinase of Lactobacillus casei subsp. casei IFPL 731 towards the ?s1-casein-(1?23)-fragment

P. FERNANDEZ DE PALENCIA, c. PELAEZ AND M.C. MARTIN-HERNANDEZ. 1997. The specificity of the bound and free forms of Lactobacillus casei subsp. casei IFPL 731 proteinase towards the αs1-casein-(1–23)-fragment has been studied. The use of the chelant agent EDTA for the extraction of the proteinase affects its specificity compared to either the use of lysozyme and mutanolysin or the whole-cell proteinase form. This gives a different pattern of the αs1-casein-fragment hydrolysis, as observed by HPLC. The effect of different chemical agents on the specific activity of the proteinase also varies depending on the method used to release the proteinase.

Recommended Name: Lactocepin

European Journal of BiochemistryVolume 250, Issue 1 p. 4-5 EC 3.4.21.96Free Access Recommended Name: Lactocepin First published: 23 June 2004 https://doi.org/10.1111/j.1432-1033.1997.01_11.xAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 Visser, S., Robben, A.J.P.M & Slangen, C.J. (1991) Specificity of a cell-envelope-located proteinase (PIII-type) from Lactococcus lactis subsp. cremoris AM1 in its action on bovine beta-casein. Appl. Microbiol. Biotechnol. 35, 477– 483 2 Monnet, V., Ley, J.P. & Gonzalez, S. (1992) Substrate specificity of the cell envelope-located proteinase of Lactococcus lactis subsp. lactis NCD0763. Int. J. Biochem. 24, 707– 718 3 Exterkate, F.A., Alting, A.C. & Bruinenberg, P.G. (1993) Diversity of cell envelope proteinase specificity among strains of Lactococcus lactis and its relationship to charge characteristics of the substrate-binding region. Appl. Environ. Microbiol. 59, 3640– 3647 4 Pritchard, G.G. & Coolbear, T. (1993) The physiology and biochemistry of the proteolytic system in lactic acid bacteria. FEMS Microbiol Rev. 12, 179– 206 Volume250, Issue1November (II) 1997Pages 4-5 ReferencesRelatedInformation

Proteolytic Activity of Lactobacillus casei Subsp. casei IFPL 731 in a Model Cheese System

The role played by the cell-envelope proteinase and the aminopeptidase activity of Lactobacillus casei subsp. casei IFPL 731 isolated from goat's milk cheese has been dilucidated, adding the cell w...