Genetics of the proteolytic system of lactic acid bacteria

Abstract The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococi, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

Abstract: The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.     

PepS from Streptococcus thermophilus. A new member of the aminopeptidase T family of thermophilic bacteria.

The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products. Streptococcus thermophilus is widely used in the dairy industry. In comparison with the model lactic acid bacteria Lactococcus lactis, S. thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS). To understand how S. thermophilus grows in milk, we purified and characterized this aminopeptidase. PepS is a monomeric metallopeptidase of approximately 45 kDa with optimal activity in the range pH 7.5-8.5 and at 55 degrees C on Arg-paranitroanilide as substrate. PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N-terminus. From the N-terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions. The deduced amino-acid sequence of the PepS gene has high identity (40-50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S. thermophilus as a new member of this family. In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products' flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds.     

Peptidases and amino acid catabolism in lactic acid bacteria

The conversion of peptides to free amino acids and their subsequent utilization is a central metabolic activity in prokaryotes. At least 16 peptidases from lactic acid bacteria (LAB) have been characterized biochemically and/or genetically. Among LAB, the peptidase systems of Lactobacillus helveticus and Lactococcus lactis have been examined in greatest detail. While there are homologous enzymes common to both systems, significant differences exist in the peptidase complement of these organisms. The characterization of single and multiple peptidase mutants indicate that these strains generally exhibit reduced specific growth rates in milk compared to the parental strains. LAB can also catabolize amino acids produced by peptide hydrolysis. While the catabolism of amino acids such as Arg, Thr, and His is well understood, few other amino acid catabolic pathways from lactic acid bacteria have been characterized in significant detail. Increasing research attention is being directed toward elucidating these pathways as well as characterizing their physiological and industrial significance.     

Casein degradation and amino acid liberation in milk by two highly proteolytic strains of lactic acid bacteria.

Amongst 100 isolates of lactic acid bacteria, Lactobacillus bulgaricus and Streptococcus feacalis proved to be highly proteolytic as demonstrated by starch gel electrophoresis and liberation of various amino acids. Both these organisms hydrolysed casein differently to produce different concentrations of amino acids.     

The proteotytic systems of lactic acid bacteria

Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The proteolytic system consists of an extracellularly located serine-proteinase, transport systems specific for di-tripeptides and oligopeptides (> 3 residues), and a multitude of intracellular peptidases. This review describes the properties and regulation of individual components as well as studies that have led to identification of their cellular localization. Targeted mutational techniques developed in recent years have made it possible to investigate the role of individual and combinations of enzymes in vivo. Based on these results as well as in vitro studies of the enzymes and transporters, a model for the proteolytic pathway is proposed. The main features are: (i) proteinases have a broad specificity and are capable of releasing a large number of different oligopeptides, of which a large fraction falls in the range of 4 to 8 amino acid residues; (ii) oligopeptide transport is the main route for nitrogen entry into the cell; (iii) all peptidases are located intracellularly and concerted action of peptidases is required for complete degradation of accumulated peptides.     

Bioactive peptides encrypted in milk proteins: proteolytic activation and thropho-functional properties

The bioactivities of peptides encrypted in major milk proteins are latent until released and activated by enzymatic proteolysis, e.g. during gastrointestinal digestion or food processing. The proteolytic system of lactic acid bacteria can contribute to the liberation of bioactive peptides. In vitro, the purified cell wall proteinase of Lactococcus lactis was shown to liberate oligopeptides from - and -caseins which contain amino acid sequences present in casomorphins, casokinines, and immunopeptides. The further degradation of these peptides by endopeptidases and exopeptidases of lactic acid bacteria could lead to the liberation of bioactive peptides in fermented milk products. However, the sequences of practically all known biologically active peptides can also be cleaved by peptidases from lactic acid bacteria. Activated peptides are potential modulators of various regulatory processes in the body: Opioid peptides are opioid receptor ligands which can modulate ab sorption processes in the intestinal tract, angiotensin-I-converting enzyme (ACE)-inhibitory peptides are hemodynamic regulators and exert an antihypertensive effect, immunomodulating casein peptides stimulate the activities of cells of the immune system, antimicrobial peptides kill sensitive microorganisms, antithrombotic peptides inhibit aggregation of platelets and caseinophosphopeptides may function as carriers for different minerals, especially calcium. Bioactive peptides can interact with target sites at the luminal side of the intestinal tract. Furthermore, they can be absorbed and then reach peripheral organs. Food-derived bioactive peptides are claimed to be health enhancing components which can be used for functional food and pharmaceutical preparations.     

Proteinase genes of cheese starter cultures.

The proteolytic enzymes of lactococci are of eminent importance for milk fermentations. By the combined action of proteinases and peptidases milk protein is degraded to peptides and amino acids which are required for cell growth and contribute to the organoleptic properties of the foods. The importance of the proteolytic system for dairy product quality has resulted in an increased fundamental research of the enzymes and genes involved. Proteinase plasmids have been identified and plasmid stability problems offered an explanation for the apparent instability of proteolysis in certain strains of lactococci. Chromosomal integration has recently been used to stably anchor the proteinase genes in the chromosome of Lactococcus lactis. The structural proteinase genes of a number of strains have been cloned and sequenced, and some of the properties of the enzymes they specify will be discussed. The product of a second gene is necessary for the activation of the proteinase, a proteinase maturation process that is unique in the bacterial world.     

Hydrolysis of sequenced beta-casein peptides provides new insight into peptidase activity from thermophilic lactic acid bacteria and highlights intrinsic resistance of phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified beta-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the beta-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the beta-casein hydrolysate as the substrate at pH 5.5 and 24 degrees C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing beta-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the beta-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

Proteolytic enzymes of dairy starter cultures

Abstract The synthesis of proteolytic enzymes by starter bacteria is a fundamental requirement for rapid acid production in milk fermentations. These organisms possess a number of proteinases and peptidases which act in concert to hydrolyse milk protein to the free amino acids required for cell growth. The same enzymes have an important secondary role in cheese ripening contributing to rheological and organoleptic changes. A highly complex mixture of both enzymes and substrates is present. The strategic location of these enzymes, in the cell wall and membrane structures and in the cytoplasm, governs enzyme access to the substrates and is central to both roles. An overview of the above topics is presented.     

Protein degradation within mitochondria: versatile activities of AAA proteases and other peptidases

Cell survival depends on essential processes in mitochondria. Various proteases within these organelles regulate mitochondrial biogenesis and ensure the complete degradation of excess or damaged proteins. Many of these proteases are highly conserved and ubiquitous in eukaryotic cells. They can be assigned to three functional classes: processing peptidases, which cleave off mitochondrial targeting sequences of nuclearly encoded proteins and process mitochondrial proteins with regulatory functions; ATP-dependent proteases, which either act as processing peptidases with regulatory functions or as quality-control enzymes degrading non-native polypeptides to peptides; and oligopeptidases, which degrade these peptides and mitochondrial targeting sequences to amino acids. Disturbances of protein degradation within mitochondria cause severe phenotypes in various organisms and can lead to the induction of apoptotic programmes and cell-specific neurodegeneration in mammals. After an overview of the proteolytic system of mitochondria, we will focus on versatile functions of ATP-dependent AAA proteases in the inner membrane. These conserved proteolytic machines conduct protein quality surveillance of mitochondrial inner membrane proteins, mediate vectorial protein dislocation from membranes, and, acting as processing enzymes, control ribosome assembly, mitochondrial protein synthesis, and mitochondrial fusion. Implications of these functions for cell-specific axonal degeneration in hereditary spastic paraplegia will be discussed.     

Multiple-peptidase mutants of Lactococcus lactis are severely impaired in their ability to grow in milk.

To examine the contribution of peptidases to the growth of lactococcus lactis in milk, 16 single- and multiple-deletion mutants were constructed. In successive rounds of chromosomal gene replacement mutagenesis, up to all five of the following peptidase genes were inactivated (fivefold mutant): pepX, pepO, pepT, pepC, and pepN. Multiple mutations led to slower growth rates in milk, the general trend being that growth rates decreased when more peptidases were inactivated. The fivefold mutant grew more than 10 times more slowly in milk than the wild-type strain. In one of the fourfold mutants and in the fivefold mutant, the intracellular pools of amino acids were lower than those of the wild type, whereas peptides had accumulated inside the cell. No significant differences in the activities of the cell envelope-associated proteinase and of the oligopeptide transport system were observed. Also, the expression of the peptidases still present in the various mutants was not detectably affected. Thus, the lower growth rates can directly be attributed to the inability of the mutants to degrade casein-derived peptides. These results supply the first direct evidence for the functioning of lactococcal peptidases in the degradation of milk proteins. Furthermore, the study provides critical information about the relative importance of the peptidases for growth in milk, the order of events in the proteolytic pathway, and the regulation of its individual components.     

Proteolytic activity of sourdough bacteria

Summary Proteolytic activity during the fermentation of sourdough results in an increase in amino acid content. The proteolysis is caused by flour enzymes, microbial enzymes of flour and by sourdough bacteria. The results indicate that the lactic acid bacteria of sourdough are important for proteolytic activity during the fermentation of sourdough. This proteolytic activity depends on the species of bacteria. Homo- and heterofermentative sourdough bacteria effect different amino acid spectra. Qualitative and quantitative differences in sulphur-containing, cyclic and hydroxy amino acids have been observed. The proteolytic process can be influenced by fermentation conditions, especially the temperature. A lesser effect is observed in the dough yield (flour-water relationship). From experiments with different strains and species of lactic acid bacteria, it is concluded that only one third of the proteolytic activity in sourdough is based on proteases from the flour.Publication-No. 5592 of Federal Research Centre for Cereal and Potato Processing, Detmold, Federal Republic of GermanyPaper presented at the Third International Conference Rye and Triticale, Poznan, Poland, 13–14 May 1987     

Insight into the bovine milk peptide LPcin‐YK3 selection in the proteolytic system of Lactobacillus species

Antimicrobial peptides are class of small, positively charged peptides known for their broad‐spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin‐YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100‐fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide‐binding protein A and LPcin‐YK3 peptide. Given that degradation related to the LPcin‐YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin‐YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide‐binding protein A in respect of LPcin‐YK3 peptide.     

Hydrolysis of Sequenced β-Casein Peptides Provides New Insight into Peptidase Activity from Thermophilic Lactic Acid Bacteria and Highlights Intrinsic Resistance of Phosphopeptides

The peptidases of thermophilic lactic acid bacteria have a key role in the proteolysis of Swiss cheeses during warm room ripening. To compare their peptidase activities toward a dairy substrate, a tryptic/chymotryptic hydrolysate of purified β-casein was used. Thirty-four peptides from 3 to 35 amino acids, including three phosphorylated peptides, constitute the β-casein hydrolysate, as shown by tandem mass spectrometry. Cell extracts prepared from Lactobacillus helveticus ITG LH1, ITG LH77, and CNRZ 32, Lactobacillus delbrueckii subsp. lactis ITG LL14 and ITG LL51, L. delbrueckii subsp. bulgaricus CNRZ 397 and NCDO 1489, and Streptococcus thermophilus CNRZ 385, CIP 102303, and TA 060 were standardized in protein. The peptidase activities were assessed with the β-casein hydrolysate as the substrate at pH 5.5 and 24°C (conditions of warm room ripening) by (i) free amino acid release, (ii) reverse-phase chromatography, and (iii) identification of undigested peptides by mass spectrometry. Regardless of strain, L. helveticus was the most efficient in hydrolyzing β-casein peptides. Interestingly, cell extracts of S. thermophilus were not able to release a significant level of free proline from the β-casein hydrolysate, which was consistent with the identification of numerous dipeptides containing proline. With the three lactic acid bacteria tested, the phosphorylated peptides remained undigested or weakly hydrolyzed indicating their high intrinsic resistance to peptidase activities. Finally, several sets of peptides differing by a single amino acid in a C-terminal position revealed the presence of at least one carboxypeptidase in the cell extracts of these species.     

Growth Response and Modifications of Organic Nitrogen Compounds in Pure and Mixed Cultures of Lactic Acid Bacteria from Wine

The interactions between the proteolytic X₂L strain of Oenococcus oeni and the non-proteolytic 12p strain of Pediococcus pentosaceus were assayed. The characteristics of cell growth, protein degradation, and amino acid production of both strains were determined in pure and mixed cultures. O. oeni showed poor cell growth and greater ability in the release of amino acids to the extracellular medium, whereas P. pentosaceus showed a higher yield in cell production with a decrease in the amino acid concentration in the medium. P. pentosaceus especially consumed essential amino acids for growth, and O. oeni released several of the essential amino acids important for growth of P. pentosaceus. In the mixed culture, mutualism was observed. The higher activity of the proteolytic system of O. oeni in mixed culture produced an increase in cell growth and in the amount of essential amino acids released. These findings provide new knowledge about the metabolic interactions between lactic acid bacteria isolated from wine when proteins are degraded in mixed bacterial populations.     

Class II antimicrobial peptides from lactic acid bacteria

Strains of lactic acid bacteria (LAB) produce a wide variety of antibacterial peptides. More than fifty of these so-called peptide bacteriocins have been isolated in the last few years. They contain 20-60 amino acids, and are cationic and hydrophobic in nature. Several of these bacteriocins consist of two complementary peptides. The peptide bacteriocins of LAB are inhibitory at concentrations in the nanomolar range, and cause membrane permeabilization and leakage of intracellular components in sensitive cells. The inhibitory spectrum is limited to gram-positive bacteria, and in many cases to bacteria closely related to the producing strain. Among the target organisms are food spoilage bacteria and pathogens such as Listeria, so that many of these antimicrobial peptides could have a potential as food preservatives as well as in medical applications.     

Analysis of MHC class II antigen processing by quantitation of peptides that constitute nested sets

Peptides associated with class II MHC molecules are of variable length because in contrast to peptides associated with class I MHC molecules, their amino and C termini are not constrained by the structure of the peptide interaction with the binding site. The proteolytic processing events that generate these peptides are still not well understood. To address this question, peptides extracted from HLA-DR*0401 were analyzed using two types of mass spectrometry instrumentation. This enabled identification of >700 candidate peptides in a single analysis and provided relative abundance information on 142 peptides contained in 11 nested sets of 3-36 members each. Peptides of 12 residues or less occurred only at low abundance, despite the fact that they were predicted to fully occupy the HLA-DR*0401 molecule in a single register. Conversely, the relative abundance of longer species suggested that proteolytic events occurring after MHC binding determine the final structure of most class II-associated peptides. Our data suggest that C-terminal residues of these peptides reflect the action of peptidases that cleave at preferred amino acids, while amino termini appear to be determined more by proximity to the class II MHC binding site. Thus, the analysis of abundance information for class II-associated peptides comprising nested sets has offered new insights into proteolytic processing of MHC class II-associated peptides.     

Complete amino acid analysis of peptides and proteins after hydrolysis by a mixture of Sepharose-bound peptidases

Incubation with a mixture of Sepharose-bound peptidases was shown to result in the quantitative release of amino acids from certain peptides and S-aminoethylated proteins. Subtraction of the low background values of amino acids generated by the enzymes enables amino acid ratios of corticotrophin-(1-24)-tetracosapeptide to be determined with a standard deviation on repeat digestions of 3-5%. Good values were obtained for amino acids that are completely or partially destroyed on acid hydrolysis, i.e. tryptophan, tyrosine, serine, asparagine and glutamine. Experiments with peptides containing d-amino acids showed that the enzyme mixture is stereospecific and could therefore be used to detect the presence of d-residues in peptides. The enzyme mixture completely hydrolyses peptide fragments obtained after Edman degradation and should therefore be useful for determining sequences of peptides containing acid-labile amino acid residues. The activities of the bound enzymes were unaltered over a period of 7 months and they provide a simple, reproducible procedure for the quantitative determination of amino acids in peptides and proteins containing l-amino acids.     

Neuropeptidases regulating gonadal function

The generation and metabolism of bioactive peptides involves a series of highly ordered proteolytic events. This post-translational processing can occur either within the cell, at the cell surface or after secretion. In the central nervous system a number of extracellular peptidases have been implicated in the regulated processing of peptides, particularly in the regulation of neuroendocrine function. The aim of this study has been to identify the peptidases involved in the metabolism of gonadotropin-releasing hormone (GnRH) and to characterize the factors and the mechanisms by which the activity of these peptidases are regulated. We have shown that both prolylendopeptidase and the thimet oligopeptidase EC 3.4. 24.15 are involved in GnRH metabolism and that both oestrogen and thiol-based reductants could be involved in the physiological regulation of their activities.