Proteolytic systems of lactic acid microorganisms : a review

The primary objective of this review is to analyze and summarize the existing scientific information about 
the structure features, formation conditions and properties of proteinases and peptidases of lactic acid microorganisms, which are widely used in the production of dairy products.


Introduction
At production and sale of food and dairy products, in particular, organoleptic properties (taste, smell, consistency) of the finished product are the main factors, which guarantee a high demand level among consumers.Special taste and smell, typical for each product, are provided with various food substances (proteins, fats, carbohydrates) and their cleavage products [1][2][3][4].A significant amount of taste and flavoring substances in dairy products are formed as a result of proteolysis of milk proteins [1,2,5,6].In particular, proteolysis actively occurs in the production of fermented milk products (kefir, koumiss, cottage cheese, etc.).And very intensively -in the production of hard cheeses, in the aging of which there are biochemical changes in milk proteins.Cleavage of proteins and amino acids with enzymes of lactic acid and propionic acid bacteria promotes the enrichment of dairy products with soluble in water nitrogen-containing and nitrogen-free compounds, and as a result, the product obtains necessary consistency, taste and smell [7][8][9][10][11][12][13][14].
In addition to providing organoleptic properties, which are important for the consumer and demand in the market, the formation of a large number of peptides with different types of biological activity occurs also in the process of proteolysis of milk proteins in the production of dairy products [15][16][17].Among the casein proteolysis products, bioactive peptides, having opioid affect, antihypertensive and immunomodulating properties, ability to influence blood coagulation processes, transport of calcium ions in the intestine etc., were revealed [18][19][20][21][22][23][24].Inhibitors of angiotensin converting enzyme, peptides with opioid and bactericidal action, immunomodulating and hypocholesterolemic, as well as peptides, affecting intestinal motility, were found among bioactive peptides from milk whey proteins [25][26][27].It was defined that β-actoglobulin is the precursor of all these types of bioactive peptides, except for immunomodulating peptides.Among bioactive peptides, formed from α-lactalbumin, there are no peptides with hypocholesterolemic action and peptides affecting intestinal motility, and only two types of biological activity are inherent in peptides with lactoferrin (bactericidal and immunomodulating).
It is known that proteolysis of proteins occurs gradually under the influence of lactic acid microorganisms [13].The casein proteins are the most sensitive to proteolytic enzymes.First the casephosphate complex decomposes into high molecular weight polypeptides, then medium and low molecular weight peptides and amino acids dominate among the products of proteolysis.The majority transformations during the initial stages of proteolysis occur under the influence of extracellular and cell-wall-bound proteinases, and more profound transformations of the peptides -under the influence of membrane and intracellular peptidases of lactic acid bacteria.[28][29][30][31][32][33].
Lactic acid bacteria (lactococci and lactobacilli), which are a part of different types of starter preparations for dairy products, are auxotrophs, that is, their ability to develop in a dairy medium depends on the activity of the proteolytic system, which ensures the liberation of essential amino acids during the cleavage of proteins of the casein complex, used by microorganisms in the synthesis of proteins [32,33].The proteolytic system of lactic acid bacteria consists of three parts: -proteinases, which that provide the initial cleavage of casein to peptides with formation of a large number of oligopeptides [32]; -peptidases, which cleavage peptides to amino acids [30]; -transport system, which provides transfer of proteolysis products through the cytoplasmic membrane [12,33].
It is known that proteinases function outside microbial cells, which produce them, and peptidases -in cells of lactic acid bacteria.
Proceeding from the aforesaid, the purpose of this work is to analyze and summarize the existing scientific information about the structure features, formation conditions and biochemical properties of proteinases and peptidases of lactic acid microorganisms, which are widely used in the production of dairy products.

Material and methods
The proteolysis of milk proteins, occured by lactic acid microorganisms, is an investigation object of this review article.Scientific articles as well as theses and monographs of microbiology, biochemistry and dairy science have been analysed.Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis.Literature referenced in this review article was obtained from searches from bibliographic information in CAB abstracts, AGRICOLA, SciFinder, Google Scholar, PubMed, ScienceDirect database and Web of Science.

Proteinases
Localization of proteinases.Proteinases of lactic acid microorganisms are monomeric serine proteinases with molecular weight 180 000-190 000 Dа (Table 1), which are connected with the bacterial cell wall and are called extracellular proteinases or in abbreviated form PrtP. Primary PrtP structure and the structure of gene, which encodes their synthesis [12,34], was defined for most types of lactic acid microorganisms.Thus, in lactococci and Lb.paracasei cell-wall-bound proteinase includes 1902 amino acid residues in Lb. delbruеckii -1946, and in Lb. lactis -1962.Primary PrtP structure in different lactococci is identical for 98% and is identical for 95% with Lb. paracasei.The analysis of the primary structure of proteinases shows their similarity with subtilisins, which are also serine proteinases with similar catalytic domains [35].Wherein, N-end part of the formed enzyme contains a catalytic domain with several conserved amino acid residues, which participate in the catalytic process and substrate binding.The next segment shows no similarity to proteins with similar functions and, evidently, is responsible for placing the catalytic domain on the surface of the cell wall [35].С-terminal part of cell-wall-bound proteinases is similar to that found in many Gram-positive bacteria and includes signal sequence and α-helix section, connected with the membrane [36].
Extracellular localization of proteinases is possible in cases of microorganisms placing in the solutions without calcium [37], or when microorganism cells are exposed to enzyme lysozyme affect [38].In the first case, the enzyme is formed with less molecular weight (165000 Dа) in comparison with lysozyme affect (180000 Da), which can happen due to auto-proteolysis.
In the laboratory of biochemistry of dairy products of Ternopil Ivan Puluj National Technical University, it has been obtained the evidence of the existence of cell-wall-bound extracellular proteases in St. salivarius ssp thermophilus (strain 9 1 ) during proteolysis of purified casein fractions by microorganism cells [39].
Substrate specificity of proteinases.Some authors defined, that cell-wall-bound proteinases are characterized with wide substrate specificity [37,40,41].Those sectors, on which proteolysis uniquely occurs, were not defined on the basis of analysis of many products of proteolysis.Only those sectors were found, which were sensitive mainly to the affect of different types of proteinases.It should be noted, that di-and tripeptides, as well as free amino acids are formed in small quantities under affect of cell-wall-bound proteinases of lactic acid bacteria on  S -, and -caseins.However, many slightly larger peptides are formed (4-8 amino acid residues), which contain all essential amino acids, necessary for normal growth of lactic acid bacteria.All products of proteolysis, formed due to the affect of cell-wall-bound proteinases, are located in the medium, outside the bacterial cell.
By the specificity of the effect on the fractions of the casein complex of milk cell-wallbound proteinases of lactic acid microorganisms are divided into 2 types -РІ and РІІІ.
Cleavage of β-casein fraction with proteinases.Recently they conduct intensive research for the purpose of detailed study of caseins proteolysis products with cell-wallbound proteinases of lactobacilli.It was established that β-caseins are the most sensitive to the affect of these enzymes.Specificity of the proteinases activity at incubation the purified casein fraction with intact bacterial cells was studied in the early works [42].At the same time it was proved, that only cell-wall-bound proteinase shows proteolytic action.Later in the experiments in vitro they used purified enzymes obtained from various strains of lactic acid bacteria, and the appropriate fraction of casein.In particular, they investigated the influence of cell-wall-bound proteinase of lactic acid microorganisms L. lactis and Lb.helveticus on -casein [43].They separated the products of -casein proteolysis by liquid chromatography method, purified and installed the primary structure by Edman.The first results showed that only part of -casein is cleavageted under affect of cell-wall-bound proteinase.In this case, large fragments are mainly formed.Further research, using liquid chromatography method under high pressure and mass spectrometry, allowed to analyze more than 95% peptides, formed at -casein cleavageting with proteinase of РІ type [40].More than 100 peptides, consisting of 4-30 amino acid residues were found.Most peptides contained 4-10 residues.
It was established that half of the peptides is formed from С-terminal part of -casein molecule.Analysis of products of -casein proteolysis, formed under the affect of proteinase of the type РІІІ, allowed to identify thirteen identical links, which are always cleavageted with proteinases of the type РІІІ and РI, and six links, which are often cleavageted with cell-wall-bound proteinases of bacteria of different strains.The aforesaid peptides are formed mainly from С-terminal part of -casein molecule.In addition to the proteinases, typical for Lb.helveticus and L. lactis in Lb. helveticus СР790, much smaller cell-wall-bound proteinase (45000 Da), which belongs to the class of serine proteinases and shows specificity in relation to -casein [43], was also revealed.
Cleavage of  S1 -i  S2 -casein fractions occurs mainly due to the affect of РІІІ-type proteinases or mixed-type proteinases.Proteinases РI do not hydrolyze the fractions of  Scaseins [44].25 main oligopeptides, half of which is formed as a result of С-terminal part cleavageting, were identified among the products of proteolysis, [41,45].Besides, the number of small peptides, formed from sectors, adjoining to peptide connections, sensitive to proteinase, were identified.
The analysis of specificity of proteinases Exterkate F.A., Albing A.C., Bruinenberg P.G. [34] was conducted on the fragment of α S1 -casein, including amino acid residues 1-28.In this case, they used proteinases, extracted from sixteen different L. lactis strains.Based on the results of these studies, they separated proteinase of lactococcus into 7 groups, which differed in the specificity of cleavageting of the aforesaid fragment of α S1 -casein.Comparison of amino acid sequences responsible for substrate binding, of proteases with different specificity showed, that their specificity is caused by minor genetic variations of the structural proteinase gene.The primary structure of the catalytic domain is conservative not only in lactococci, but also in lactobacilli with cell-wall-bound proteinases.So, in Lb. paracasei two substitutions of amino acid residues in this domain were found, and in Lb. delbrueckii -three substitutions [46].In proteinase Lb. helveticus (strain L89) the same specificity as in the proteinase lactococci was revealed.Differences between them were due to different ratios of proteolysis products [47].
Cleavage of -casein fraction with proteinases.Products of proteolysis of -casein were studied under the influence of cell-wall-bound proteinase of different types in the number of strains of lactococcus L. lactis [41,48,49].Cleavage of -casein causes the formation of large number of small peptides predominantly from С-terminal part of molecule.Most peptide connections are constantly subjected to hydrolysis with all types of proteinases, however, the peptides were also found, which are formed under affect of certain types of specific proteinases.

Peptidases
Peptidases -are the enzymes of hydrolase class, which cleavage one by one amino acid from the carboxyl or amine end from peptide molecules.There are many works in the scientific literature, devoted to structure, properties and specificity of peptidases, main results of which are systematized in the Table 2. Peptidases localization.The question of the peptidases localization in the cells of lactic acid bacteria has been changed since the time of its rising.In earlier works it was written about their localization on cell membranes and even outside the cells [13].At present most researchers tend to think about intracellular localization of all peptidases of lactic acid microorganisms [50][51][52][53].The absence of signal sequences and anchor sections for fixation on the membrane in all peptidases, for which the primary structure was established, can be the evidence of this.Besides, transport system of oligopeptides of lactic acid bacteria can provide entering of large peptides, formed with casein proteolysis, into the cell.And there is no need to cleavage peptides outside the cell or on cell membranes.

Substrate specificity of peptidases.
In accordance with the effect on substrates, we can distinguish -aminopeptidases, hydrolyzing the peptide connection, formed by the amino group of the polypeptide chain and the carboxyl group of the terminal amino acid, -carboxypeptidases, acting on the peptide connection, formed by the carboxyl group of the polypeptide chain and the amino group of the final amino acid, -dipeptidases, which provide hydrolysis of dipeptides.
None of the peptidases with carboxypeptidase activity were revealed among large number of lactic acid bacteria peptidases, different by specificity.PepN, PepС, РерА are referred to the aminopeptidases, found in lactic acid microorganisms.In addition to aminopeptidase, dipeptidases РерD and PepV [50,52] were revealed in lactobacilli.
Aminopeptidase N (PepN).Study of genes PepN in different bacteria showed a high level of their identity [50], at the same time the primary structure PepN is homologous to aminopeptidase N in mammals [54].PepN can cleavage off N-end amino acids in di-and tripeptides.However, dipeptides, which contain proline residue in the first or second positions, are not cleavageted with PepN, whereas such connection in tripeptides undergoes hydrolysis [55,56].PepN dipeptides better the hydrolyses, in which N-end amino acid residue is arginine.Dipeptides, containing lysine and leucine in the first position, are hydrolyzed less [57].The enzyme activity increases with increasing of hydrophobicity Сend amino acid residue of dipeptide Аrg-Х.PepN with Lb. helveticus has similar properties with respect to dipeptides Ala-Х and Lei-Х [55].
The affect of PepN on oligopeptides was studied in several works [56,57].PepN ability to cleavage oligopeptides, including from 4 to 14 amino acid residues, is shown on the example of using products of trypsin hydrolyzate of -casein as a substrate.It was found that hexapeptide is the optimal substrate for PepN [57].Aminopeptidase N with Lb. helveticus is capable of hydrolyze the peptides, containing up to 10 amino acids, at that, the proline residue may be in the first position.It is also known that the enzyme is capable to sptil the end tyrosine from the fragment -CN f 193-209, containing 16 amino acid residues [58].
Regulation of PepN expression in L. lactis depends on the bacterial strain, as well as from the nutrient medium [50].At lactococci growth in milk the PepN activity is higher than at growth on artificial nutrient media.It is known that the dipeptide Pro-Lei reduces PepN expression in L. lactis МG1363 [59].They used the mutants of Lb. helveticus and L. lactis with deletion of the gene рepN [58,60] in order to clarify the physiological role of peptidase N. Slight reduction of lactobacilli growth in the dairy medium was revealed, whereas difference in their growth was not revealed on the artificial complex nutrient medium.Similarly, mutants and wild strains L. lactis grow on the artificial nutrient medium, however they grow much more slowly in milk media.
Aminopeptidase С (PepС) was defined from many strains of lactic acid bacteria [50,61].They established high activity of PepС at cleavageting the peptide connections, created with basic (Arg, Gis, Liz), acidic (Glu, Asp), hydrophobic (Ala, Lei) and aromatic (Fen) amino acids.At the same time the connections, formed with the proline of the type: Pro-рNA, Pro-NAP, Х-Pro-рNA, Х-Pro-NAP, remained discontinuous.Structural modeling of PepС with L.lactis showed that С-terminal residues, participating in the interaction of -carboxyl group PepС and -amino group of the substrate [62], are the part of the enzyme active center.This was also confirmed at studying of the mutants without Сterminal residue РерС.The mutants L. lactis with deletion of the gene РерС did not lag behind in growth in nutrient medium, but in milk they decreased by 10% [60].
Aminopeptidase А (РерА) is able to cleavage off acidic N-terminal amino acid residues, to hydrolyse well Glu-and Аsp-рNА and much less Glu-and Аsp-NAP [63,64].РерА is able to cleavage off N-terminal Glu and Аsp in peptides of different sizes (from 2 to 10 residues).The mutants L. lactis, in which there is no РерА, somewhat lag behind in growth at lag-phase, but in the final version they reach the same concentration as in wild strains.
The original aminopeptidase РерР, which releases N-terminal amino acids from the peptides of the type Х-Pro-Pro-(Y) n [68,69], was found only in lactococci L. lactis.РерР showed the highest activity for pentapeptides, including from 3 to 9 amino acid residues.PepР specificity towards the next N-terminal amino acids (Х): Arg, Met, Liz, Lei and Tir was established.Difference in the growth rate in artificial and dairy medium of the mutants with deletion gene РерР and wild strains L. lactis is small.
Dipeptidases.Dipeptidase РерD has a wide specificity, but does not hydrolyze АА-рNA, dipeptides, containing proline residues, and dipeptides with N-terminal remains of glycine.In contradistinction to РерD, peptidase РерV is more important for lactic acid bacteria growth.It was shown on the example of L. lactis, that strains, which didn't contain РерV, lagged behind in growth in 22% [70].
Proline-iminopeptidase (РерІ), which cleavages off N-terminal proline residue in peptides, is common among lactic acid bacteria.[70,71].РерІ shows hydrolytic activity to peptides of type Pro-Х, where Х may be a hydrophobic residue (Ala, Ile, Lei, Val), acidic (Glu) or aromatic residue (Fen, Tir).Use of peptides of different sizes as substrates showed, that РерІ mainly cleavages off N-terminal proline in di-and tripeptide (rarely in tetrapeptide, for example, Prо-Fen-Gli-Liz), but not in pentapeptides [72].By its specificity РерІ of lactococci and lactobacilli differ between themselves: РерІ of lactococcimetalopeptidase, and РерІ of lactobacilli -serine.The absence of proline-iminopeptidase in the mutants with deletion gene РерІ does not affect their growth in complex artificial nutrient medium.Wherein The time of doubling of microorganisms in dairy medium is increased by 9%.
Peptidase, which cleavages off N-terminal amino acid, when the second position is the proline residue -prolidase (РерQ) [53,73].Prolidase is enzyme, which hydrolyses dipeptide Х-Pro.However, not all of the prolidase are dipeptidases, and they are capable to hydrolyze not all substrates of the type Х-Pro.First of all РерQ hydrolyses dipeptides, which in the first position contain residues of hydrophobic (Ala, Ile, Lei, Val), main (GIS), aromatic (Fen, Tir) and sulfur-containing (Меt) amino acids.Some prolidases show an incomprehensible high ability to cleavage peptides without residues of proline, or contained it in the first position (Pro-Ala, Pro-Pro, Pro-Val).In a dairy medium the strains Lb. helveticus, with РерQ deficit, developed by 13% slower.Study of bacteria strains, in which PepQ functions normally, and strains without this enzyme showed, that almost 100% dipeptides Меt-Pro, Lei-Pro and Fen-Pro are hydrolyzed with РерQ participation.
Tripeptidases.Tripeptidase РерТ, separated from L. lactis, hydrolyses tripeptides, except peptides of the type Х-Prо-Y.They are not able to hydrolyze di-, tetra-or large peptides [74].Other tripeptidases are characterized by greater ability to cleavage tripeptides with hydrophobic and aromatic amino acid residues [75,76].Physiological role of РерТ was studied little.It is only known that РерТ absence in lactococci delays their growth in milk [60].
Peptidase РерО cleavages oligopeptides, which include from 5 to 35 amino acid residues [76][77][78].Like thermolysin, РерО hydrolyses peptide connections, are formed with leucine and phenylalanine.Though РерО cleavages a series of casein fragments, the native proteins of the casein complex are not hydrolyzed.Growth of mutants with deletion gene РерО and wild strains Lb. helveticus were examined in various nutrient medium [77].They observed a slight lag in growth of mutants L.lactis, using milk as a nutrient medium [60].
One more peptidase (РерF), which cleavages the oligopeptides, was found only in лактококки [79].РерF hydrolyzes the oligopeptides, which include from 5 to 17 amino acid residues [79].РерF shows the highest cleavageting ability for substrates, consisting of 8 or 9 residues.РерF hydrolyses three connections, releasing peptides from 3 to 5 residues in the fragment АKТG (f 1-24).Lack of activity of РерF towards -chain of insulin (30 residues), glucagon (29 residues) and the fragment АKТG (f 1-24) allows you to determine the limits of substrates size (less than 24 residues).РерF doesn't cleavage native proteins of the casein complex of proteinase.This enzyme plays an important role in the processes of lactococci growth in a dairy medium.Generation time of the mutants with lack of РерF increases by 16% [9].

Conclusion
Based on the analysis of scientific sources, it is established that microorganisms widely used in technologies of dairy products, can produce a number of proteases (proteinases and peptidases), which play important role in proteolytic processes of industrial production of protein dairy products -primary cleavageting of the casein complex proteins and release of amino acids from exogenous peptides.Proteinases function outside microbial cells, which produce them, and peptidases -in the cells of lactic acid bacteria.In most cases, the mutants, deficient in each of the peptidases, do not differ in their growth from wild strains of lactobacilli in complex artificial nutrient media, and in a dairy medium they significantly lagged behind in growth.
It is recommended to use systematized characteristics of proteinases and peptidases of appropriate microorganisms with the purpose of providing quality organoleptic parameters of dairy products as well as the formation of biologically active peptides in the process of selecting the species composition of starter cultures.