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Proteolytic System of Streptococcus thermophilus
1Departamento de Biotecnología Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186. Col. Vicentina, México D.F. 09340, México. CP 09340, México, Ciudad de México, 2Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Hidalgo, Mineral de la Reforma, Hidalgo, México
J. Microbiol. Biotechnol. 2018; 28(10): 1581-1588
Published October 28, 2018 https://doi.org/10.4014/jmb.1807.07017
Copyright © The Korean Society for Microbiology and Biotechnology.
Abstract
Keywords
Introduction
The proteolytic system of lactic acid bacteria (LAB) has been extensively studied using genetic and biochemical methods. Recently, research has been focused on the production of bioactive peptides by specific action of the proteolytic system over milk proteins during its fermentation [1, 2]. In addition, several studies have been carried out in order to prove that the proteolytic systems of lactococci and lactobacilli hold a close relation [3, 4], while the proteolytic system of
-
Fig. 1.
Streptococcus thermophilus proteolytic system FOR. Graphic presentation of the proposed proteolytic system ofStreptococcus thermophilus , its function and regulation. Model follows that proposed by Kujiet al . (1996). The system is divided into three steps. The first step is the breakdown of proteins by a proteinase PrtS, which is anchored at cell wall by sortase A (SrtA). The second step is transport of peptides and oligopeptides, the transport of peptides is integrated by the Dpp system with five proteins (DppA, DppB, DppC, DppD, and DppE) regulated by ATP and proton formation (NAD+); the oligopeptides transport is carried out by Ami system integrated in a operon system whit seven proteins (Ami1, Ami2, Ami3, AmiC, AmiD, AmiE, and AmiF) regulated by ATP and activate by sulfur amino acid presence. The transcriptional regulatory protein ComR associated to Ami system is regulated by the peptide ComS. Finally, third step is the cut of peptides by fourteen intracellular peptidases, where five (PepO, PepS, PepX, PepN, and PepC) have different characteristics and specificity to those of other lactic acid bacteria.
It has been clearly stated that many lactic acid bacteria are multi-auxotrophic for amino acids [6, 7]. These requirements are specific for each strain, for example, some strains of
Due to low activity of peptidases of
Cell Wall Proteinase of Lactic Acid Bacteria
The degradation of caseins by LAB is triggered by an extracellular proteinase linked to the cell wall called PrtP in lactococci and lactobacilli [1, 4, 17, 18]. In the case of
PrtP is classified according to its specificity in PI and PIII. It is a monomeric serine proteinase weighing between 180 and 190 kDa, although Laan and Konings [21] found some fractions associated with this proteinase during its isolation, suggesting that this fraction is monomeric only under certain conditions where the medium has neutral pH. The gene that encodes for PrtP has been cloned and sequenced from some LAB. Primary structure of this enzyme consists of around 1,902 amino acids for lactococci and 1,946 to 1,962 amino acids for lactobacilli species [3]. Proteinases of different
In a study on the proteolytic capacity of LAB, Shihata and Shah [23] observed that some streptococcal species showed higher proteolytic activity than other lactic acid bacteria, including lactobacilli and bifidobacteria. These results can be better explained with the requirements of glutamic acid and methionine, which are nearly five to six times higher for
PrtS is anchored to the cell wall of
Specificity of Streptococcus thermophilus Proteinase PrtS
The products derived from the action of proteinases in milk are mainly from caseins αS1 and β, which are the most abundant proteins. These proteins are preferred by the proteolytic system of LAB in over 80% of serum proteins [1, 5]. In the case of β-casein (BCN), some studies have revealed that the action of both PrtP and PrtS, can generate more than 100 oligopeptides with sizes from 4 to 30 residues, without any indications of the existence of di- and tri-peptides but with trace concentrations of phenylalanine. Many peptides are generated from region 60-105 of this protein [1, 22, 29]. It has been observed in recent studies, that residues generated by the hydrolysis of sodium caseinate by S. thermophillus 4F44 and S. termophilus LMD-9-ΔsrtA PrtS, are peptides with different biological functions such as antihypertensive, anti-inflammatory, antioxidant, etc. [20, 30].
Regarding κ-casein (KCN), the oligopeptides are generated from the region 96-106 [3]. BCN and KCN can be degraded by both types of PrtP of lactococci, PI and PIII; nevertheless, in the case of α-casein (ACN) degradation can only occur through proteinase PIII and some proteinases with intermediate specificity between PI and PIII, since PII is unable to hydrolyze caseins α-S1 and α-S2. Nearly 23 oligopeptides coming from C-terminal end of ACN have been found [31].
In addition, specificity of PrtS of
Despite the previous information, not all the
Amino Acid and Peptide Transport System
The transport of amino acids and peptides, through the cell membrane, is the second stage in the proteolytic system of LAB. This step includes a great number of subsystems for certain amino acids [1, 3, 5, 18]. In the case of lactococci and lactobacilli, at least 10 different amino acid transport systems with a high specificity for amino acids with similar structures have been found, for example: Glu/Gln, Leu/Ile/Val, Ser/Thr, Ala/Gly, and Lys/Arg [3]. These systems can be regulated by ATP hydrolysis in the cases of Glu/Gln, Asn, and Pro/Gly [18], or by protons in the case of Leu/Val/Ile, Ala/Gly, Ser/Thr, and Met, while the Arg/Orn is regulated by a concentration gradient in a passive transport [1].
The transport system of
Di- and Tri-Peptide Transport System
Di- and tri-peptide transport system is related to the transport of essential amino acids [42]. Studies on transport of hydrophilic di- and tripeptides in lactobacilli and lactococci have shown that this mechanism is regulated by the generation of protons. Dipeptide transport is carried out through an enzyme called DtpT that belongs to a family of enzymes called the PRT family. This group of enzymes is characterized by allowing the transport of hydrophilic di-and tri-peptides, which are unrelated to the peptide transporters in other bacteria belonging to the ABC family. Through the analysis of the genomic sequence encoding the DtpT enzyme, it is known that this group of enzymes requires ATP hydrolysis to perform its function [3].
Studies of the genome sequence of some strains of
A genomic DNA fragment of 5.8 kb of
Oligopeptide Transport System
The transport system of oligopeptides in LAB, particularly of lactobacilli and lactococci, has been one of the most studied [29]. It consists of five proteins (OppA, B, C, D, and F), belonging to the ABC family which are ATP-dependent, as in the case of the proteins from the transport systems of amino acids and peptides [13]. This system has not been fully characterized, but it is known for its ability to transport oligopeptides with up to 12 amino acids into the cell [44].
In contrast, the oligopeptide transport system of
The operon structure of the Ami system makes it more efficient than the Opp operon in terms of amino acid transport into the cell [46]. This higher efficiency compensates for the lack of a proteinase such as PrtP present in other LAB [19]. It has been shown that in the Ami system, long-chain peptides with a negative charge cannot be transported into the cell. On the other hand, peptide chains between 1,000 and 3,000 Da with a net positive charge and hydrophobic peptides can be transported more efficiently [13]. This system is complemented with two proteins bound to the cell wall (AmiC and AmiD). When the operon is activated, as occurs when sulfur amino acids are present in the medium, these proteins change to an α-helix conformation. This change allows them to act as a peptide binding permease, permitting the passing of peptides of up to 23 amino acids through the cell membrane. The energy required for their transport is also generated by the Ami operon through two ATP protein donors: AmiE and AmiF [43].
The existence of a gene regulatory system associated with the Ami system has been demonstrated. The gene regulatory mechanism involves the production, secretion and transport of ComS and this peptide regulates the activity of the ComR protein, which is in turn a natural regulator of the
Peptidases of S. thermophilus
Once the peptides have been transported into the microorganism, they must be fractionated to release the essential amino acids. This action is performed through a complex system of intracellular peptidases and amino-peptidases. Several studies have been conducted to isolate and identify enzymes that participate in this system. Their sequences, specificity and physicochemical characteristics have been described [29]. The proteolytic system of
In contrast, aminopeptidase of
Chapot-Chartier
Aminopeptidase C is usually a thiol peptidase of about 50 kDa in all the LAB [54], which hydrolyzes dipeptides with Ala, Leu or Lys in the N-terminal position while being incapable of hydrolyzing hexapeptides. A notable difference in terms of the quaternary structure is the organization of the hexamer shown by the PepC of
Additionally, there is no associated hydrophobic sequence as it is found in the extracellular enzymes [60]. PepS can participate in the hydrolysis of peptides with high concentration of hydrophobic amino acids and thus releases smaller peptides with aromatic amino acids [61-63].
PepX isolated from LAB and other bacteria, yeast, and eukaryotes has been classified as serine proteinase based on the inhibitory effect of several compounds [64, 67-70]. The activation or inactivation of peptidases has been studied because it activates cheese ripening and yoghurt fermentation. Giannoglou
The proteolytic system of
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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Related articles in JMB

Article
Review
J. Microbiol. Biotechnol. 2018; 28(10): 1581-1588
Published online October 28, 2018 https://doi.org/10.4014/jmb.1807.07017
Copyright © The Korean Society for Microbiology and Biotechnology.
Proteolytic System of Streptococcus thermophilus
Gabriela Mariana Rodríguez-Serrano 1, Jose Mariano Garcia-Garibay 1, Alma Elizabeth Cruz-Guerrero 1, Lorena del Carmen Gomez-Ruiz 1, Alexis Ayala-Nino 2, Araceli Castaneda-Ovando 2 and Luis Guillermo Gonzalez-Olivares 2*
1Departamento de Biotecnología Universidad Autónoma Metropolitana-Iztapalapa, Av. San Rafael Atlixco No. 186. Col. Vicentina, México D.F. 09340, México. CP 09340, México, Ciudad de México, 2Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Hidalgo, Mineral de la Reforma, Hidalgo, México
Abstract
The growth of lactic acid bacteria (LAB) generates a high number of metabolites related to
aromas and flavors in fermented dairy foods. These microbial proteases are involved in
protein hydrolysis that produces necessary peptides for their growth and releases different
molecules of interest, like bioactive peptides, during their activity. Each genus in particular
has its own proteolytic system to hydrolyze the necessary proteins to meet its requirements.
This review aims to highlight the differences between the proteolytic systems of Streptococcus
thermophilus and other lactic acid bacteria (Lactococcus and Lactobacillus) since they are
microorganisms that are frequently used in combination with other LAB in the elaboration of
fermented dairy products. Based on genetic studies and in vitro and in vivo tests, the
proteolytic system of Streptococcus thermophilus has been divided into three parts: 1) a serine
proteinase linked to the cellular wall that is activated in the absence of glutamine and
methionine; 2) the transport of peptides and oligopeptides, which are integrated in both the
Dpp system and the Ami system, respectively; according to this, it is worth mentioning that
the Ami system is able to transport peptides with up to 23 amino acids while the Opp system
of Lactococcus or Lactobacillus transports chains with less than 13 amino acids; and finally, 3)
peptide hydrolysis by intracellular peptidases, including a group of three exclusive of
S. thermophilus capable of releasing either aromatic amino acids or peptides with aromatic
amino acids.
Keywords: Proteolysis, lactic acid bacteria, Streptococcus thermophilus
Introduction
The proteolytic system of lactic acid bacteria (LAB) has been extensively studied using genetic and biochemical methods. Recently, research has been focused on the production of bioactive peptides by specific action of the proteolytic system over milk proteins during its fermentation [1, 2]. In addition, several studies have been carried out in order to prove that the proteolytic systems of lactococci and lactobacilli hold a close relation [3, 4], while the proteolytic system of
-
Figure 1.
Streptococcus thermophilus proteolytic system FOR. Graphic presentation of the proposed proteolytic system ofStreptococcus thermophilus , its function and regulation. Model follows that proposed by Kujiet al . (1996). The system is divided into three steps. The first step is the breakdown of proteins by a proteinase PrtS, which is anchored at cell wall by sortase A (SrtA). The second step is transport of peptides and oligopeptides, the transport of peptides is integrated by the Dpp system with five proteins (DppA, DppB, DppC, DppD, and DppE) regulated by ATP and proton formation (NAD+); the oligopeptides transport is carried out by Ami system integrated in a operon system whit seven proteins (Ami1, Ami2, Ami3, AmiC, AmiD, AmiE, and AmiF) regulated by ATP and activate by sulfur amino acid presence. The transcriptional regulatory protein ComR associated to Ami system is regulated by the peptide ComS. Finally, third step is the cut of peptides by fourteen intracellular peptidases, where five (PepO, PepS, PepX, PepN, and PepC) have different characteristics and specificity to those of other lactic acid bacteria.
It has been clearly stated that many lactic acid bacteria are multi-auxotrophic for amino acids [6, 7]. These requirements are specific for each strain, for example, some strains of
Due to low activity of peptidases of
Cell Wall Proteinase of Lactic Acid Bacteria
The degradation of caseins by LAB is triggered by an extracellular proteinase linked to the cell wall called PrtP in lactococci and lactobacilli [1, 4, 17, 18]. In the case of
PrtP is classified according to its specificity in PI and PIII. It is a monomeric serine proteinase weighing between 180 and 190 kDa, although Laan and Konings [21] found some fractions associated with this proteinase during its isolation, suggesting that this fraction is monomeric only under certain conditions where the medium has neutral pH. The gene that encodes for PrtP has been cloned and sequenced from some LAB. Primary structure of this enzyme consists of around 1,902 amino acids for lactococci and 1,946 to 1,962 amino acids for lactobacilli species [3]. Proteinases of different
In a study on the proteolytic capacity of LAB, Shihata and Shah [23] observed that some streptococcal species showed higher proteolytic activity than other lactic acid bacteria, including lactobacilli and bifidobacteria. These results can be better explained with the requirements of glutamic acid and methionine, which are nearly five to six times higher for
PrtS is anchored to the cell wall of
Specificity of Streptococcus thermophilus Proteinase PrtS
The products derived from the action of proteinases in milk are mainly from caseins αS1 and β, which are the most abundant proteins. These proteins are preferred by the proteolytic system of LAB in over 80% of serum proteins [1, 5]. In the case of β-casein (BCN), some studies have revealed that the action of both PrtP and PrtS, can generate more than 100 oligopeptides with sizes from 4 to 30 residues, without any indications of the existence of di- and tri-peptides but with trace concentrations of phenylalanine. Many peptides are generated from region 60-105 of this protein [1, 22, 29]. It has been observed in recent studies, that residues generated by the hydrolysis of sodium caseinate by S. thermophillus 4F44 and S. termophilus LMD-9-ΔsrtA PrtS, are peptides with different biological functions such as antihypertensive, anti-inflammatory, antioxidant, etc. [20, 30].
Regarding κ-casein (KCN), the oligopeptides are generated from the region 96-106 [3]. BCN and KCN can be degraded by both types of PrtP of lactococci, PI and PIII; nevertheless, in the case of α-casein (ACN) degradation can only occur through proteinase PIII and some proteinases with intermediate specificity between PI and PIII, since PII is unable to hydrolyze caseins α-S1 and α-S2. Nearly 23 oligopeptides coming from C-terminal end of ACN have been found [31].
In addition, specificity of PrtS of
Despite the previous information, not all the
Amino Acid and Peptide Transport System
The transport of amino acids and peptides, through the cell membrane, is the second stage in the proteolytic system of LAB. This step includes a great number of subsystems for certain amino acids [1, 3, 5, 18]. In the case of lactococci and lactobacilli, at least 10 different amino acid transport systems with a high specificity for amino acids with similar structures have been found, for example: Glu/Gln, Leu/Ile/Val, Ser/Thr, Ala/Gly, and Lys/Arg [3]. These systems can be regulated by ATP hydrolysis in the cases of Glu/Gln, Asn, and Pro/Gly [18], or by protons in the case of Leu/Val/Ile, Ala/Gly, Ser/Thr, and Met, while the Arg/Orn is regulated by a concentration gradient in a passive transport [1].
The transport system of
Di- and Tri-Peptide Transport System
Di- and tri-peptide transport system is related to the transport of essential amino acids [42]. Studies on transport of hydrophilic di- and tripeptides in lactobacilli and lactococci have shown that this mechanism is regulated by the generation of protons. Dipeptide transport is carried out through an enzyme called DtpT that belongs to a family of enzymes called the PRT family. This group of enzymes is characterized by allowing the transport of hydrophilic di-and tri-peptides, which are unrelated to the peptide transporters in other bacteria belonging to the ABC family. Through the analysis of the genomic sequence encoding the DtpT enzyme, it is known that this group of enzymes requires ATP hydrolysis to perform its function [3].
Studies of the genome sequence of some strains of
A genomic DNA fragment of 5.8 kb of
Oligopeptide Transport System
The transport system of oligopeptides in LAB, particularly of lactobacilli and lactococci, has been one of the most studied [29]. It consists of five proteins (OppA, B, C, D, and F), belonging to the ABC family which are ATP-dependent, as in the case of the proteins from the transport systems of amino acids and peptides [13]. This system has not been fully characterized, but it is known for its ability to transport oligopeptides with up to 12 amino acids into the cell [44].
In contrast, the oligopeptide transport system of
The operon structure of the Ami system makes it more efficient than the Opp operon in terms of amino acid transport into the cell [46]. This higher efficiency compensates for the lack of a proteinase such as PrtP present in other LAB [19]. It has been shown that in the Ami system, long-chain peptides with a negative charge cannot be transported into the cell. On the other hand, peptide chains between 1,000 and 3,000 Da with a net positive charge and hydrophobic peptides can be transported more efficiently [13]. This system is complemented with two proteins bound to the cell wall (AmiC and AmiD). When the operon is activated, as occurs when sulfur amino acids are present in the medium, these proteins change to an α-helix conformation. This change allows them to act as a peptide binding permease, permitting the passing of peptides of up to 23 amino acids through the cell membrane. The energy required for their transport is also generated by the Ami operon through two ATP protein donors: AmiE and AmiF [43].
The existence of a gene regulatory system associated with the Ami system has been demonstrated. The gene regulatory mechanism involves the production, secretion and transport of ComS and this peptide regulates the activity of the ComR protein, which is in turn a natural regulator of the
Peptidases of S. thermophilus
Once the peptides have been transported into the microorganism, they must be fractionated to release the essential amino acids. This action is performed through a complex system of intracellular peptidases and amino-peptidases. Several studies have been conducted to isolate and identify enzymes that participate in this system. Their sequences, specificity and physicochemical characteristics have been described [29]. The proteolytic system of
In contrast, aminopeptidase of
Chapot-Chartier
Aminopeptidase C is usually a thiol peptidase of about 50 kDa in all the LAB [54], which hydrolyzes dipeptides with Ala, Leu or Lys in the N-terminal position while being incapable of hydrolyzing hexapeptides. A notable difference in terms of the quaternary structure is the organization of the hexamer shown by the PepC of
Additionally, there is no associated hydrophobic sequence as it is found in the extracellular enzymes [60]. PepS can participate in the hydrolysis of peptides with high concentration of hydrophobic amino acids and thus releases smaller peptides with aromatic amino acids [61-63].
PepX isolated from LAB and other bacteria, yeast, and eukaryotes has been classified as serine proteinase based on the inhibitory effect of several compounds [64, 67-70]. The activation or inactivation of peptidases has been studied because it activates cheese ripening and yoghurt fermentation. Giannoglou
The proteolytic system of
Conflict of Interest
The authors have no financial conflicts of interest to declare.
Fig 1.

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