PepS from Streptococcus thermophilus

Abstract

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 <45 kDa with optimal activity in the range pH 7.5±8.5 and at 55 8C 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.Keywords: aminopeptidase T family; lactic acid bacteria; peptidase; Streptococcus thermophilus. Aminopeptidases (EC 3.4.11) are ubiquitous enzymes, frequently observed in animals, plants and microorganisms. As exopeptidases, they catalyse the release of free amino acids from peptides. They are involved in many different functions in the cell, such as protein maturation, protein turnover, hydrolysis of regulatory peptides, nitrogen nutrition, modulation of gene expression etc. and, consequently, are considered essential enzymes.Aminopeptidases, and more generally the proteolytic system of lactic acid bacteria (LAB) ± which are used as starters in food fermentation processes ± are essential for the nitrogen nutrition of LAB and also for the development of the organoleptic properties of dairy products (texture and flavour). Indeed, LAB are auxotrophic for several amino acids and therefore depend highly on their proteolytic system to hydrolyse milk proteins (caseins) into assimilable nitrogen compounds (small peptides and free amino acids). This proteolytic system has been studied extensively in Lactococcus lactis which is the best known species among LAB. The proteolytic cascade of casein utilization in this bacterium is now well known. This multienzymatic system is composed of a cell-wall bound proteinase, responsible for the first step of casein hydrolysis, as well as several peptide and amino-acid transport systems, and of various intracellular peptidases, mainly aminopeptidases, which are involved in further hydrolysis of the peptides produced from caseins after their transport into the cell (for...

The Thermophilic, Homohexameric Aminopeptidase ofBorrelia burgdorferiIs a Member of the M29 Family of Metallopeptidases

“…In addition to considerable sequence identity with members of the M29 family, TAP Bb is thermophilic. Its optimal temperature for activity was observed at 60°C, identical to that for the aminopeptidases of G. stearothermophilus and S. thermophilus (3,14) and similar to that determined for T. aquaticus aminopeptidase (70°C) (35). In contrast to these microorganisms, B. burgdorferi is not thermophilic.…”

“…None of the aminopeptidases of the M29 family described to date is hexameric: aminopeptidase T of Thermus aquaticus and Bacillus stearothermophilus are homodimeric enzymes (3,37), and PepS of Streptococcus thermophilus is monomeric (14). The hexameric quaternary structure of TAP Bb is required for activity, but the mechanism of this structural dependence is not currently known.…”

“…To date, in addition to bacteria, genes encoding M29 aminopeptidases have been identified in archea (27) and plants (EMBL AJ004922). However, only aminopeptidases from T. aquaticus, Thermus thermophilus, G. stearothermophilus, and S. thermophilus have been conclusively characterized as members of this family (14,36,42). Among the features that justify this classification are thermophilicity, high conservation of C termini, and no significant identity with other known aminopeptidases.…”

Impaired Growth Rates in Milk of Lactobacillus helveticus Peptidase Mutants Can Be Overcome by Use of Amino Acid Supplements

“…Quantification of free amino acids and small peptides in UFMP against Gly standard curves was accomplished using the Cd-ninhydrin assay (method D; 9) with modifications (12). The concentration of the unhydrolyzed UFMP sample was 580 M (44 g/ml) Gly standard equivalents.…”

Bioactive Casein Phosphopeptides in Dairy Products as Nutraceuticals for Functional Foods

“…Other peptidases with more specific substrate specificities include PepA, which liberates Nterminal acidic residues from 3-to 9-residue long peptides; PepP, which prefers tripeptides carrying proline in the middle position; PepR and PepI, which act on dipeptides containing proline in the penultimate position; PepQ, which cleaves dipeptides carrying proline in the second position; and PepS, which shows preference for peptides containing two to five residues with Arg or aromatic amino acid residues in the N-terminal position [20][21]23] (Figure 2). This proteolytic system is able to support LAB growth to high cell densities (10 9 -10 10 cfu/mL) in milk containing only small amounts of hydrolytic products that are transportable into the cells for assimilation.…”