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°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.Lactic acid bacteria (LAB) are widely used as starters in fermented milk products due to their properties of milk acidification and flavor development. For these applications, their capacity to grow fast in milk is of major importance. LAB are fastidious microorganisms and require an exogenous source of amino acids or peptides for optimal growth. As milk is poor in these low-molecular-weight compounds, their growth largely depends on their proteolytic system to achieve hydrolysis of caseins (65). The cell envelope proteinase (CEP) is the key enzyme of this process since it is the only enzyme capable of initiating the breakdown of caseins into oligopeptides. The latter are then transported into the bacteria and further degraded by a complex set of intracellular peptidases (12).The cell envelope proteinases of lactococci, and to a lesser extent those of lactobacilli, have been the subject of intensive biochemical and genetic investigation (for a review, see reference 37). Lactococcal proteinase PrtP is synthesized as an inactive preproenzyme and maturated via an autoproteolytic process involving a chaperone lipoprotein PrtM, and it is anchored to the cell wall. The hydrolysis specificity of CEPs determined on caseins or casein peptides varies among strains, and several classifications have been proposed (5,18,21,22,24,40,66). The differences observed in substrate specificity are only due...

A peptidase showing a high level of specificity towards dipeptides of the X-Pro type was purified to homogeneity from the cell extract of Lactobacillus casei subsp. casei IFPL 731. The enzyme was a monomer having a molecular mass of 41 kDa. The pH and temperature optima were 6.5 to 7.5 and 55؇C, respectively. Metal chelating agents completely inhibited enzyme activity, indicating that the prolidase was a metalloenzyme. The Michaelis constant (K m) and V max for several proline-containing dipeptides were determined.

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...

Proteolysis and flavor development were monitored in Cheddar cheese made from milk inoculated with Lactobacillus casei ssp. casei IFPL 731 (104 cfu/mL cheesemilk) or Propionibacterium shermanii NCDO 853 at three different levels (105, 106, and 107 cfu/mL cheesemilk). The pH and chemical composition of the experimental cheeses were not affected by the addition of L. casei or Prop. shermanii. Cheeses inoculated with L. casei or Prop. shermanii at low and medium levels received the best scores for flavor development and body. However, the addition of Prop. shermanii at a high level caused a sweet and nutty flavor. Urea-PAGE, WSN, and RP-HPLC showed minor differences in proteolysis between the control and experimental cheeses. Major differences were found at the amino acid level. In general, the content of amino acid increased as the Propionibacterium inoculum increased in the experimental cheeses. Besides, a decrease in the content of hydrophobic peptides was observed in cheeses inoculated with Prop. shermanii at medium and high levels. The increase in amino acid content was not apparent when L. casei was added to cheese, possibly due to the lack of cell lysis. There is an optimum inoculum level of Prop. shermanii above which the flavor of Cheddar cheese resembles that of Swiss-type cheese. Keywords: Propionibacterium shermanii; Lactobacillus casei ssp. casei; adjunct cultures; Cheddar cheese; proteolysis