Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions.

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

Expression of a heterologous glutamate dehydrogenase gene in Lactococcus lactis highly improves the conversion of amino acids to aroma compounds.

The first step of amino acid degradation in lactococci is a transamination, which requires an alpha-keto acid as the amino group acceptor. We have previously shown that the level of available alpha-keto acid in semihard cheese is the first limiting factor for conversion of amino acids to aroma compounds, since aroma formation is greatly enhanced by adding alpha-ketoglutarate to cheese curd. In this study we introduced a heterologous catabolic glutamate dehydrogenase (GDH) gene into Lactococcus lactis so that this organism could produce alpha-ketoglutarate from glutamate, which is present at high levels in cheese. Then we evaluated the impact of GDH activity on amino acid conversion in in vitro tests and in a cheese model by using radiolabeled amino acids as tracers. The GDH-producing lactococcal strain degraded amino acids without added alpha-ketoglutarate to the same extent that the wild-type strain degraded amino acids with added alpha-ketoglutarate. Interestingly, the GDH-producing lactococcal strain produced a higher proportion of carboxylic acids, which are major aroma compounds. Our results demonstrated that a GDH-producing lactococcal strain could be used instead of adding alpha-ketoglutarate to improve aroma development in cheese.

Genetic and biochemical characterization of a high-affinity betaine uptake system (BusA) in Lactococcus lactis reveals a new functional organization within bacterial ABC transporters.

The cytoplasmic accumulation of exogenous betaine stimulates the growth of Lactococcus lactis cultivated under hyperosmotic conditions. We report that L. lactis possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters. Through transposon mutagenesis, a mutant deficient in betaine transport was isolated. We identified two genes, busAA and busAB, grouped in an operon, busA (betaine uptake system). The transcription of busA is strongly regulated by the external osmolality of the medium. The busAA gene codes for the ATP-binding protein. busAB encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the Bacillus subtilis betaine-binding protein, OpuAC. BusA of L. lactis displays a high affinity towards betaine (K(m) = 1.7 microM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of L. lactis to be under dual control at the biochemical and genetic levels. A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of L. lactis. The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.

Deletion of the four C-terminal residues of PepC converts an aminopeptidase into an oligopeptidase.

The aminopeptidase PepC is a cysteine peptidase isolated from lactic acid bacteria. Its structural and enzymatic properties closely resembles those of the bleomycin hydrolases, a group of cytoplasmic enzymes isolated from eukaryotes. Previous biochemical and structural data have shown that the C-terminal end of PepC partially occupies the active site cleft. In this work the substrate specificity of PepC was engineered by deletion of the four C-terminal residues. The mutant PepCDelta432-435 cleaved peptide substrates as an oligopeptidase while the aminopeptidase specificity was totally abolished. The substrate size dependency indicated that PepCDelta432-435 possesses an extended binding site able to accommodate four residues of the substrate on both sides of the cleaved bond. The activity of PepCDelta432-435 towards tryptic fragments of casein revealed a preference for peptides with hydrophobic amino acids at positions P2 and P3 and for Gly, Asn and Gln at position P1. PepCDelta432-435 was shown to be highly sensitive to the thiol peptidase inhibitors leupeptin or E64 which are inefficient towards the wild-type PepC. In conclusion, deletion of the four C-terminal residues in PepC produces a new enzyme with properties resembling those of an endopeptidase from the papain family.

Catalytic properties of the cysteine aminopeptidase PepC, a bacterial bleomycin hydrolase.

PepC is a cytoplasmic thiol aminopeptidase widely conserved among lactic acid bacteria. PepC from Lactococcus lactis shares 35-38% identity with aminopeptidases of eukaryotic origins: the yeast and mammalian bleomycin hydrolases (BLMase). In this work we investigated the hydrolytic activity of PepC towards various substrates: bleomycin A2, aminoacyl-p-nitroanilides (pNA) and peptides. First, we found the bleomycin hydrolase activity of lactococcal PepC and measured similar kinetics parameters to those reported for the mammalian BLMase. Second, the results obtained on aminoacyl-pNA confirmed the capacity of the enzyme to release a broad range of amino acids and the pH activity profile suggests the presence of an ionic interaction between the enzyme and the free alpha-amino group of the substrate. Third, the aminopeptidase activity measured on peptide substrates revealed that PepC possesses an extended binding site which interacts with the peptidic backbone of the substrate. The hydrolytic efficiency is highly dependent on the length of the peptide, optimal for tetrapeptides and further enhanced by the presence of hydrophobic residues in the P' positions of the substrate. These enzymatic properties are of importance for the design of specific inhibitors and the biological function of the bleomycin hydrolases.

Specificity of milk peptide utilization by Lactococcus lactis.

To study the substrate specificity of the oligopeptide transport system of Lactococcus lactis for its natural substrates, the growth of L. lactis MG1363 was studied in a chemically defined medium containing milk peptides or a tryptic digest of alpha s2-casein as the source of amino acids. Peptides were separated into acidic, neutral, and basic pools by solid-phase extraction or by cation-exchange liquid chromatogrpaphy. Their ability to sustain growth and the time course of their utilization demonstrated the preferential use of hydrophobic basic peptides with molecular masses ranging between 600 and 1,100 Da by L. lactis MG1363 and the inability to use large, acidic peptides. These peptide utilization preferences reflect the substrate specificity of the oligopeptide transport system of the strain, since no significant cell lysis was inferred. Considering the free amino acid content of milk and these findings on peptide utilization, it was demonstrated that the cessation of growth of L. lactis MG1363 in milk was due to deprivation of leucine and methionine.

Characterization of cspB, a cold-shock-inducible gene from Lactococcus lactis, and evidence for a family of genes homologous to the Escherichia coli cspA major cold shock gene.

Upon temperature downshift, the major cold shock protein CspA is highly induced in Escherichia coli. This protein being conserved in other bacteria, we used a PCR-based approach with a pair of degenerate primers derived from highly conserved regions of the CspA-related proteins to evidence the presence of at least three related genes in Lactococcus lactis. One of them, cspB, was cloned and sequenced. It encodes a 66-residue protein which possesses 60% sequence identity with E. coli CspA. Following a cold shock from 30 to 15 degrees C, the level of the cspB mRNA transcript increased, as shown by Northern blot hybridization. In addition, induction of cspB-directed beta-galactosidase activity was observed. These results indicate that the L. lactis cspB gene is cold shock inducible.

An aminotransferase from Lactococcus lactis initiates conversion of amino acids to cheese flavor compounds.

The enzymatic degradation of amino acids in cheese is believed to generate aroma compounds and therefore to be involved in the complex process of cheese flavor development. In lactococci, transamination is the first step in the degradation of aromatic and branched-chain amino acids which are precursors of aroma compounds. Here, the major aromatic amino acid aminotransferase of a Lactococcus lactis subsp. cremoris strain was purified and characterized. The enzyme transaminates the aromatic amino acids, leucine, and methionine. It uses the ketoacids corresponding to these amino acids and alpha-ketoglutarate as amino group acceptors. In contrast to most bacterial aromatic aminotransferases, it does not act on aspartate and does not use oxaloacetate as second substrate. It is essential for the transformation of aromatic amino acids to flavor compounds. It is a pyridoxal 5'-phosphate-dependent enzyme and is composed of two identical subunits of 43.5 kDa. The activity of the enzyme is optimal between pH 6.5 and 8 and between 35 and 45 degrees C, but it is still active under cheese-ripening conditions.

Experimental evidence for the essential role of the C-terminal residue in the strict aminopeptidase activity of the thiol aminopeptidase PepC, a bacterial bleomycin hydrolase.

PepCs isolated from lactic acid bacteria and bleomycin hydrolases of eukaryotic organisms are strict aminopeptidases which belong to the papain family of thiol peptidases. The structural basis of the enzymic specificity of the lactococcal PepC has been investigated by site-directed mutagenesis. The deletion of the C-terminal residue (Ala-435) abolished the aminopeptidase activity, whereas this deletion led to a new peptidase specificity. The enzymic properties of wild-type and mutant PepCs demonstrate that the terminal alpha-carboxy group plays a key role in the strict aminopeptidase activity.

Purification, crystallization, and preliminary X-ray analysis of PepX, an X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis.

The X-prolyl dipeptidyl aminopeptidase PepX, a serine peptidase isolated originally from Lactococcus lactis subsp lactis NCDO 763, was cloned and overproduced in Escherichia coli. The enzyme was isolated in its active form in two purification steps. Crystals of PepX were grown by the hanging drop vapor diffusion method using polyethyleneglycol 4000 as precipitant at pH 5.0. The crystals are orthorhombic with cell dimensions a = 92.8 A, b = 102.6 A, and c = 101.6 A, space group P2(1)2(1)2, and probably contain one monomer of 87.5 kDa in the asymmetric unit. The crystals, very stable under X-rays, diffract to at least 2.2 A and are suitable for high-resolution structural analysis.

Oligopeptides are the main source of nitrogen for Lactococcus lactis during growth in milk.

The consumption of amino acids and peptides was monitored during growth in milk of proteinase-positive (Prt+) and -negative (Prt-) strains of Lactococcus lactis. The Prt- strains showed monophasic exponential growth, while the Prt+ strains grew in two phases. The first growth phases of the Prt+ and Prt- strains were in same, and no hydrolysis of casein was observed. Also, the levels of consumption of amino acids and peptides in the Prt+ and Prt- strains were similar. At the end of this growth phase, not all free amino acids and peptides were used, indicating that the remaining free amino acids and peptides were unable to sustain growth. The consumption of free amino acids was very low (about 5 mg/liter), suggesting that these nitrogen sources play only a minor role in growth. Oligopeptide transport-deficient strains (Opp-) of L. lactis were unable to utilize oligopeptides and grew poorly in milk. However, a di- and tripeptide transport-deficient strain (DtpT-) grew exactly like the wild type (Opp+ Dtpt+) did. These observations indicate that oligopeptides represent the main nitrogen source for growth in milk during the first growth phase. In the second phase of growth of Prt+ strains, milk proteins are hydrolyzed to peptides by the proteinase. Several of the oligopeptides formed are taken up and hydrolyzed internally by peptidases to amino acids, several of which are subsequently released into the medium (see also E.R.S. Kunji, A. Hagting, C.J. De Vries, V. Juillard, A.J. Haandrikman, B. Poolman, and W.N. Konings, J. Biol. Chem. 270:1569-1574, 1995).(ABSTRACT TRUNCATED AT 250 WORDS)

Separation and identification of hydrophilic peptides in dairy products using FMOC derivatization.

Small hydrophilic di- and tripeptides from food products are not separated by reversed-phase high-performance liquid chromatography (RP-HPLC). A simple method using precolumn derivatization with 9-fluorenylmethoxycarbonyl chloride (FMOC) of hydrophilic peptides followed by RP-HPLC separation is presented. Peptides can subsequently be identified by Edman degradation after deprotection of the peptide derivatives with piperidine. Fifteen peptides (ten dipeptides, four tripeptides and one tetrapeptide) were sequenced from the water-soluble fraction of an enzyme-modified cheese model. Some synthetic peptides (Ile-Asn, Val-Thr, Ala-Pro, Val-Gln, Thr-Gln and Gly-Gly) corresponding to purified peptides were sensory tested.

Preparation of bacterial X-prolyl dipeptidyl aminopeptidase and its stabilization by organic cosolvents.

To obtain large amounts of the X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis subsp lactis (PepX, E.C. 3.4.14.5), PepX was purified from a commercial L. lactis cell extract. The enzyme was purified in only three steps and the last one was performed by HPLC on a C4 reverse-phase column using acetonitrile as an eluent. Despite its high molecular mass (175 kDa), the enzyme was recovered with a good activity yield (75%). Advantages and drawbacks of this technique compared to the classical ones are discussed. The stability of the enzyme in aqueous solutions and in the presence of 10 water-miscible solvents was also investigated. PepX was found to be stabilized by dimethyl sulfoxide, triglyme, and glycerol.

Biochemical and genetic characterization of PepF, an oligopeptidase from Lactococcus lactis.

Lactococcus lactis possesses a complex proteolytic system which is essential for its growth in milk. We characterized one of the peptidases of this system, oligopeptidase PepF, together with its structural gene. PepF hydrolyzed peptides containing between 7 and 17 amino acids with a rather wide specificity. It was purified to homogeneity. The N-terminal sequences of PepF and of peptides resulting from tryptic digestion of PepF were determined and used to design degenerate oligonucleotides which served to amplify a DNA fragment internal to pepF. This fragment was used as a probe to screen a lactococcal genomic library in Escherichia coli and to clone the entire gene pepF. The gene coded for a 70 kDa protein and was located on a 55-kilobase lactose-protease plasmid. A motif His-Glu-X-X-His, characteristic of metallopeptidases was evidenced. Two regions of PepF were found similar, first to a stretch of 43 amino acids around the zinc-binding site of several other peptidases, second to a stretch of 33 amino acids well conserved among creatine and arginine kinases. Preliminary results suggest the presence of a second copy of pepF.

Purification and characterization of a general aminopeptidase (St-PepN) from Streptococcus salivarius ssp. thermophilus CNRZ 302.

A general aminopeptidase (St-PepN) was purified from an intracellular extract of Streptococcus salivarius ssp. thermophilus CNRZ 302 by ion-exchange chromatography and hydrophobic interaction chromatography. Gel electrophoresis of the purified enzyme in denaturing or nondenaturating conditions showed a single protein band. The enzyme is a monomer with a molecular mass of 97 kDa. Its activity is maximal at pH 7 and 36 degrees C and is completely abolished by CuCl2 and ZnCl2. The enzyme is strongly inhibited by metal-chelating reagents, such as EDTA and o-phenanthroline, which suggests that St-PepN is a metalloenzyme. The enzyme showed activity toward p-nitroanilide derivatives or dipeptides and tripeptides and showed a preference for hydrophobic or basic amino acids at the N-terminal position. Longer peptide chains, such as the B-chain of insulin, glucagon, or peptides generated by the hydrolysis of caseins, were degraded, too. The sequence of the first 21 residues of the mature enzyme was determined and showed high homology with that of the aminopeptidase PepN isolated from Lactococcus lactis ssp. cremoris Wg2. The properties of the enzyme are compared with those of corresponding enzymes of other species of lactic acid bacteria.

Gene cloning and characterization of PepC, a cysteine aminopeptidase from Streptococcus thermophilus, with sequence similarity to the eucaryotic bleomycin hydrolase.

Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe-beta-naphthylamide substrate. The gene encoding one of these aminopeptidases was cloned from a total DNA library of S. thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid. The wild-type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe-beta-naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate. One clone was selected which was shown to express an aminopeptidase, identified as a PepC-like enzyme on the basis of cross-reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase. The gene was further subcloned and sequenced. A complete open reading frame coding for a 445-residue (50414 Da) polypeptide was identified. 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene. High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase. In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site. The product of the cloned pepC gene was overproduced in E. coli and was purified from a cellular extract. Purification to homogeneity was achieved by two-step ion-exchange chromatography. Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity. The enzyme has a molecular mass of 300 kDa suggesting an hexameric structure. On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC-type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases.

Xaa-Pro-dipeptidyl-aminopeptidase from Lactococcus lactis catalyses kinetically controlled synthesis of peptide bonds involving proline.

Xaa-Pro-dipeptidyl-aminopeptidase (EC 3.4.14.5) from Lactococcus lactis (PepX) was used, for the first time, as a catalyst in kinetically controlled synthesis of peptide bonds involving proline. PepX had amidase and esterase activities in addition to peptidase activity. Thus amide and ester derivatives of X-Pro peptides could be employed as acyl donors. PepX showed a broad specificity for the residue in position P'1, accepting a large variety of amino acid amides, esters, peptides as well as free amino acids as nucleophiles. This also indicated that it was not necessary to protect the C-terminus of the nucleophile. The major factors controlling yield, e.g. pH, an excess of nucleophile, ionic strength and type of carboxyl protecting and activating groups, were evaluated. Under optimum reaction conditions (pH 8.5, high excess of nucleophile over acyl donor and moderate ionic strength) the selectivity of the reaction ranged from 5 to 99% depending on the structure of the nucleophile and the acyl donor. Our work contributes to the elucidatation of the mechanism of aminolysis reactions catalysed by an aminopeptidase.

Crystallization and preliminary X-ray analysis of PepC, a thiol aminopeptidase from Lactoccocus lactis homologous to bleomycin hydrolase.

Crystals of the recombinant thiol aminopeptidase PepC, from Lactoccocus lactis, have been obtained using the hanging-drop method of vapor diffusion from ammonium sulfate solutions. Crystals are rhombohedral, the space group is R32, a = 175.2 A, c = 94.5 A (hexagonal setting). The asymmetric unit probably contains one monomer of a hexameric molecule-arrangement of 300 kDa which exhibits the crystallographic point group of symmetry 32. The crystals diffract to at least 3 A resolution.

Reversible enzymic protection of the alpha-amino group of amino acid derivatives using an aminopeptidase A.

In previous papers we have reported that an aminopeptidase A (EC 3.4.11.7) purified from Staphylococcus chromogenes was able to catalyse the introduction of L-malic acid at the N-terminus of Tyr and Phe derivatives. We now show that this enzyme can be used for selective alpha-amino protection of derivatives of probably all amino acids, except Gly and Pro, by the malyl group. The following L-malyl derivatives were synthesized in thermodynamically controlled reactions with yields ranging from 4 to 47%: L-malyl-Tyr-OEt, -ALA-OMe, -Ser-OEt, -Lys-OEt, -Phe-OMe, -Met-NH2, -Glu-MH2, Arg-NH2, -Tye-NH2, -Val-NH2, -Ala-Phe and -Ala-Phe-NH2 (OEt and OMe are ethyl and methyl esters respectively). The reactions were monitored by reverse-phase h.p.l.c.; the products were quantified by amino acid analysis, and their structure was confirmed by m.s. No synthesis was obtained with Gly and Pro derivatives as nucleophiles. The effects of pH, temperature, enzyme concentration, nucleophile concentration, reaction time and addition of an organic co-solvent were studied. An important shift towards synthesis was obtained by carrying out the reactions at 55 degrees C in the presence of 55% organic co-solvent Triglyme (2,4,8,11-tetraoxadodecane) [8-60-fold increase in Ksyn. ([product] [acyl-donor]-1 [nucleophile]-1)].

Design of (omega-N-(O-acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline-specific peptidases.

A novel class of competitive, acylating inhibitors for the proline-specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N-terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting Ki values in the microM range. Boc-protected (omega-N-hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching Ki values of 30 nM to 60 microM. Additionally, alpha-N-BOC-(omega-N-hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time-dependent reaction.