WO1994016082A1 - X-prolyl-dipeptidyl-aminopeptidase tiree de lactobacillus delbruckii ssp. lactis, acides nucleiques la codant et son utilisation dans des procedes de preparation d'aliments fermentes - Google Patents

X-prolyl-dipeptidyl-aminopeptidase tiree de lactobacillus delbruckii ssp. lactis, acides nucleiques la codant et son utilisation dans des procedes de preparation d'aliments fermentes Download PDF

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WO1994016082A1
WO1994016082A1 PCT/EP1993/003711 EP9303711W WO9416082A1 WO 1994016082 A1 WO1994016082 A1 WO 1994016082A1 EP 9303711 W EP9303711 W EP 9303711W WO 9416082 A1 WO9416082 A1 WO 9416082A1
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leu
pro
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gly
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Elke Meyer-Barton
Jürgen Robert KLEIN
Bernhard Henrich
Roland Plapp
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COMMUNAUTE ECONOMIQUE EUROPEENNE (CEE)
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0328—Enzymes other than milk clotting enzymes, e.g. lipase, beta-galactosidase

Definitions

  • the invention relates to X-Prolyl-dipeptidyl-aminopeptidase (PepX) from Lactobacillus delbruckii ssp. lactis or polypeptides derived thereof, and more particularly recombinant PepX from said Lactobacillus strain or recombinant polypeptides derived thereof, which can be used in fermented foodstuff preparation processes.
  • PepX X-Prolyl-dipeptidyl-aminopeptidase
  • the invention also relates to processes for preparing the PepX from said Lactobacillus strain or said derived polypeptides, which are in a state of biological purity such that they can be used in processes for the preparation of fermented foodstuff.
  • the invention also relates to nucleic acids coding for PepX from said Lactobacillus strain or said derived polypeptides, and to cellular hosts containing said nucleic acids and their use in processes for the preparation of fermented foodstuff. Furthermore, the invention relates to processes for the preparation of fermented foodstuff and kits using the PepX from said Lactobacillus strain or said derived polypeptides, and/or said cellular hosts.
  • recombinant polypeptides it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation of a corresponding DNA sequence under the control of appropriate regulation elements within a efficient cellular host. Consequently the expression “recombinant polypeptides” such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
  • the term “recombinant” indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host.
  • biologically pure or biological purity or “in a substantially pure form” means on the one hand a grade of purity such that the polypeptide or recombinant polypeptide can be used for the preparation of fermented foodstuff and on the other hand the absence of contaminants, more particularly of natural contaminants.
  • lactobacilli Rapid growth of lactobacilli in milk depends of their ability to utilize milk proteins as an external source for essential amino acids. As the concentrations of small peptides and free amino acids in milk are rather low, lactobacilli dispose of various proteolytic and peptidolytic activities which are involved in the degradation of casein (1 , 17, 31).
  • the PepX is capable of releasing X-Pro dipeptides from the N-terminus of oligopeptides.
  • the structure of the amino acid proline influences the conformation of peptides and restricts the cleavage of proline-containing peptides by common exo- and endopeptidases.
  • casein has an extraordinarily high content of proline residues (16,7 % for ⁇ -casein) (8), the enzyme is supposed to be indispensable for efficient casein utilization.
  • the enzyme was purified from Lactobacillus lactis, Streptococcus thermophilus (22), Lactobacillus delbruckii ssp. bulgaricus (2, 5, 23), Lactobacillus helveticus (13), Lactobacillus acidophilus (5),
  • An aspect of the invention is to provide a new family of nucleic acids coding for new proteins and polypeptides which can be used for the preparation of fermented foodstuff.
  • Another aspect of the invention is to provide cellular hosts transformed with said nucleic acids which can be used for the preparation of fermented foodstuff, and more particularly as starter organisms in the fermentation of milk.
  • Another aspect of the invention is to provide nucleic acids coding for the peptidic chain of biologically pure recombinant polypeptides which enable their preparation on a large scale.
  • Another aspect of the invention is to provide new proteins and polypeptides which can be used for the preparation of fermented foodstuff.
  • Another aspect of the invention is to provide processes for the preparation of fermented foodstuff, and more particularly cheese.
  • the invention relates to a protein in a substantially pure form, as produced and excreted by Lactobacillus delbruckii ssp. lactis and capable of releasing proline-containing dipeptides (or X-Pro dipeptides) from the N-terminus of oligopeptides present in the culture medium of said lactobacilli, or fragments thereof having said releasing X-Pro dipeptides activity.
  • the invention relates more particularly to the protein (PepX) as defined above, such as produced by the strain of Lactobacillus delbruckii ssp. lactis WS87, deposited at the Deutsche Sammlung von Mikroorganismen (DSM) under the number 7290 on October 15, 1992, the polypeptidic sequence of said PepX being represented on figure 5 or figure 6.
  • PepX is also represented by SEQ ID NO 2 in the sequence listing which follows.
  • Said PepX represented by SEQ ID NO 2 is more particularly characterized in that: - its isoelectric point calculated from the nucleotide sequence represented by SEQ ID NO 1, is 4.96, and its isoelectric point determined by preparative isoelectric focusing after ammonium sulfate fractionation of crude extract of said protein, is 3.9,
  • the PepX according to the invention is more particularly characterized in that it is obtained from a cell extract of said Lactobacillus delbruckii ssp. lactis WS87 by the following four-step procedure:
  • the degree of purity of the enzyme obtained by applying the process above-described is approximately of 98% .
  • the invention also relates to polypeptides derived from the PepX from said Lactobacillus strain defined above, said derived polypeptides corresponding to fragments of the polypeptide sequence represented by SEQ ID NO 2, or muteins (which differ from said protein by addition and/or substitution and/or suppression of one or several amino acid) thereof, provided that said fragments and muteins are capable of releasing X-Pro dipeptides.
  • Proteins or polypeptides derived thereof according to the invention are in the form of a monomer or a polymer, more particularly a dimer or a trimer.
  • Preferred proteins or polypeptides derived thereof according to the invention are in the form of a monomer.
  • the invention also relates to nucleic acids coding for proteins or polypeptides such as defined above.
  • the invention relates more particularly to the nucleic acid sequence represented by SEQ ID NO 1 coding for PepX, the amino acid sequence of this latter protein being represented under the nucleotide sequence coding for PepX in SEQ ID NO 1.
  • Nucleic acids according to the invention are more particularly characterized in that: - they comprise all or part of the nucleotidic sequence represented on figure 5 or figure 6, and corresponding to SEQ ID NO 1, coding for the PepX from said Lactobacillus strain, or its complementary sequence,
  • the hybridization above-mentionned can be performed with membranes like HeybondTM-N (Amersham) or Hybridization Transfer Membrane (Micron Separations Inc.) under conditions as described by the supplier.
  • the invention also relates to recombinant nucleic acid containing at least one of the nucleic acids according to the invention, inserted in a heterologous nucleic acid.
  • the invention relates more particularly to recombinant vectors comprising a vector sequence, notably of the type plasmid (such as plasmids originating from lactic acid bacteria), cosmid or phage, and a nucleic acid according to the invention, in one of the non-essential sites for their replication.
  • a vector sequence notably of the type plasmid (such as plasmids originating from lactic acid bacteria), cosmid or phage, and a nucleic acid according to the invention, in one of the non-essential sites for their replication.
  • Preferred recombinant vectors according to the invention are those containing in one of their non essential sites for their replication, necessary elements to promote the expression of polypeptides according to the invention in a cellular host and possibly a promoter recognized by the polymerase of the cellular host, particularly an inducible promoter and possibly a signal sequence and/or an anchoring sequence.
  • Recombinant vectors can contain the elements enabling the expression by E. coli or lactic acid bacteria including lactococcus and, especially, lactobacilli of the thermophilic groups, of a nucleic acid according to the invention inserted in the vectors, to demonstrate product formation.
  • Particularly preferred recombinant vectors are constructed from replicons isolated from lactobacilli, especially from thermopilic Lactobacillus species.
  • the invention also relates to cellular hosts which are transformed by a recombinant vector such as defined above, and comprising the regulation elements enabling the expression of the nucleotide sequence coding for a protein according to the invention in these hosts.
  • Preferred cellular hosts are chosen from among bacteria such as E. coli. or from among lactic acid bacteria including lactococcus and, especially, lactobacilli of the thermophilic group, transformed by a vector according to the invention.
  • probes i.e. cloned or synthetic oligonucleotides
  • These probes can be from 25 nucleotides up to the total length of the pepX gene.
  • These oligonucleotides can also be used as amplification primers in the PCR technique (PCR, Mullis and Faloona, Methods in Enzymology, vol. 155, p. 335, 1987) to generate specific enzymatically amplified fragments and/or as probes to detect fragments amplified between bracketing oligonucleotide primers.
  • the invention also relates to expression products of nucleic acids expressed by transformed cellular hosts such as described above.
  • the invention also relates to a process for preparing a protein such as defined above comprising the following steps:
  • polypeptides of the invention can be prepared according to the classical techniques in the field of peptide synthesis.
  • the synthesis can be carried out in homogeneous solution or in solid phase.
  • polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Sheppard in their book entitled “Solid phase peptide synthesis” (IRL Press, Oxford, New York, Tokyo, 1989).
  • the invention also relates to a process for preparing the nucleic acids according to the invention.
  • a suitable method for chemically preparing the single-stranded nucleic acids can be carried out according to the automatic ⁇ -cyanoethyl phosphoramidite method of DNA synthesis described in Bioorganic Chemistry 4; 274-325 (1986).
  • the material which is obtained at the end of the DNA synthesis can be used as such.
  • a suitable method for chemically preparing the double-stranded nucleic acids comprises the following steps: - DNA synthesis of one sense oligonucleotide using the automatic ⁇ -cyanoethyl phosphoramidite method above cited, and DNA synthesis of one antisense oligonucleotide using either the above-mentioned automatic ⁇ -cyanoethyl phosphoramidite method, or enzymatic transcription of the sense- strand using a specific primer hybridizing to the 3 '-end of the sense strand,
  • a method for the chemical preparation of nucleic acids with lengths greater than 100 nucleotides - or base pairs, in the case of double-stranded nucleic acids - comprises the following steps: - assembling the synthesized oligonucleotides, provided at their ends with different restriction sites, the sequences of which are compatible with the succession of amino acids in the natural peptide, according to the principle described by Urdea et al. in Proc. Nat. Acad. Sci. USA 80; 7461-7465 (1983),
  • the invention also relates to antibodies themselves formed against the polypeptides according to the invention, more particularly by immunization of appropriate animals with said polypeptides and recovery of antibodies thus formed.
  • the invention also relates to any antibody of the invention labeled by an appropriate label of the enzymatic, fluorescent or radioactive type.
  • the invention relates more particularly to the use of the polypeptides described above for the determination of the exact localization of PepX in Lactobacillus delbruckii ssp. lactis, rendering possible experiments performed by immunoblotting after cell fractionation and by electron microscopy of immunogold labeled peptidases.
  • the invention also relates to fermentation processes comprising a step of treatment of material to be fermented with:
  • lactis an appropriate amount of at least one of the transformed cellular host such as described above, and/or - an appropriate amount of lactobacilli, such as Lactobacillus delbruckii ssp. lactis.
  • the invention also relates to a process for the preparation of fermented foodstuff, and more particularly of cheese, which comprises a step of treatment of food material to be fermented, such as milk, with: - an appropriate amount of at least one of the protein according to the invention, and/or
  • lactobacilli such as Lactobacillus delbruckii ssp. lactis.
  • the process for the preparation of fermented foodstuff described above can also comprise a step of treatment of food material to be fermented, such as milk, with other species and strains susceptible to be used as starter organisms in fermentation processes, and more particularly lactic acid bacteria susceptible to produce a PepX protein, such as Lactococcus, Streptococcus and
  • the fermentation processes according to the invention are more particularly characterized in that they can be used as fermentation processes for the obtention of hard cheeses, such as Emmentaler type cheese (South Germany, Switzerland).
  • the invention also relates to foodstuff, and more particularly cheeses, such as obtained by fermentation processes as described above.
  • Figure 1 represents the purification steps visualized on SDS-Gel.
  • Lane 1 marker proteins; a myosin (205 kDa), b ⁇ -galactosidase (116 kDa), c phosphorylase B (97.5 kDa), d bovine serum albumin (66 kDa), e ovalbumin (45 kDa);
  • lane 2 crude extract of Lactobacillus delbruckii ssp. lactis WS87 (DSM 7290);
  • lane 3 ammonium sulfate fractionation; lane 4; HIC-fraction; lane 5: Mono Q-fraction; lane 6: L-Ala-L-Pro-EAH-Sepharose-fraction.
  • Figure 2 :
  • Figure 2a represents the effect of temperature on the activity of PepX.
  • the x-axis corresponds to the temperature (°C), and the y-axis corresponds to the relative activity (%).
  • Figure 2b represents the effect of pH on the activity of PepX.
  • the x-axis corresponds to the pH, and the y-axis corresponds to the relative activity
  • Figure 3 represents the localization of pepX on the plasmid pJK433, and subcloned fragments in pUC18.
  • Figure 4 represents the localization of pepX on the plasmid pJK433, and subcloned fragments in pUC18.
  • Figure 4 represents the overexpression of PepX in E. coli.
  • Lane 1 crude extract of JM109;
  • lane 2 crude extract of JM109 (pLG339);
  • lane 3 crude extract of JM109 (pJK433);
  • lane 4 marker proteins, a myosin (205 kkDa), b ⁇ -galactosidase (116 kDa), c phosphorylase B (97.5 kDa), d bovine serum albumin (66 kDa), e ovalbumin (45 kDa), f carbonic anhydrase (29 kDa).
  • Figure 5 represents the nucleotide sequence (corresponding to SEQ ID NO 1) and deduced amino acid sequence (in the one-letter code) of PepX. the restriction sites used for subcloning and complementation experiments are indicated. Putative ribosome binding sites (RBS) and potential promotor regions are underlined. The convergent arrows above DNA sequence indicate the putative transcription terminator. Asterisk, stop codon. N-terminal amino acid sequence determined on the purified protein is over lined.
  • Figure 6 Figure 6 represents the nucleotide sequence and deduced amino acid sequence (in the three-letter code) of PepX (corresponding to SEQ ID NO 2).
  • Figure 7 represents the nucleotide sequence and deduced amino acid sequence (in the three-letter code) of PepX (corresponding to SEQ ID NO 2).
  • Figure 7 represents a computer alignment of the amino acid sequences of the PepX from Lactobacillus lactis ssp. lactis WS87 (lbpepX), Lactococcus lactis ssp. cremoris (llxpdap, 21), and Lactococcus lactis ssp. lactis (llpepxp,
  • Figure 8 represents the hydropathy plot of PepX according to Kyte and Doolittle (15).
  • Bacterial strains and plasmids are listed in Table 1. Lactobacillus delbruckii ssp. lactis was grown in MRS medium (20) at 42 °C. Escherichia coli was grown in LB medium (3) at 37°C with shaking. When needed, kanamycin (40 ⁇ g/ml), ampicilin (200 ⁇ g/ml), or tetracycline (12.5 ⁇ g/ml) was added to the culture medium.
  • Lb. delbruckii was grown at 42 °C for 15 - 20 h in 10 1 MRS broth. Cells were harvested by centrifugation at 11.000 g for 10 min at 4°C, and washed twice with 50 mM Tris hydrochloride buffer pH 7.2. By grinding with aluminium oxide at 4°C cells were disrupted and then suspended in 50 mM Tris hydrochloride buffer pH 7.2 (1.0 - 1.5 ml per g wet cells). Aluminium oxide was removed from the homogenate by centrifugation at 11,000 g and 4°C for 10 min.
  • Absorbed proteins were extracted from aluminium oxide by washing with 50 mM Tris hydrochloride buffer pH 7.2 (0.5 - 0.75 ml per g wet cells), before the cell debris was down by ultracentrifugation at 139,000 g and 4°C for
  • E. coli was grown overnight at 37 °C with shaking in 100 ml LB medium. Cells were harvested by centrifugation at 7,000 g for 10 min. The pellet was washed once with 20 ml 50 mM Tris hydrochloride buffer pH 7.5, suspended in 50 mM Tris hydrochloride buffer pH 7.5 with an end volume of the suspension of 1 ml. After disruption by ultrasonication (Bandelin electronic, type UW 60) cell debris were removed at 52,000 g and 4°C for 1 h.
  • PepX was routinely tested in microtiter plates with L-Ala-L-Pro-p-nitroanilide (Bachem, Bubendorf, Switzerland) as substrate.
  • the reaction mixture contained 0.2 ml Tris hydrochloride pH 7.2. 10 ⁇ l substrate
  • the cell extract was fractionated by salting out at 4°C with solid ammonium sulfate from 40 to 60 % saturation. After gently mixing for 1 h at 4°C the precipitate formed was collected by centrifugation at 21,000 g and 4°C for 30 min and dissolved in a minium amount of 50 mM Tris hydrochloride pH 7.2.
  • the undialysed ammonium sulfate fraction was applied at 4 °C to a 26 by 1.5 cm column of Octyl-Sepharose 4B (Pharmacia, Uppsala, Sweden) equilibrated with 50 mM Tris hydrochloride pH 7.2 in 1 M ammonium sulfate.
  • the enzyme was eluted (28.5 ml/h) with a linear salt gradient between equilibration buffer and distilled water.
  • the fractions with the highest specific activity were pooled, dialysed against 20 mM Tris hydrochloride pH 7.2, and concentrated by passage through a PM30 Diaflo membrane (Amicon, Beverly).
  • step 2 The pooled fractions of step 2 were applied to Mono Q HR 5/5 (Pharmacia) equilibrated with 20 mM Tris hydrochloride pH 7.2. Proteins were eluted at 1 ml/min with a two-step linear NaCl gradient: 0 to 0.25 M NaCl for 6 min, 0.25 to 0.5 M NaCl for 30 min.
  • Carbodiimide coupling of L-Ala-L-Pro to EAH-Sepharose 4B was based on a procedure recommended by the supplier. After coupling of the ligand a columm was packed (Bio-Rad, Richmond, Calif. ; gel bed 7 by 1 cm) and equilibrated at 4°C with 20 mM Tris hydrochloride buffer pH 7.2. The dialysed fraction of step 3 was applied. The columm was washed with equilibration buffer and eluted (12 ml/h) with a linear salt gradient of 0 to 0.5 M NaCl.
  • Non-denaturing polycrylamide gel electrophoresis (12 %) was carried out according to Davis (9).
  • Activity staining was carried out by incubation of individual gels with 10 ml stainig solution (1.5 % agar, 0.1 mM C0CI2,
  • the molecular weight of the native enzyme was estimated by gel filtration on Fractogel TSK HW-55(S) (Merck, Darmstadt, Germany ; 2.5 by 74 cm).
  • 5'-[ ⁇ -35s]dATP was purchased from Amersham-Buchler (Buckinghamshire, United Kingdom), the T7 DNA polymerase sequencing kit was obtained from Pharmacia. DNA and amino acid sequence analysis were performed using the Microgenie sequence analysis software package (25).
  • Table 2 summarizes the purification of PepX from Lb. delbruckii ssp. lactis WS87 to electrophoretic purity by use of a four-step procedure including ammonium sulfate fractionation, hydrophobic interaction chromatography on
  • FIG. 1 shows SDS-PAGE analysis of the enzyme fractions during purification. The final purification was approximately 200-fold, and recovery was 4.6 % .
  • SDS-PAGE and gelfiltration on HW TSK-55 S
  • the enzyme is a monomer with a molecular mass of 95 kDa (SDS-PAGE) and 84 kDa (gel filtration).
  • Optimal PepX activity in crude extract was measured at pH of 7.0, and temperatures of 46-50 °C (figures 2a and 2b respectively). Denaturation occurs at pH values below 4 and at temperatures above 55 °C.
  • the enzyme belongs to the class of serine proteases since it is inhibited by 3,4-dichloroisocoumarine and phenylmethanesulphonyl fluoride, specific inhibitors for serine proteases.
  • the N-terminal amino acid sequence detected by microsequencing in gasphase is overlined in figure 5.
  • PepX Overexpression in E. coli
  • the PepX gene designated pepX, was very efficiently expressed in the E. coli host strain from its authentic promotor. Overproduction could be visualized on SDS-PAGE : PepX constituted more than 50% of the cytoplasmatic proteins (figure 4).
  • the plasmid pJK433 exhibited only limited stability in the heterologous E. coli system. Even in the presence of kanamycin, which selects for the presence of the plasmid, PepX activity was completely lost after approximately 100 generations.
  • PepX on the 6.3 kb insert of pJK433 was determined by cleavage with appropriate restriction enzymes, religation, and subsequent complementation experiments (figure 3). Deletion of a 0.8 kb Sphl fragment, and of a 0.5 kb _S__mHI-_5glII fragment, respectively, resulted in loss of Gly-L- Pro-naphthylamide cleaving activity of the respective E. coli ER1562 transformants. Regions flanking the partial 4.8 kb Sphl fragment, on the other hand, were found to be dispensable to the functional integrity of the pepX gene.
  • the nucleotide sequence of a 3,215 bp region was determined, and an open reading frame was found that starts at position 288 (AUG) and stops at the ochre termination codon (TAA) at position 2,664.
  • Figure 5 shows the complete nucleotide sequence, the open reading frame detected encodes a protein of 792 amino acids with a calculated molecular mass of 88,449 Da. Upstream of the ORF, there are several alternative stretches of A's and T's, the AT-content is 65.5 % (position 1 - 287). It is less clear-cut, however, to assign the expression signals.
  • the GC content of the DNA fragment sequenced is
  • N-terminal amino acids of the protein specified by the ORF agree with those determined by amino acid sequencing of the purified enzyme.
  • BRADFORD M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Analytical Biochem. 72: 248-254.
  • LAEMMLI U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nauture 227: 680-685.
  • E. coli JM109 recAl, endAl, gyrA96, thi, hsdRU 35 sup ⁇ A , relAl, ⁇ ⁇ , A(lac-proAB), Pharmacia [F ⁇ traO36, proAB, / ⁇ dq Z ⁇ M15] (Uppsala, Sweden)
  • LACTOBACILLUS DELBRUCKII SSP LACTIS, NUCLEIC ACIDS CODING FOR THE SAME AND ITS USE IN FERMENTED FOODSTUFF PREPARATION PROCESSES (iii) NUMBER OF SEQUENCES: 2
  • MOLECULE TYPE DNA (genomic)
  • CAG GAC ATG AAG CAG CCA AGC AAG CTG GAA GCC GGC CAG TTC GTC GAC 2504
  • TTTATCGCAA ACAAAGTAAA AGGCTGTCGC GGACGTACCG CTGACAGCCT TTTTGTGTTC 2763

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Abstract

L'invention concerne une protéine produite et excrétée par Lactobacillus delbrückii ssp. lactis et capable de libérer des dipeptides de X-Pro à partir de la N-terminaison d'oligopeptide présent dans le milieu de culture desdits lactobacillis, ou fragments de ceux-ci présentant ladite activité de libération de dipeptides de X-Pro. L'invention concerne également l'utilisation de ladite protéine ou de fragment de celle-ci dans la préparation d'aliment fermenté, et notamment de fromage.
PCT/EP1993/003711 1992-12-30 1993-12-29 X-prolyl-dipeptidyl-aminopeptidase tiree de lactobacillus delbruckii ssp. lactis, acides nucleiques la codant et son utilisation dans des procedes de preparation d'aliments fermentes Ceased WO1994016082A1 (fr)

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
WO1996032468A3 (fr) * 1995-04-11 1996-11-28 Merck & Co Inc Processus biologique de production de composes a base de dipeptides
EP0754752A3 (fr) * 1995-07-21 1997-05-21 Ajinomoto Kk Dipeptidyl-peptidase IV et procédé de préparation
US6168939B1 (en) 1995-03-07 2001-01-02 Masahiro Sasaki Endopeptidase produced by Lactobacillus helveticus

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Title
ELKE CHRISTEL MEYER-BARTON ET AL.: "Cloning and sequence analysis of the X-prolyl-dipeptidyl-aminopeptidase gene (pepX) from Lactobacillus delbrückii ssp. lactis DSM7290", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 40, no. 1, October 1993 (1993-10-01), pages 82 - 89 *
EMBL Database entry Accesion number S32244; 22 November 1993 MEYER, E. ET AL. *
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HIROSHI MIYAKAWA ET AL.: "Purification and characterization of an X-prolyl dipeptidyl aminopeptidase from Lactobacillus delbrueckii ssp. bulgaricus LBU-147", JOURNAL OF DAIRY SCIENCE, vol. 74, no. 8, August 1991 (1991-08-01), CHAPAIGN, ILLINOIS US, pages 2375 - 2381 *
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WILHEM BOCKELMANN ET AL.: "Purification and characterization of the X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus acidophilus", INT. DAIRY JOURNAL, vol. 1, 1991, pages 51 - 66 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6168939B1 (en) 1995-03-07 2001-01-02 Masahiro Sasaki Endopeptidase produced by Lactobacillus helveticus
WO1996032468A3 (fr) * 1995-04-11 1996-11-28 Merck & Co Inc Processus biologique de production de composes a base de dipeptides
US6204008B1 (en) 1995-04-11 2001-03-20 Merck & Co., Inc. Bioprocess for production of dipeptide based compounds
EP0754752A3 (fr) * 1995-07-21 1997-05-21 Ajinomoto Kk Dipeptidyl-peptidase IV et procédé de préparation
US5811278A (en) * 1995-07-21 1998-09-22 Ajinomoto Co., Inc. Dipeptidyl peptidase IV from Xanthomonas maltophilia and process for producing the same

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