Classifications

• • 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)
  • C12N9/485—Exopeptidases (3.4.11-3.4.19)
• • 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; MAKING THEREOF
  • A23C21/00—Whey; Whey preparations
  • A23C21/02—Whey; Whey preparations containing, or treated with, microorganisms or enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
  • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
  • A23J3/343—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins
  • A23J3/344—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of dairy proteins of casein
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J3/00—Working-up of proteins for foodstuffs
  • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
  • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
  • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
  • A23J3/346—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of vegetable proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
  • A23L29/06—Enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/18—Peptides; Protein hydrolysates
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/185—Vegetable proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/19—Dairy proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/195—Proteins from microorganisms
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/01—Hydrolysed proteins; Derivatives thereof
  • A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
  • A61K38/018—Hydrolysed proteins; Derivatives thereof from animals from milk
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/4813—Exopeptidases (3.4.11. to 3.4.19)
• • C—CHEMISTRY; METALLURGY
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  • 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)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
  • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/11—Aminopeptidases (3.4.11)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
  • C12Y304/24—Metalloendopeptidases (3.4.24)
  • C12Y304/24027—Thermolysin (3.4.24.27)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2300/00—Processes
  • A23V2300/08—Denaturation, e.g. denaturation of protein

Definitions

• the present invention relates to a composition comprising aminopeptidases, more particularly a composition which comprises tetrahedral aminopeptidases, also called "TET aminopeptidases”.
• long peptides also called “complex” are naturally present in the food consumed. These long peptides are generally resulting from an insufficient degradation process and are sometimes toxic, which generates intolerances or allergies to foods containing these peptides and also reduces their nutritional efficiency. These peptides also determine the taste qualities and texture of many foods such as breads, cheeses, cured meats, etc.
• gluten intolerance is probably one of the most widespread food intolerances. It is caused by a family of complex proteins called “gliadin” which is part of the gluten composition, these proteins sometimes carrying a peptide called “immunodominant” which causes an allergic reaction in people sensitive or intolerant to gluten.
• proteolytic enzymes in many processes to degrade gluten and make it less immunogenic. They are essentially endoproteases that allow cleavage of proteins, and long peptides, into fragments. These endoproteases are particularly used in the production processes in pastries, cheese, biscuits, but also in the production of fruit or beer juices, and even in the production of protein hydrolysates for special feeding. These proteases usually originate from bacteria or fungi and their exact nature is usually kept confidential.
• proteases currently used are that they do not allow the production of peptides sufficiently short for all the toxic parts of the food peptides are removed or the peptides can be effectively digested.
• the incomplete digestion of gluten induces the presence of the "immunodominant" peptide in the digestive system and ultimately causes the symptoms of Celiac disease.
• the invention aims to remedy all these drawbacks.
• the invention therefore relates to a composition comprising at least a first aminopeptidase and at least a second aminopeptidase, said first and second aminopeptidases being different from one another, said first and second aminopeptidases being derived from extremophilic microorganisms, said first and second aminopeptidases being aminopeptidases of the family of tetrahedral aminopeptidases or TET aminopeptidases, said first aminopeptidase representing up to 40% by weight relative to the total weight of the composition, and if said first and second aminopeptidases are different from PhTET2 and PhTET3, then said first aminopeptidase represents up to 50% by weight relative to the total weight of the composition.
• the inventors have surprisingly found that the use of a composition comprising at least two TET aminopeptidases was capable of degrading peptides, especially in a mixture, and exerting a synergistic effect going beyond the additive effect of the individual activities. of each of the TET proteins. Indeed, instead of finding an overall activity of the composition corresponding to the combination of the activities of the different TET aminopeptidases present in the composition, the inventors have found a different overall activity that can be modulated by the physicochemical conditions of the reaction medium or by the interactions / interferences between the various TET aminopeptidases of the composition.
• aminopeptidase is meant an enzyme having an amino acid cleavage activity by the end of the peptides, polypeptides or proteins.
• peptide is meant in the invention a chain of amino acids comprising at least 2 amino acids. The peptides can be obtained either from the degradation of proteins or from chemical syntheses.
• polypeptide is meant in the invention an amino acid chain larger than an amino acid chain of a peptide and obtained from the degradation of proteins and not from a chemical synthesis. Peptides and polypeptides may have a biological function. However, peptides and polypeptides can not perform this function alone as part of a cellular process.
• protein in the invention a molecule containing a chain of amino acids having a biological function and that is naturally found in an organism. This biological function is part of a natural process of the cell.
• the composition therefore comprises two aminopeptidases different from each other in that they each have a different amino acid sequence with respect to each other. In other words, the aminopeptidases have amino acid sequences that diverge by at least one amino acid. In other words, the sequences of the two aminopeptidases diverge from one another by one or more amino acids.
• the tetrahedral aminopeptidases or TET aminopeptidases used in the composition of the invention are isolated from extremophilic microorganisms and more particularly from marine extremophilic microorganisms. These tetrahedral aminopeptidases or TET aminopeptidases belong to the M42 and M18 metallo aminopeptidase families according to the MEROPS classification. In other words, the TET aminopeptidases are in the form of enzymatic complexes comprising 12 subunits which have the particularity of self-assembly in buildings forming typical structures in tetrahedron. Such a form contributes to the high stability of the enzyme. By “metallo aminopeptidases” is meant that these aminopeptidases all have in common the presence of at least one metal ion within the active site.
• extremeophilic microorganisms living organisms, invisible to the naked eye, which can be observed only with the aid of a microscope. These microorganisms can take various forms of life among which include bacteria, microscopic fungi, archaebacteria, protists, microscopic green algae, plankton animals, planarians, amoebas or even viruses. "Extremophile” refers to an organism whose normal living conditions are generally deadly conditions for most other organisms. These living conditions can be high or low temperature, extreme pressures, salinity, acidity or alkalinity of the environment in which the organism lives, a presence of radioactivity or a lack of oxygen or light.
• aminopeptidases TET are distinguished from other aminopeptidases in that they can be very specific for certain types of amino acids. Therefore, it is possible with the compositions according to the invention to associate different TET aminopeptidases, in order to degrade peptides or so-called complex proteins into sufficiently short peptides. Therefore, it is possible to degrade all the toxic parts so that they can be effectively digested. Such a final result is obtained thanks to an aminopeptidase activity specific to the composition used. This aminopeptidase activity is established with regard to the polypeptide content of the substrate and the parts that it is desirable to modify, or even totally degrade.
• TET aminopeptidases associated according to their specificity for certain amino acids and their interaction / interference with each other concerning their activity.
• certain TET aminopeptidases exhibit, under certain conditions, a better activity for the degradation of peptides enriched in a particular type of amino acid than other TET aminopeptidases of which it is known in the art. state of the art that they are specific for this type of amino acid.
• the different combinations of TET aminopeptidases are not a simple addition of the known activities of the TET aminopeptidases that are associated in the composition.
• the first aminopeptidase may represent up to 40% by weight relative to the total weight of the composition.
• up to 40% relative to the total weight of the composition it is meant that the first aminopeptidase may represent 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%. %, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
• the second aminopeptidase may therefore represent at least 60% by weight relative to the total weight of the composition, which means 60%, 61%, 62%, 63%, 64%.
• the first and second TET aminopeptidases may be in proportions ranging from 1: 99 by weight relative to the total weight of the composition up to 40: 60 by weight relative to the total weight of the composition, depending on the weight of the first aminopeptidase relative to the total weight of the composition.
• the first aminopeptidase and the second aminopeptidase are not PhTET2 and PhTET3
• the first aminopeptidase may represent up to 50% by weight relative to the total weight of the composition.
• up to 50% relative to the total weight of the composition is meant in the invention that the first aminopeptidase may represent 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%
• said first and second aminopeptidases are different from PhTET2 and PhTET3
• said first and second aminopeptidases comprise, consist essentially or consist of amino acid molecules whose sequences have less than 65% identity with the sequence PhTET2 (SEQ ID NO: 2) or the sequence PhTET3 (SEQ ID NO: 3).
• % identity between two sequences is meant that when these two amino acid sequences are aligned to compare them, by any means known to those skilled in the art, these two sequences have portions of sequence whose sequences in amino acids are identical. The set of all these portions makes it possible to establish the percentage of identity between the two sequences.
• the second aminopeptidase TET may represent at least 50% by weight relative to the total weight of the composition, this which means 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%
• said at least one first aminopeptidase and said at least one second aminopeptidase are selected from aminopeptidases of the group consisting of: PhTETI, PhTET2, PhTET3, PhTET4 and MjTET.
• PhTETI is represented by the amino acid sequence SEQ ID NO: 1, PhTET2 by the amino acid sequence SEQ ID NO: 2, PhTET3 by the amino acid sequence SEQ ID NO: 3, PhTET4 by the amino acid sequence SEQ ID NO: 4 and MjTET by the amino acid sequence named SEQ ID NO: 5.
• the first and second pairs of TET aminopeptidases of a composition according to the invention can be chosen from the list of pairs consisting of: PhTETI -PhTET2, PhTETI -PhTET3, PhTETI -PhTET4, PhTETI -MjTET, PhTET2 -PhTET3, PhTET2-PhTET4, PhTET2-MjTET, PhTET3-PhTET4, PhTET3-MjTET, PhTET4-MjTET.
• said aminopeptidases comprise, consist essentially or consist of the amino acid molecules of sequences SEQ ID NO: 1 to SEQ ID NO: 5 respectively, or proteins having an aminopeptidase activity, said proteins comprising, consisting essentially or consisting of amino acid molecules whose sequences have at least 65% identity with one of SEQ ID NO: 1 to SEQ ID NO: 5.
• % identity with one of the sequences SEQ ID NO: 1 to SEQ ID NO: 5" is meant for the invention that the proteins have an amino acid sequence which, when it is aligns with one of SEQ ID NO: 1 (PhTEH sequence), SEQ ID NO: 2 (PhTET2 sequence), SEQ ID NO: 3 (PhTET3 sequence), SEQ ID NO: 4 (PhTET4 sequence) or SEQ ID NO: 5 (sequence MjTET) to compare the two sequences, allows to observe portions of sequence whose amino acid sequences are identical from one sequence to another.
• the proteins considered are those having an amino acid sequence whose percentage of identity with one of the sequences SEQ ID NO: 1 to SEQ ID NO: 5 is one of those described above and having a aminopeptidase activity, i.e. an amino acid cleavage activity by the N-terminus of the polymers.
• the dodecamérisation interface it is an area where the interactions are less well defined, it involves polar interactions, hydrophobic, hydrogen bonds and a salt bridge.
• the presence of the a5 helix at the interface between the monomers in a suitable conformation associated with the presence of residues with a long side-chain loaded on the latter seems to be decisive.
• the active site belongs to the characteristics of a TET and peptidases of the family M42, it is defined by the residues following: H65XD67, D181, E213E214, E / D236 and H319 in PhTET3 and implies the coordination of two metal ions. These ions are, in general, Co 2+ or Zn 2+ ions, but some structures have been characterized with different ions, the structure of Q11 Z05_CYTH3 has for example been characterized with active sites carrying Fe 2+ ions.
• said first aminopeptidase represents up to 10% by weight relative to the total weight of the composition, in particular up to 5% by weight relative to the total weight of the composition.
• said first aminopeptidase represents 50% by weight relative to the total weight of the composition, provided that said first and second aminopeptidases are different from PhTET2 and PhTET3.
• these two TET aminopeptidases may be in equimolar proportions, provided that said first and second aminopeptidases are different from PhTET2 and PhTET3.
• the composition of the invention comprises at least one third aminopeptidase, said third aminopeptidase being an aminopeptidase of the family of tetrahedral aminopeptidases or TET aminopeptidases.
• the triplets may be chosen from the list of triplets consisting of: PhTET 1 -PhTET2-PhTET3, PhTET1-PhteT2-PhTET4, PhTET1-PhteT2-MjTET, PhTET 1-PhteT3-PhTET4, PhTET 1 -PhTET3-MjTET, PhTET1 -PhTET4-MjTET, PhTET2-PhTET3-PhTET4, PhTET2-PhTET3-MjTET, PhTET2-PhTET4-MjTET, PhTET3-PhTET4-MjTET.
• the quadruplets may be chosen from the list of quadruplets consisting of: PhTET 1 -PhTET2-PhTET3-PhTET4, PhTET 1 -PhTET2-PhTET3-MjTET, PhTET2-PhTET3-PhTET4 -MjTET, PhTET 1 -PhTET3-PhTET4-MjTET.
• the quintuplet may be PhTETI-PhTET2-PhTET3-PhTET4-MjTET.
• the first TET aminopeptidase can represent up to 40% by weight relative to the total weight of the composition, the at least 60% by weight relative to the total weight of the remaining composition that can be distributed between the second TET aminopeptidase and the third aminopeptidase TET.
• the composition may be in such proportions that the first aminopeptidase TET represents up to 50% by weight relative to the total weight of the composition, less than 50% by weight relative to the total weight of the remaining composition that can be distributed between the second aminopeptidase TET and the third aminopeptidase TET.
• the composition consists of a first TET aminopeptidase, a second TET aminopeptidase and a third TET aminopeptidase, and according to which the proportions by weight relative to the total weight.
• the proportions by weight relative to the total weight. of the composition of the first, second and third aminopeptidases are the following: 50/25/25, 40/30/30, 40/40/20, 10/10/80 or 10/20/70.
• said first, second and third aminopeptidases are in equimolar or substantially equimolar proportions.
• equimolar or substantially equimolar proportions it is meant that the quantities of the first, second and third aminopeptidases are the same or substantially the same, ie they are very close to each other. Such precision makes it possible to precisely define the aminopeptidase activity of a composition and thus to better adapt to the peptide to be degraded.
• the composition further comprises an endopeptidase, especially thermolysin, in particular thermolysin of sequence SEQ ID NO: 6.
• Thermolysin is an endopeptidase belonging to the family of metalloproteinases. Thermolysin is the most stable member of a family of metalloproteinases produced by various species of Bacillus. Unlike many proteins that undergo conformational changes during heating and denaturation, thermolysin undergoes no major conformational changes up to at least 70 ° C. Thermolysin therefore remains stable and active at temperatures where TET proteins are the most active.
• endopeptidase an enzyme capable of cleaving the bonds between non-terminal amino acids of a peptide, a polypeptide or a protein.
• composition has a broader aminopeptidase activity thanks to the possibility of cleaving non-terminal amino acids.
• the invention also relates to a use of a composition according to the invention for modifying all or part of the polypeptide content of a substrate comprising peptides, polypeptides and / or proteins.
• modification of all or part of the polypeptide content of a substrate is meant at least one modification of a portion of the polypeptide content of a substrate.
• the polypeptide content of the substrate is different from the polypeptide content of the product obtained after contacting with the composition according to the invention.
• the modification of all the contents corresponds to the integral degradation of the three proteins.
• the modification of part of this content may correspond to the integral degradation of one of the three proteins or to the partial modification of a protein, of two proteins out of the three, or even of the three proteins of the substrate.
• Those skilled in the art are able to determine whether a protein content is fully or partially modified.
• composition according to the invention is used to modify all or part of the polypeptide content of a substrate which comprises peptides, polypeptides and / or proteins.
• a substrate which comprises peptides, polypeptides and / or proteins.
• These peptides, polypeptides and / or proteins undergo the action of TET aminopeptidases forming all or part of the composition and are thus modified into short peptides, which allows the elimination of their toxic part and a better digestion of the peptide content of the peptide. product obtained after contacting the polypeptide content of the substrate with the composition.
• the substrate comprises at least peptides, polypeptides and / or proteins of gluten and / or whey.
• composition according to the invention modifies the peptides, polypeptides and / or proteins of gluten or whey, these two protein assemblies comprising peptides, polypeptides and / or proteins. which are the cause of food intolerances and allergies for end consumers.
• gluten consists mainly of two families of proteins: gliadins and glutenins. These proteins are insoluble in water and give to the dough, obtained after rehydration of the flour, viscoelastic properties exploited in the agro-food field to give a certain structure to the products.
• Whey contains most of the milk's water. It consists of 94% water, 4 to 5% lactose, soluble protein (9% dry matter), and mineral salts.
• Whey proteins have a real nutritional value because of their high composition of essential amino acids. The most important are b-lactoglobulin (b-LG), a-lactalbumin (a-LA), bovine immunoglobulin (IgG), bovine serum albumin (BSA) and bovine lactoferrin (LF).
• b-LG b-lactoglobulin
• a-LA a-lactalbumin
• IgG bovine immunoglobulin
• BSA bovine serum albumin
• LF bovine lactoferrin
• the substrate comprises at least one of the following proteins: gliadin, b-lactoglobulin, Ga-lactalbumin, immunoglobulins, serum albumin and lactoferrin.
• composition according to the invention allows the modification of these proteins which are the cause of intolerances and food allergies for end consumers.
• said aminopeptidases forming all or part of the composition are used simultaneously, separated or spread over time.
• aminopeptidases simultaneously, separated or spread over time, allows the adaptation of the overall activity of the composition to the polypeptide content of the substrate on which the composition is used. Indeed, it is possible to use the various aminopeptidase activities of the composition, functions among others aminopeptidases, their proportion and their interaction with each other in terms of activity, in order to adapt the action of modifying the substrate polypeptide content, in the weather.
• the aminopeptidases forming the composition can be used simultaneously. As such, they may, for example, be added at the same time in a medium comprising the substrate to be modified. Aminopeptidases can also be used separately.
• a portion of the aminopeptidases may be added to one end of the container while another portion is added to another end of the container.
• Such a solution can be envisaged with the aim of obtaining a homogeneous distribution within the container of the added aminopeptidases more rapidly.
• This solution can also be considered when an aminopeptidase introduced at one end of the container requires a certain period of adaptation within the medium before being operational for bringing it into contact with other aminopeptidases added at another end of the container.
• Aminopeptidases can also be used over time. Thus, it is possible to add aminopeptidase for a period of time to modify the polypeptide content of the substrate.
• the TET proteins may be attached to a support, in particular a column, silica or magnetic beads, or any other suitable support for the attachment of proteins.
• the enzymes may be used in the form of cross-linked enzyme crystals or CLECs (for cross-linked enzymes crystals).
• the invention therefore relates to a composition in which one or more aminopeptidases, preferably all the aminopeptidases of the composition, are in the form of crosslinked crystals.
• the invention thus relates to a composition in which one or more aminopeptidases, preferably all of the aminopeptidases of the composition, are immobilized, in particular in the form of crosslinked crystals.
• the invention also relates to a method for modifying all or part of the polypeptide content of a substrate comprising peptides, polypeptides and / or proteins, said method comprising a contacting step: • of said substrate with
• said at least one first aminopeptidase and said at least one second aminopeptidase being capable of being activated at a temperature above 80 ° C, and optionally comprising, prior to said contacting step, a step of denaturing the polypeptides of said substrate.
• contacting step is meant a step in which the polypeptide content of the substrate and the TET aminopeptidases of the composition can interact with each other, with a view to modifying the polypeptide content of the polypeptide. substrate.
• a first aminopeptidase and said at least second aminopeptidase that can be activated at a temperature greater than 80 ° C is meant that the aminopeptidases can pass from a stage in which they do not cleave the amino acids of the peptides, polypeptides and / or substrate proteins at a stage where they cleave these amino acids.
• the polypeptides of the substrate may previously undergo a denaturation step, for example propanol denaturation, which has the effect of promoting their modification by the aminopeptidases of the composition.
• a denaturation step for example propanol denaturation
• These polypeptides are those which naturally have a tertiary structure. Therefore, organic solvents such as propanol causes destruction of non-covalent bonds, for example the internal hydrogen bonds of the polypeptides. Such bonds stabilize the tertiary structure of the polypeptides. Such destruction therefore causes destabilization, unfolding, or even denaturation of the polypeptides.
• the invention also relates to a food compound that can be obtained by the method of the invention.
• the invention also relates to a food compound comprising at least one of the following proteins in modified form: gliadin, b-lactoglobulin, Ga-lactalbumin, immunoglobulins, serum albumin and lactoferrin, said food compound further comprising a composition according to the invention. 'invention.
• FIGS. 1A, 1B and 1C are superimpositions of chromatograms resulting from reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of synthetic peptide 2 incubated for 15 min with PhTET3 (FIG. 1 A and curves A) and PhTET2 (FIG. 1B and curves C).
• the control sample ie the peptide incubated without peptidase, is represented in curve B.
• FIG. 1C represents the superposition of the 3 chromatograms;
• FIGS. 2A and 2B are superimpositions of the chromatograms resulting from the reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of synthetic peptide 4 incubated for 15 min with PhTET4 (FIG. 2A) and synthetic peptide 1 incubated for 15 min. min with PhTETI ( Figure 2B);
• RP-HPLC reverse phase chromatography
• FIGS. 3A, 3B, 3C and 3D are superimpositions of chromatograms resulting from reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of the synthetic peptide incubated for 15 min with PhTET3 (FIG. 3A and curves A) with MjTET (FIG. 3B and curves B) or with a mixture of 10% PhTET3 / 90% MjTET (FIG. 3C and curves C).
• the control sample ie the peptide incubated without peptidase, is represented at the curves D.
• FIG. 3D represents the superposition of the 4 chromatograms;
• FIGS. 4A, 4B, 4C and 4D are chromatogram overlays resulting from analysis by reverse phase chromatography (RP-HPLC) of the hydrolysis of the synthetic peptide incubated for 15 min with PhTET3 (FIG. 4A and curves A) with MjTET (FIG. 4B and curves B) or with a mixture of 5% PhTET3 / 95% MjTET (FIG. 4C and curves C).
• the control sample ie the peptide incubated without peptidase, is represented at the curves D.
• FIG. 4D represents the superposition of the 4 chromatograms;
• FIGS. 5A, 5B and 5C are chromatogram overlays resulting from reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of the synthetic peptide incubated for 15 min at different temperatures at 40.degree. A) or 60 ° C (curves B) with PhTET3 ( Figure 5A) with MjTET ( Figure 5B) or with a mixture of 5% PhTET3 / 95% MjTET ( Figure 5C).
• the control sample ie the peptide incubated without peptidase, is not visible in FIGS. 5A, 5B and 5C;
• FIGS. 6A, 6B and 6C are superimpositions of chromatograms resulting from reverse phase chromatography (RP-HPLC) analysis of the kinetics of the hydrolysis of the synthetic peptide 7 incubated for 5, 15 or 30 min with PhTET4 (FIG. 6A), with MjTET (FIG. 6B) or with a mixture of 50% PhTET4 / 50% MjTET (FIG. 6C).
• Curves A 5 min
• curves B 15 min
• curves C 30 min.
• the control sample that is the peptide incubated without peptidase, is represented in curves D;
• FIG. 7 represents a superposition of chromatograms resulting from the reverse phase chromatography (RP-HPLC) analysis of the kinetics of the hydrolysis of the synthetic peptide 7 incubated for 30 min with MjTET (curve A) or with a mixture of 50 % of PhTET4 / 50% of MjTET (curve B). The sample controls either the peptide incubated without peptidase, is shown in curve C;
• FIG. 8 represents a chromatogram resulting from the analysis of the whey protein hydrolyzate by reverse phase chromatography (RP-HPLC);
• Figure 10A shows incubation with PhTET2 alone (curve A).
• Figure 10B shows the incubation with PhTET3 alone (curve B).
• Figure 10C shows the incubation with PhTET2 and PhTET3 in equimolar amount (curve C);
• FIG. 11 represents a superposition of the chromatograms resulting from the analysis of the whey protein hydrolyzate after incubation with PhTET2 and PhTET3 in equimolar quantity (A curves) compared with different TET aminopeptidase compositions.
• Figure 11A shows incubation with PhTET2 alone (curve B) and a mixture of 90% PhTET2 and 10% PhTET3 (curve B1).
• Figure 11B shows the incubation with PhTET3 alone (curve C) and a composition of 10% PhTET2 and 90% PhTET3 (curve C1).
• Figure 11C shows the incubation with a composition of 90% PhTET2 and 10% PhTET3 (curve B1) and with a composition of 10% PhTET2 and 90% PhTET3 (curve C1);
• FIG. 13A represents the incubation with PhTET4 alone (curve A).
• Figure 13B shows the incubation with MjTET alone (curve B).
• Figure 13C shows the incubation with the composition of PhTET4 and MjTET in equimolar amount (curve C);
• FIG. 14 represents a superposition of the chromatograms obtained in reverse phase HPLC (ZORBAX SB-300 C18 column).
• Curve A sample of whey incubated in the presence of thermolysin.
• Curve B sample of whey incubated in the presence of thermolysin and TET aminopeptidases.
• Curve C whey sample incubated alone. The column used here makes it possible to analyze small peptides, so the "whole" proteins of the pup are not visible.
• FIG. 15 shows a superposition of the chromatograms resulting from the reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of the synthetic peptide 7 incorporated in a casein hydrolyzate incubated with PhTET3 (FIG.
• RP-HPLC reverse phase chromatography
• FIG. 16 represents a superposition of the chromatograms resulting from the analysis of gluten samples: non-incubated control (curve A), incubated without enzymes (curve B), incubated with the PhTETI, PhTET2 and PhTET3 enzymes in equimolar quantity (line C). After 2 hours of incubation, we note the decrease of several peaks of absorbance reflecting the significant degradation of several gluten proteins;
• FIG. 17 represents a superposition of the chromatograms resulting from the reverse phase chromatography (RP-HPLC) analysis of the hydrolysis of gluten samples.
• Curve A sample of total gluten incubated in the presence of thermolysin.
• Curve B total gluten sample incubated in the presence of thermolysin and TET PhTETI, PhTET2 and PhTET3 aminopeptidases.
• Fig. 18 shows a superposition of the chromatograms resulting from RP-HPLC analyzes of the control samples of the new whey protein hydrolyzate used for each mix.
• Fig. 19 shows a superposition of the chromatograms resulting from RP-HPLC analysis of the samples after hydrolysis of the new whey protein hydrolyzate by different mixture of TET (mix 1 to 5).
• FIG. 20 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by the mixture # 1 (mix 1) of TET (70% PhTET2, 15% PhTET3, 15% PhTET4).
• FIG. 21 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by the mixture # 2 (mix 2) of TET (70% PhTET 2, 15% PhTET4, 15% MjTET).
• FIG. 22 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by the mixture # 3 (mix 3) of TET (90% MgTET, 10% PhTET4).
• FIG. 23 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by the mixture # 4 (mix 4) of TET (70% MgTET, 15% PhTET 1, 15% PhTET3).
• FIG. 24 represents a superposition of the chromatograms resulting from the analysis in RP-HPLC of hydrolysis of peptides of whey protein hydrolyzate by mixture # 5 (mix 5) of TET (70% MjTET, 15% PhTET3, 15% PhTET4).
• FIG. 25 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of the peptides of the whey protein hydrolyzate by the mixture # 6 (TET4 - 1 or mix 6) of TET (100% PhTET4).
• FIG. 26 represents a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by mixtures # 1 and # 2 of TET.
• FIGS. 27 and 28 show a superposition of the chromatograms resulting from RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate mixtures # 1 and # 2 of TET, compared with PhTET2 ("TET 2 ").
• the standard is a compound that does not react in the experiment and whose response to the signal is very close to the measured products.
• the internal standard chosen is tryptophan.
• the internal standard is added to the concentration indicated in the sample prior to its RP-HPLC analysis.
• the reaction medium is then incubated for various times at the desired temperatures (40 ° C. or 60 ° C.) with stirring (500 rpm).
• the tubes are then placed in ice to stop the hydrolysis reaction.
• reaction medium 80 ⁇ l of the reaction medium are added to 320 ⁇ l of a solution composed of 2% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA).
• ACN 2% acetonitrile
• TFA trifluoroacetic acid
• the samples are then centrifuged at 10,000 g for 10 min before being transferred to vials prior to injection on an RP-HPLC column for analysis.
• the reaction media of these activity tests were also analyzed by mass spectrometry in order to precisely identify the size of the observed hydrolysis products. This made it possible to identify the different peaks observed on the chromatograms and to optimally follow the hydrolysis processes.
• Whey represents the liquid fraction obtained after coagulation of milk, it is a by-product obtained especially in the cheese industry. It contains about 10% of protein which is divided into 5 main families: b-lactoglobulin (50%), Galactalbumin (20%), immunoglobulins (10%), bovine serum albumin (10%) and lactoferrin (2.8%). In the present case, these different proteins are hydrolysed and the resulting peptides are used as a model substrate.
• a cow's milk solution is incubated in the presence of thermolysin (Sigma®) at a final concentration of 100 ⁇ g / ml for 2 h at 60 ° C. with shaking (500 rpm). After hydrolysis, the solution is incubated for 15 min at 95 ° C to inactivate the thermolysin. The whey protein hydrolyzate is then aliquoted and stored at -20 ° C until use.
• reaction medium 80 ⁇ l of the reaction medium are added to 320 ⁇ l of a solution composed of 2% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA).
• ACN 2% acetonitrile
• TFA trifluoroacetic acid
• phase A is composed of 0.1% TFA and 2% ACN in In the case of water, phase B contains 0.1% TFA and 80% ACN in water.
• the adsorbed proteins are then eluted at 1 mL / min with a linear 0-50% B phase gradient and are detected by measuring their absorbances at 280 nm for peptide studies or 214 nm for other studies. Protein peaks are identified and analyzed using TotalChrom software version 6.3.1 (Perkin Elmer®).
• the TET enzymes used in these experiments are metalloamino peptidases of the M42 family (MEROPS). They all belong to the same enzyme family and have a very strong structural identity in them. On the other hand, they are different enzymes with different specificities.
• PhTETI is a glutamyl aminopeptidase
• PhTET2 is a leucyl aminopeptidase with significant residual activity towards some of the unfilled hydrophobic and polar residues
• PhTET3 is a lysyl aminopeptidase
• PhTET4 is a strict glycyl aminopeptidase
• MjTET is a leucyl aminopeptidase with significant activity towards hydrophobic and positively charged residues.
• MjTET is also the only one to have hydrolysis activity towards aromatic residues.
• Peptide 4 enriched with wisteria
• Table 1 List of synthetic peptides used during the study.
• Each of these peptides carries an enriched N-ter end. Then follows 4 residues which compensate for the effect of the enriched zone (in particular for the solubility of the peptide).
• the C-terminal end is conserved and has the same series of 7 residues chosen for their absorbance at 280 nm and their solubility: YTSWNSE (SEQ ID NO: 7).
• the aminopeptidase PhTET3 has an optimal activity against positively charged residues, it is classified as lysyl-aminopeptidase having a residual activity against leucine, methionine, glutamine and aspartate. However, during the tests carried out on the synthetic peptides, an important activity of PhTET3 was observed on the peptide enriched in hydrophobic residues, the peptide 2 (FIG. 1A).
• PhTET3 has a greater activity on the peptide enriched in hydrophobic residues than PhTET2 ( Figure 1 C). Indeed, during the 15 minutes of the activity test, the first residues of the peptide were hydrolyzed more rapidly in the presence of PhTET3 than in the presence of PhTET2.
• TET aminopeptidase compositions show the possibility offered by the compositions according to the invention in terms of hydrolysis modulation, allowing the appropriate case to modify the peptides in a fashion. substrates rather than destroying them completely.
• the hydrolysis of the first residue of the substrate peptide is accelerated by the addition in the composition of a peptidase which, again, did not exhibit optimum activity for it. It is noted that by specifically modulating the type of TET aminopeptidase, their ratio, the temperature or the pH, the modification method makes it possible to enrich a peptide mixture in one of the observed hydrolysis intermediates (in this case peptide pep-2 enriched at 15 min in Figure 6B).
• the whey represents the liquid fraction obtained after coagulation of the milk. It contains approximately 10% of protein which is divided into 5 major families: 3-lactoglobulin (50%), Ga-lactalbumin (20%), immunoglobulins (10%), bovine serum albumin (10%) and lactoferrin (2.8%).
• the substrate used in the tests that follow is a whey protein hydrolyzate whose preparation is explained above.
• it is analyzed by reverse phase chromatography on an HPLC system.
• the chromatogram resulting from the analysis of the control hydrolyzate is shown in FIG.
• PhTET2 / PhTET3 Compositions 50/50%, 90/10% and 10/90%)
• PhTET2 and PhTET3 are first measured when the aminopeptidases are used alone (FIG. 10). It is observed that not all peptides are supported by TET aminopeptidases, this being due to the fact that the two TET aminopeptidases used have different and marked substrate specificities. Since the peptide mixture used for this experiment is a complex mixture, differences in the degree of hydrolysis of the different peptides supported by the aminopeptidases are also observed. Only part of the chromatogram is shown for clarity, but the results presented are observable throughout the chromatogram.
• FIG. 12 the chromatograms obtained with 3 different ratios of PhTET2 and PhTET3 compositions.
• the "equimolar" mixture corresponding to 50% of each of the TETs, it is also visible in FIG. 10.
• the chromatograms shown in dashed lines correspond to different ratios of each TET aminopeptidase, 90% of the one and 10% of the other and vice versa. It is thus noted that the hydrolysis profiles are modified, meaning that a variation in the proportions of each TET aminopeptidase in the mixture modifies the degree of hydrolysis of the peptides in solutions.
• a superposition of all the different chromatograms is presented in FIG. 12 and makes it possible to clearly visualize the possible modulation in the hydrolysis of the peptides.
• the whey was brought to 4 5 C before being fractionated into 1 ml samples stored at -20 ° C.
• thermolysin After incubation with thermolysin (FIG. 14), a large number of small peptides present in the sample are observed as a result of the hydrolysis of whey proteins by thermolysin.
• thermolysin and TET the vast majority of these peptides was degraded.
• enrichment of a few peptides which represent the degradation products related to the activity of TET aminopeptidases was remarkable to note.
• the synthetic peptide 7 was incorporated into a complex peptide mixture, a casein hydrolyzate (Sigma). After incubation with a composition of aminopeptidases PhTET3, MjTET and PhTET4, the reaction medium was analyzed by RP- HPLC. The results of the various experiments carried out are presented in FIG. 15. The peptide can be identified without ambiguity and its first stages of degradation could be observed. The complexity of the environment has made the analysis of short fragments more complex.
• the peptide of interest intact pep-0 is also totally absent when the peptide mixture is incubated in the presence of the composition of the three TET aminopeptidases (FIG. 15D).
• the pep-1 peptide itself has been hydrolysed since its concentration has decreased significantly compared to the experiment with PhTET4 alone.
• PhTET1 / PhTET2 / PhTET3 composition (33/33/33%)
• gluten is a mixture of different proteins classified into two major families, glutenin and gliadins. Some gliadins carry a peptide called "immunodominant" that causes an allergic reaction in people sensitive or intolerant to gluten, this syndrome is better known as celiac disease.
• composition of the PhTETI, PhTET2 and PhTET3 aminopeptidases alone, that is to say without the addition of endopeptidase, made it possible to reduce the concentration of the proteins carrying the immunodominant peptide in a sample of total gluten solubilized in a 50% solution of propanol.
• PhTET 1 / PhTET2 / PhTET3 / thermoiysin composition After incubation of the gluten with the endoprotease thermolysin, there is shown in Figure 17 a large number of small peptide in the sample resulting from the highly efficient hydrolysis of gluten proteins by the endoprotease.
• PhTETI, PhTET2 and PhTET3 aminopeptidases are integrated into the composition, a decrease of the vast majority of the absorbance peaks is noted, which reflects a hydrolysis of all these peaks by the aminopeptidases.
• reaction medium 80 ⁇ l of the reaction medium are added to 320 ⁇ l of a solution composed of 2% acetonitrile (ACN) and 0.1% trifluoroacetic acid (TFA).
• ACN 2% acetonitrile
• TFA trifluoroacetic acid
• Phase A is composed of 0.1% TFA and 2% ACN in water
• phase B contains 0.1% TFA and 80% ACN in water.
• the adsorbed proteins are then eluted at 1 mL / min with a linear 0-50% B phase gradient and are detected by measuring their absorbances at 280 nm for peptide studies or 214 nm for other studies. Protein peaks are identified and analyzed using TotalChrom software version 6.3.1 (Perkin Elmer®). The "windows" of the chromatograms (FIGS.
• Figures 18 to 28 show a superposition of the chromatograms resulting from RP-HPLC analyzes.
• This whey protein hydrolyzate is incubated with different mixtures of TET.
• the superposition of the chromatograms resulting from RP-HPLC analysis of the samples after hydrolysis of the new whey protein hydrolyzate by different TET mixtures are shown in FIG. 19.
• FIGS. 20 to 28 are shown various chromatograms obtained after RP-HPLC analysis of the reaction media after the hydrolysis by the different mixtures. from TET.
• the legend of the mix number and its composition is indicated in the legend.
• FIG. 26 represents one of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of peptides of the whey protein hydrolyzate by mixtures # 1 and # 2 of TET (Mix # 1: 70% PhTET2, 15% PhTET3, 15% PhTET4, Mix # 2: (70% PhTET2, 15% PhTET4, 15% MjTET).
• PhTET2 The majority peptidase here is PhTET2, present in both mixes up to 70%. This predominant presence of a peptidase explains the similarity that can be observed between chromatograms. Although very similar, they are not identical. The only difference here is the presence in one case of PhTET3 and in the other of MjTET.
• PhTET3 and MjTET peptidases have a similar behavior in peptide context. It appears here that there are still significant differences and that these two peptidases can not be used one in place of the other.
• the major peptidase here PhTET2 strongly influences the general peptide profile. This is very clear in the present case, especially from FIGS. 27 and 28 with a superposition of the chromatograms resulting from the RP-HPLC analysis of the hydrolysis of the peptides of the whey protein hydrolyzate by the mixtures # 1 and # 2 of TET (Mix # 1: 70% PhTET2, 15% PhTET3, 15% PhTET4, Mix # 2: (70% PhTET2, 15% PhTET4, 15% MjTET) compared each respectively all at PhTET2), since in both cases, the peptide profile after hydrolysis with the peptidase alone is very close to that obtained after hydrolysis by the mixture of peptidases.
• Crystals of the PhTET3 protein were obtained using the hanging drops method on ComboPlate 24-well plates of the Greiner Bio-One brand.
• 1 mL of mother liquor is deposited in the well of the crystallization plate, the drop is formed on silanized coverslips by mixing 1.5 ⁇ L of protein solution PhTET3 concentrated at 20 ° C. mg / mL and 1.5 ul of mother liquor.
• a crosslinking solution is prepared from the mother liquor implemented with final 1% glutaraldehyde (v / v). Drops of 1 ⁇ l of crosslinking solution are deposited on silanized lamellae, the different crystals obtained earlier are then transferred into these drops. The crosslinking is obtained by incubation for 1 night, the crosslinked crystals are then harvested and placed in drops of the initial mother liquor pending their use. The crosslinked crystal obtained has a larger dimension of at least 0.5 mm.
• a chromogenic substrate in this case Lys-pNA.
• This substrate has been selected in particular because the PhTET3 enzyme has a maximum of activity against the amino acid lysine.

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