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WO2023149512A1 - Préparation enzymatique - Google Patents

Préparation enzymatique Download PDF

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Publication number
WO2023149512A1
WO2023149512A1 PCT/JP2023/003428 JP2023003428W WO2023149512A1 WO 2023149512 A1 WO2023149512 A1 WO 2023149512A1 JP 2023003428 W JP2023003428 W JP 2023003428W WO 2023149512 A1 WO2023149512 A1 WO 2023149512A1
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WO
WIPO (PCT)
Prior art keywords
protein
component
enzyme preparation
deamidase
enzyme
Prior art date
Application number
PCT/JP2023/003428
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English (en)
Japanese (ja)
Inventor
杏匠 酒井
Original Assignee
天野エンザイム株式会社
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Publication of WO2023149512A1 publication Critical patent/WO2023149512A1/fr

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Classifications

    • 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
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/52—Adding ingredients
    • A23L2/66—Proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Preparation or treatment thereof
    • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/132—Amines having two or more amino groups, e.g. spermidine, putrescine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
    • 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)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/14—Prodigestives, e.g. acids, enzymes, appetite stimulants, antidyspeptics, tonics, antiflatulents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • 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)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • 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
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the present invention relates to an enzyme preparation of protein deamidase with excellent thermostability.
  • Protein deamidase is used as a food additive enzyme, and utilizes the action of converting glutamine residues in food proteins into glutamic acid residues to improve the solubility, emulsification properties, Improves physical functions such as foam properties and gelation properties. Therefore, protein deamidase is expected to be used in various applications including foods and beverages (Patent Document 1). In particular, with the recent rise in health consciousness, the consumption of plant proteins is accelerating, and protein deamidase has a high potential value in plant protein processing applications. On the other hand, protein deamidase has a problem of poor thermostability (Non-Patent Document 1).
  • an object of the present invention is to provide an enzyme preparation of protein deamidase with improved thermostability.
  • Section 1 An enzyme preparation comprising (A) a protein deamidase and (B) a guanidinium compound and/or a polyamine.
  • Section 2. Item 2. The enzyme preparation according to Item 1, wherein the guanidinium compound is arginine and/or argininamide.
  • Item 3. Item 3. The enzyme preparation according to Item 1 or 2, wherein the polyamine is spermidine.
  • Section 4. Item 4. The enzyme preparation according to any one of Items 1 to 3, wherein the content of the component (B) per 1 U of the component (A) is 0.5 ⁇ mol or more.
  • Item 5. Item 5.
  • Item 6. The enzyme preparation according to any one of Items 1 to 5, wherein the protein deamidase is protein glutaminase.
  • Item 7. Item 7. The enzyme preparation according to any one of Items 1 to 6, which is a liquid preparation.
  • Item 8. (P) A method for deamidating a protein, comprising the step of treating a protein with (A) a protein deamidating enzyme in the presence of (B) a guanidium compound and/or a polyamine to allow the deamidation reaction to proceed.
  • Item 10. Item 10. The production method according to Item 9, wherein the reaction mixture is prepared by mixing the (P) component with an enzyme preparation containing the (A) component and the (B) component.
  • Item 11. Item 10. The production method according to Item 9, wherein the reaction mixture is prepared by mixing a mixture containing the component (P) and the component (B) with the component (A).
  • Item 12. Item 12. The production method according to any one of items 9 to 11, wherein the deamidation reaction is carried out at a temperature higher than the optimum temperature of the component (A).
  • Item 13 The production method according to any one of items 9 to 12, wherein the component (P) is vegetable protein and/or animal protein.
  • Item 14 Deamidation reaction of the component (P) in a reaction mixture containing (P) a protein-containing raw material for food or drink or a pharmaceutical raw material, (A) a protein deamidase, and (B) a guanidium compound and/or a polyamine.
  • thermostability an enzyme preparation of protein deamidase with improved thermostability, a protein deamidation method in which the thermostability of protein deamidase is high, and deamidated protein preparation using the same A manufacturing method is provided.
  • the enzyme preparation of the present invention comprises (A) a protein deamidase (hereinafter also referred to as “(A) component”) and (B) a guanidium compound and/or polyamine (hereinafter referred to as “(B) component ”) is included.
  • A protein deamidase
  • B a guanidium compound and/or polyamine
  • Protein deamidase The enzyme preparation of the present invention contains protein deamidase as component (A).
  • Protein deamidase is an enzyme that deamidates the amide groups of glutamine and asparagine residues in proteins without breaking peptide bonds and cross-linking the protein.
  • the protein deamidase used in the enzyme preparation of the present invention includes the following. (1) Enzymes that deamidate glutamine residues in proteins and convert them to glutamic acid (eg, protein glutaminase). (2) Enzymes that deamidate asparagine residues in proteins and convert them to aspartic acid (eg protein asparaginase). (3) Enzymes that deiminate arginine residues in proteins and convert them to citrulline (eg, arginine deiminase, protein arginine deiminase, peptidyl arginine deiminase).
  • the deamidated protein has amphipathic properties and becomes an ideal surfactant, and greatly improves the protein's emulsifying power, emulsion stability, foamability and foam stability.
  • deamidation of proteins brings about improvements in various functional properties of proteins, and the uses of the proteins increase dramatically.
  • Deimination of arginine residues in a protein also increases the hydrophobicity of the protein and changes the higher-order structure of the protein.
  • protein deamidase More specific examples of protein deamidase include Chryseobacterium genus, Flavobacterium genus, Empedobacter ( Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus protein deamidase, Luteimicrobium genus disclosed in WO2015/133590, Protein deamidase from the genera Agromyces, Microbacterium, or Leifsonia.
  • Empedobacter Empedobacter genus, Sphingobacterium genus, Aureobacterium genus, or Myroides genus protein deamidase
  • Luteimicrobium genus disclosed in WO2015/133590
  • Protein deamidase from the genera Agromyces, Microbacterium, or Leifsonia As the component (A) in the enzyme preparation of the present invention, one of these protein deamidase enzymes may be used alone, or two or more of them may be used in combination.
  • protein deamidase enzymes derived from the genus Chryseobacterium, more preferably Chryseobacterium genus are preferred from the viewpoint of further enhancing the effect of improving thermal stability by coexistence with component (B).
  • Protein glutaminase from the genus more preferably protein glutaminase from the species Chryseobacterium proteolyticum.
  • the protein deamidase can be prepared from the culture solution of the microorganism from which the above protein deamidase is derived.
  • a specific preparation method includes a method of recovering the protein deamidase from the culture solution or cells of the above microorganisms.
  • the enzyme can be separated and/or purified after previously collecting the cells from the culture solution by filtration, centrifugation, or the like, if necessary.
  • the cells were collected from the culture solution in advance, and then crushed by pressure treatment, ultrasonic treatment, or the like to expose the enzyme.
  • the enzyme can then be isolated and/or purified.
  • known protein separation and/or purification methods can be used without particular limitation.
  • Various chromatographic methods using The separated and/or purified enzyme can be pulverized by a drying method such as freeze drying or vacuum drying.
  • the protein deamidase can be used in the form of an enzymatic agent formulated by pulverizing using a suitable excipient and/or drying aid in the drying method described above.
  • the protein deamidase can also be used in the form of an enzymatic agent formulated as a liquid product by adding an appropriate additive to the separated and/or purified enzyme and sterilizing it by filtration.
  • the content of component (A) in the enzyme preparation of the present invention is not particularly limited, and may be appropriately set to the extent that the enzymatic activity of component (A) can be utilized when the enzyme preparation of the present invention is used.
  • the deamidase activity is, for example, 1 U/mL or higher, preferably 10 U/mL or higher, more preferably 25 U/mL or higher, even more preferably 50 U/mL or higher, particularly preferably 90 U/mL or higher.
  • the upper limit of the content of component (A) is, for example, 10,000 U/mL or less, preferably 1,000 U/mL or less, more preferably 500 U/mL or less, even more preferably 200 U/mL or less, particularly preferably 110 U/mL or less. mL or less.
  • 1 unit (1 U) is defined as the amount of enzyme that liberates 1 ⁇ mol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate. .
  • the enzyme preparation of the present invention contains a guanidinium compound and/or polyamine as component (B).
  • Component (B) improves the thermal stability of component (A).
  • guanidinium compounds for use in the present invention include guanidine, arginine, and argininamide. One of these guanidinium compounds may be used alone, or two or more of them may be used in combination. Among these guanidinium compounds, arginine and argininamide are preferred, and argininamide is more preferred, from the viewpoint of further enhancing the effect of improving the thermal stability of component (A).
  • the polyamine is not particularly limited as long as it is a linear aliphatic hydrocarbon having two or more amino groups. From the viewpoint of further enhancing the thermal stability improvement effect of component (A), polyamine is preferably used as component (B).
  • polyamines used in the present invention include diamines, triamines, tetraamines, pentamines, hexaamines, more specifically putrescine, cadaverine, ethylenediamine, trimethylenediamine, hexamethylenediamine, spermidine, cardine, homospermidine.
  • One of these polyamines may be used alone, or two or more of them may be used in combination.
  • component (A) includes spermidine.
  • the content ratio of the (A) component and the (B) component in the enzyme preparation of the present invention may be appropriately set according to the desired degree of thermal stability improvement effect of the (A) component.
  • the content of component (B) per 1U of component (A) is, for example, 0.5 ⁇ mol or more, and from the viewpoint of further enhancing the thermal stability improvement effect of component (A), preferably 0.8 ⁇ mol or more, More preferably 4 ⁇ mol or more, and still more preferably 9 ⁇ mol or more.
  • the upper limit of the content of component (B) per 1 U of component (A) is not particularly limited, but is, for example, 50 ⁇ mol or less, preferably 30 ⁇ mol or less, more preferably 15 ⁇ mol or less.
  • the specific content of the component (B) in the enzyme preparation of the present invention is determined according to the content of the component (A) and the above ratio. From the viewpoint of further enhancing the effect of improving thermal stability, it is preferably 0.08 M or more, more preferably 0.4 M or more, and still more preferably 0.9 M or more.
  • the upper limit of the content of component (B) in the enzyme preparation of the present invention is not particularly limited, it is, for example, 5M or less, preferably 3M or less, and more preferably 1.5M or less.
  • the enzyme preparation of the present invention may contain other enzymes, additives, solvents, and/or culture residue components as components other than the above components (A) and (B). and may or may not contain
  • enzymes include, for example, amylase ( ⁇ -amylase, ⁇ -amylase, glucoamylase), glucosidase ( ⁇ -glucosidase, ⁇ -glucosidase), galactosidase ( ⁇ -galactosidase, ⁇ -galactosidase), protease (acid protease, medium protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase other than protein glutaminase, pectinase, catalase, dexamethasone stranase, transglutaminase, pullulanase and the
  • Additives include excipients, buffers, suspending agents, stabilizers, preservatives, preservatives, pH adjusters, and the like.
  • Excipients include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol and the like.
  • Buffers include phosphate, citrate, acetate and the like.
  • Stabilizers include propylene glycol, ascorbic acid and the like.
  • Preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben and the like.
  • antiseptics examples include ethanol, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like. These additives may be contained singly or in combination of multiple types. Solvents include water. The contents of these additives and solvent may be appropriately set according to the types of these components and/or the properties of the enzyme preparation.
  • the culture residue components include components contained in the medium used for culturing the component (A) or other enzyme-producing bacteria that are optionally blended, contaminant proteins and bacterial cell components produced by the culture, and the like. mentioned. These culture residue components may be contained singly or in combination of multiple types.
  • the properties of the enzyme preparation of the present invention are not particularly limited, and examples thereof include liquid and solid forms (powder, granules, etc.). Since the enzyme preparation of the present invention is excellent in the effect of improving the thermal stability of the component (A), it is possible to effectively obtain the effect of improving the thermal stability even in a liquid form, which is inherently unstable. can. From such a point of view, a preferred example of the properties of the enzyme preparation of the present invention is liquid (liquid preparation).
  • the pH (25°C) of the liquid preparation is, for example, 6.5 to 7.5, preferably 6.8 to 7.2, more preferably 6.9 to 7. .1 can be mentioned.
  • the enzyme preparation of the present invention can be used for any use that utilizes protein deamidase activity. That is, the enzyme preparation of the present invention suppresses changes in protein properties caused by deamidation of ⁇ -amide groups and/or ⁇ -amide groups of protein glutamine residues and/or asparagine residues to form carboxyl groups. It can be used for any protein modification application of your choice. Specific examples of protein modification include enhancement of protein solubility, enhancement of water dispersibility, enhancement of emulsifying power, enhancement of emulsion stability, and the like.
  • Preferred uses of the enzyme preparation of the present invention include uses in the food and pharmaceutical fields, and more preferred uses include uses in the food field. Specifically, it is used to improve the solubility, dispersibility, and emulsifiability of animal proteins and/or vegetable proteins under weakly acidic conditions, which is the pH range of normal foods (e.g., coffee whiteners, juices, etc.). production of acidic beverages, dressings, mayonnaise, and cream); increase in solubility and dispersibility of poorly soluble vegetable proteins (for example, production of tempura flour using wheat gluten); Modification applications (e.g. for producing crackers, biscuits, cookies, pizza or pie crusts); applications for removing or reducing allergens in allergenic proteins in foods (e.g.
  • use to reduce the mineral sensitivity of proteins increase the content of soluble minerals in liquids containing proteins and minerals, and increase the absorption of minerals by the human body (e.g., high mineral (e.g. calcium)-containing beverages, minerals (e.g. calcium) absorption enhancer manufacturing use); use to reduce bitterness, use to improve protease proteolytic rate, and/or use to enhance glutamic acid content (e.g., amino acid seasoning (animal protein hydrolyzate (HAP), hydrolyzate of vegetable protein (HVP)), production of miso and soy sauce), and the like.
  • HAP animal protein hydrolyzate
  • HVP hydrolyzate of vegetable protein
  • guanidinium compounds and/or polyamines can improve the thermal stability of protein deamidase.
  • coexistence of a guanidinium compound and/or polyamine allows the enzyme to act in a stable state.
  • (P) protein (hereinafter also referred to as "(P) component") is treated with (A) protein deamidase in the presence of (B) a guanidinium compound and/or polyamine. Also provided is a method for deamidating a protein, comprising the step of allowing the deamidation reaction to proceed.
  • the protein deamidation method of the present invention can be used to produce deamidated proteins. Therefore, according to the present invention, a reaction mixture containing (P) protein, (A) protein deamidase, and (B) a guanidium compound and/or polyamine proceeds with the deamidation reaction of component (P).
  • a method for producing a deamidated protein is also provided, comprising the step of allowing. Furthermore, the method for producing a deamidated protein of the present invention can be used for producing food, beverages or pharmaceuticals.
  • the present invention provides a reaction mixture containing (P) a protein-containing raw material for food or drink or a pharmaceutical raw material, (A) protein deamidase, and (B) a guanidium compound and/or polyamine, wherein (P)
  • the present invention also provides a method for producing food, drink or pharmaceuticals, including the step of allowing the deamidation reaction of the ingredients to proceed.
  • the method for deamidating a protein of the present invention and the method for producing a deamidated protein are collectively referred to as "the method of the present invention”.
  • the protein which is the component (P) to be deamidated in the method of the present invention, may be either vegetable protein or animal protein. In the method and production method of the present invention, either vegetable protein or animal protein may be used as the (P) component, or both may be used in combination.
  • the reaction mixture can be prepared by using the enzyme preparation described in the above "1. Enzyme preparation" and mixing the enzyme preparation with the component (P). can.
  • Enzyme preparation and mixing the enzyme preparation with the component (P).
  • the component (A) contained in the enzyme preparation has excellent thermal stability, the activity of the component (A) can be efficiently enjoyed.
  • the activity of the component (A) can be exhibited stably even under the heating conditions used in the step of advancing the deamidation reaction.
  • the reaction mixture can be prepared by mixing the (A) component with the mixture containing the (P) component and the (B) component.
  • the activity of the component (A) can be exhibited stably under the heating conditions used in the step of advancing the deamidation reaction.
  • the amount of component (A) used relative to component (P) can be the amount used in known methods without any particular limitation. Also, the ratio of the amount of component (A) and component (B) used may be appropriately set according to the desired degree of thermal stability improvement effect of component (A).
  • the amount of component (B) used per 1U of component (A) is, for example, 0.5 ⁇ mol or more. More preferably 4 ⁇ mol or more, and still more preferably 9 ⁇ mol or more.
  • the upper limit of the amount of component (B) to be used per 1U of component (A) is not particularly limited, but is, for example, 50 ⁇ mol or less, preferably 30 ⁇ mol or less, more preferably 15 ⁇ mol or less.
  • reaction conditions temperature, time, pH, etc.
  • Optimum treatment conditions may be determined through preliminary experiments.
  • the temperature conditions in the step of proceeding with the deamidation reaction can be appropriately set according to the optimum temperature of the component (A).
  • the temperature conditions may be set to a temperature higher than the optimum temperature for the component (A).
  • the temperature condition a temperature at which the component (A) is deactivated when the component (B) is not coexistent is acceptable.
  • the temperature conditions are, for example, 5 to 15° C., preferably 8 to 12° C. higher than the optimum temperature of component (A).
  • the method, etc. of the present invention can include steps other than the step of proceeding with the deamidation reaction.
  • the other steps are not particularly limited as long as they are suitable for the uses described in "1-5. Applications", and include, for example, an enzyme deactivation step, a cooling step, a filtration step, a cooking step, and the like. These other steps may be performed singly or in combination of two or more steps.
  • Protein deamidase Chryseobacterium proteolyticum-derived protein glutaminase (manufactured by Amano Enzyme) was used as the protein deamidase. This protein glutaminase has an optimum temperature of 60°C and is inactivated by treatment at 70°C for 1 hour. In the following, this protein deamidase is also referred to as "PG".
  • the protein deamidase activity value was measured by the following method. 0.1 mL of sample solution containing protein deamidase was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, left at 37° C. for 10 minutes, and then treated with 0.4 M TCA. 1 mL of solution was added to stop the reaction. As a blank, 1 mL of 0.4 M TCA solution was added to 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, and 0.1 mL of sample solution containing protein deamidase was added, It was left at 37°C for 10 minutes.
  • the amount of ammonia generated in the reaction solution was measured using Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • the ammonia concentration in the reaction solution was obtained from a calibration curve representing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).
  • the activity of protein deamidase was calculated from the following formula, with the amount of enzyme that generates 1 ⁇ mol of ammonia per minute as 1 unit (1 U).
  • the reaction liquid volume is 2.1
  • the enzyme solution volume is 0.1
  • Df is the dilution ratio of the enzyme solution.
  • 17.03 is the molecular weight of ammonia.
  • Enzyme solutions containing component (A) (PG) at a final concentration of 100 U/mL with or without component (B) were prepared.
  • the pH of the prepared enzyme solution at 25°C was adjusted to 7.0.
  • the enzyme solution was subjected to heat treatment at 70° C. for a predetermined time, and the degree of thermal aggregation of the enzyme was evaluated by measuring the absorbance at 413 nm. A higher absorbance indicates a higher extent to which the enzyme forms thermal aggregates, ie, a lower thermal stability.
  • Test Example 1 0.5 M of various amino acids were added to PG (final concentration: 100 U/mL) as component (A) to prepare an enzyme solution having a pH of 7.0 at 25°C. Separately, an enzyme solution (control; Ctrl) was prepared in the same manner except that no amino acid was added. Each enzyme solution was heat-treated at 70° C. for 15 minutes, then rapidly cooled to room temperature and the absorbance at 413 nm was measured. The results for each enzyme solution are shown in FIG.
  • the arginine-added groups (0.1 M Arg, 0.5 M Arg, and 1.0 M Arg) depended on the added arginine concentration. A decrease in absorbance value was confirmed. In other words, it was found from these results that arginine, which is the component (B), improves the thermal stability of PG.
  • Example 3 0.1 M of component (B) (arginine, argininamide, or spermidine) was added to component (A), PG (final concentration: 100 U/mL), and the solution was adjusted to pH 7.0 at 25°C. was prepared. Separately, an enzyme solution (control; Ctrl) was prepared in the same manner except that component (B) was not added. Each prepared solution was heat-treated at 60° C. for 10 minutes and the absorbance at 413 nm was measured. The results are shown in FIG.
  • the results show that the stability of PG was remarkably improved when using any of arginine, argininamide, and spermidine.
  • the absorbance value of the control is 100%
  • the relative absorbance of the 0.1M arginine-added section is 70%
  • the relative absorbance of the 0.1M arginine amide-added section is 62%
  • the relative absorbance of the 0.1M spermidine-added section is 70%.
  • Relative absorbance was 45%. In other words, the effect of improving the thermal stability of PG by spermidine was remarkable.

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  • General Chemical & Material Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Le but de la présente invention est de fournir un agent sous forme de protéine désamidase présentant une stabilité thermique améliorée. Un composé guanidinium et une polyamine peuvent améliorer la stabilité thermique d'une protéine désamidase.
PCT/JP2023/003428 2022-02-03 2023-02-02 Préparation enzymatique WO2023149512A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012060409A1 (fr) * 2010-11-05 2012-05-10 味の素株式会社 Procédé de fabrication de viandes transformées, et préparation d'enzymes pour l'amélioration de viandes transformées
WO2013012022A1 (fr) * 2011-07-19 2013-01-24 中外製薬株式会社 Préparation à teneur en protéines stable renfermant de l'argininamide ou un composé analogue correspondant
WO2015064591A1 (fr) * 2013-10-28 2015-05-07 テルモ株式会社 Suspension aqueuse de protéine
JP2021520815A (ja) * 2018-04-10 2021-08-26 グリーンライト バイオサイエンシーズ インコーポレーテッドＧｒｅｅｎｌｉｇｈｔ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ． Ｔ７ｒｎａポリメラーゼバリアント
WO2021201277A1 (fr) * 2020-04-03 2021-10-07 天野エンザイム株式会社 Procédé de désamidation d'une protéine

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012060409A1 (fr) * 2010-11-05 2012-05-10 味の素株式会社 Procédé de fabrication de viandes transformées, et préparation d'enzymes pour l'amélioration de viandes transformées
WO2013012022A1 (fr) * 2011-07-19 2013-01-24 中外製薬株式会社 Préparation à teneur en protéines stable renfermant de l'argininamide ou un composé analogue correspondant
WO2015064591A1 (fr) * 2013-10-28 2015-05-07 テルモ株式会社 Suspension aqueuse de protéine
JP2021520815A (ja) * 2018-04-10 2021-08-26 グリーンライト バイオサイエンシーズ インコーポレーテッドＧｒｅｅｎｌｉｇｈｔ Ｂｉｏｓｃｉｅｎｃｅｓ，Ｉｎｃ． Ｔ７ｒｎａポリメラーゼバリアント
WO2021201277A1 (fr) * 2020-04-03 2021-10-07 天野エンザイム株式会社 Procédé de désamidation d'une protéine

Non-Patent Citations (1)

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Title
KOVACEVIC Z, DAY S H, COLLETT V, BROSNAN J T, BROSNAN M E: "Activation of hepatic glutaminase by spermine", BIOCHEMICAL JOURNAL, PUBLISHED BY PORTLAND PRESS ON BEHALF OF THE BIOCHEMICAL SOCIETY., GB, vol. 305, no. 3, 1 February 1995 (1995-02-01), GB , pages 837 - 841, XP093083350, ISSN: 0264-6021, DOI: 10.1042/bj3050837 *

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