Liquid enzyme preparation

Abstract

The present invention provides a liquid enzyme preparation containing a protein deamidase and having excellent activity stability, the liquid enzyme preparation comprising the protein deamidase together with another enzyme.　A liquid enzyme preparation comprising a protein deamidase and a protease is excellent with respect to the stability of the activity of the protein deamidase.

Description

Liquid enzyme preparation

[0001] The present invention relates to a liquid enzyme preparation. More specifically, the present invention relates to a liquid enzyme preparation containing protein deamidase, which has excellent activity stability of protein deamidase.

[0002] As described in Patent Document 1 and the like, known enzymes that deamidate the γ-amide group and β-amide group of glutamine residues and asparagine residues in proteins include protein glutaminase derived from Chryseobacterium gleum JCM2410 strain and protein glutaminase derived from Chryseobacterium proteolyticum 9670 strain. Enzyme preparations of such protein deamidating enzymes are currently only commercially available in powder form.

[0003] In general, liquid enzyme preparations are more desirable than powders in terms of dusting and handling. For this reason, techniques for formulating enzyme preparations in liquid form have been investigated for various enzymes. For example, Patent Document 2 proposes a liquid enzyme preparation characterized by containing at least one milk protein cross-linking and / or modifying enzyme in a polyol-water suspension containing 25% to 100% (w / w) polyol and having a pH value in the range of 4.4 to 5.1. Specifically, Patent Document 2 discovers that transglutaminase has low stability at pH 5.2, and based on this finding, it discloses improved stability of a transglutaminase preparation in a glycerol-water suspension at pH 4.4 to 4.8 and a transglutaminase preparation in a sorbitol-water suspension at pH 4.6. Patent Document 2 also discloses a tyrosinase preparation in a glycerol-water suspension at pH 4.6 and a protein glutaminase preparation in a glycerol-water suspension at pH 4.6.

[0004] Furthermore, Patent Document 3 describes that a useful protein composition such as a stable interferon can be produced by adjusting the dissolved oxygen concentration to 3 mg / L or less in a useful protein composition containing a useful protein, a solvent, and a compound having a thiol structure by bubbling an asphyxiating gas such as nitrogen.

[0005] International Publication No. 2010 / 029685 International Publication No. 2013 / 064736 Japanese Patent Application Laid-Open No. 2013-049650

[0006] While Patent Document 2 examines the stability of liquid enzyme preparations of transglutaminase preparations, it only discloses the formulation of liquid enzyme preparations of protein deamidase, and does not fully examine the stability of this enzyme. Furthermore, Patent Document 3 examines the antiviral activity of aqueous preparations containing canine interferon-γ, but does not even mention or, naturally, examine liquid enzyme preparations of protein deamidase. Furthermore, some enzyme preparations are composed of a combination of multiple enzymes, but the stability of protein deamidase in liquid enzyme preparations containing protein deamidase together with other enzymes is not examined.

[0007] Therefore, an object of the present invention is to provide a liquid enzyme preparation containing protein deamidase together with other enzymes, which liquid enzyme preparation has excellent activity stability of protein deamidase.

[0008] As a result of extensive research, the present inventors have found that a liquid enzyme preparation containing protein deamidase has excellent stability of activity of protein deamidase even when it contains a protease.

[0009] Furthermore, the present inventors have found that in a liquid enzyme preparation containing protein deamidase and protease, the activity stability of protein deamidase is further improved when the preparation is placed in a container whose internal space is purged with an inert gas, when the upper limit of the protease content is limited to a predetermined amount, and / or when sorbitol is added in the presence of a predetermined amount or more of protease, and that excellent activity stability of protein deamidase can be obtained even when citrate is added in the presence of a predetermined amount or more of protease.

[0010] The present invention was completed based on these findings. That is, the present invention includes the following inventions.

[0011] Item 1. A liquid enzyme preparation comprising (A) a protein deamidase and (B) a protease. Item 2. The liquid enzyme preparation according to Item 1, wherein the liquid enzyme preparation is contained in a container whose internal space is replaced with an inert gas. Item 3. The liquid enzyme preparation according to Item 1 or 2, further comprising (C) sorbitol. Item 4. The liquid enzyme preparation according to any one of Items 1 to 3, further comprising (D) a citrate salt. Item 5. The liquid enzyme preparation according to Item 3, wherein the content of the component (C) is 30 w / v % or more. Item 6. The liquid enzyme preparation according to Item 3 or 4, wherein the content of the component (D) is 1 w / v % or more. Item 7. The liquid enzyme preparation according to any one of Items 1 to 6, wherein the pH is 5.5 or higher. Item 8. The liquid enzyme preparation according to any one of Items 1 to 7, wherein the pH is 8.2 or lower. Item 9. The liquid enzyme preparation according to any one of Items 1 to 8, wherein the content of the component (A) is 0.1 to 10,000 U / ml. Item 10. Item 11. The liquid enzyme preparation according to any one of Items 1 to 9, wherein the component (A) is a protein deamidase derived from Chryseobacterium proteolyticum. Item 12. The liquid enzyme preparation according to any one of Items 1 to 11, wherein the content of the component (B) is 0.01 to 2,000 U / mL. Item 13. The liquid enzyme preparation according to Item 12, wherein the bacterial protease is a protease derived from the genus Chryseobacterium. Item 14. The liquid enzyme preparation according to Item 12, wherein the bacterial protease is a protease derived from the genus Bacillus and / or Geobacillus. Item 15. The liquid enzyme preparation according to any one of Items 1 to 14, wherein the content of the component (B) per 100 U of the component (A) is 0.002 to 400 U.

[0012] According to the present invention, a liquid enzyme preparation having excellent stability of protein deamidase activity is provided.

[0013] 1 shows the relationship between the amount of protease added (additional activity) to a liquid enzyme preparation containing protein deamidase and the residual activity of protein deamidase in the liquid enzyme preparation.

[0014] [1. Liquid Enzyme Preparation] The liquid enzyme preparation of the present invention is characterized by containing (A) protein deamidase (hereinafter also referred to as "component (A)") and (B) protease (hereinafter also referred to as "component (B)"). The liquid enzyme preparation of the present invention will be described in detail below. Furthermore, the liquid enzyme preparation of the present invention may contain (C) sorbitol (hereinafter also referred to as "component (C)") and / or (D) citrate (hereinafter also referred to as "component (D)").

[0015] [1-1. (A) Protein Deamidase] The liquid enzyme preparation of the present invention contains protein deamidase as component (A). Protein deamidase is an enzyme that exhibits the action of degrading amide group-containing side chains of proteins without cleaving peptide bonds or cross-linking of proteins, and its type, origin, etc. are not particularly limited. Furthermore, as long as the above-mentioned action is its main activity, it may also have the action of degrading amide group-containing side chains of proteins that cleave peptide bonds and cross-linking of proteins. Examples of protein deamidase include enzymes that deamidate glutamine residues in proteins and convert them to glutamic acid (e.g., protein glutaminase), and enzymes that deamidate asparagine residues in proteins and convert them to aspartic acid (e.g., protein asparaginase). More specific examples of protein deamidating enzymes include those of the genus Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, Aureobacterium, etc., which are disclosed in JP2000-50887A, JP2001-218590A, and WO2006 / 075772A1. Examples of protein deamidating enzymes include protein deamidating enzymes derived from the genus Luteimicrobium, Agromyces, Microbacterium, or Leifsonia, as disclosed in WO 2015 / 133590; and commercially available protein glutaminases derived from the genus Chryseobacterium. These protein deamidating enzymes may be used singly or in combination of two or more.

[0016] Among these protein deamidating enzymes, from the viewpoint of obtaining a liquid enzyme preparation with even more excellent activity stability, protein deamidating enzymes derived from the genus Chryseobacterium are preferred, protein glutaminases derived from the genus Chryseobacterium are more preferred, and protein glutaminases derived from the species Chryseobacterium proteolyticum are even more preferred.

[0017] Protein deamidase can be prepared from a culture solution of a microorganism from which the above-mentioned protein deamidase is derived. Specific preparation methods include a method of recovering protein deamidase from the culture solution or cells of the above-mentioned microorganism. For example, when a protein deamidase-secreting microorganism is used, the cells can be recovered from the culture solution in advance by filtration, centrifugation, or the like, as necessary, and the enzyme can then be separated and / or purified. Furthermore, when a protein deamidase-nonsecreting microorganism is used, the cells can be recovered from the culture solution in advance by pressure treatment, ultrasonic treatment, or the like, as necessary, and the enzyme can then be separated and / or purified. The method for separating and / or purifying the enzyme can be any known protein separation and / or purification method, without any particular limitation, and examples thereof include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc.

[0018] The content of component (A) in the liquid enzyme preparation of the present invention is not particularly limited, and examples thereof include 0.1 to 10,000 U / mL, 1 to 5,000 U / mL, 10 to 3,000 U / mL, or 50 to 2,000 U / mL, preferably 100 to 1,500 U / mL, more preferably 200 to 1,000 U / mL, even more preferably 300 to 900 U / mL or 350 to 700 U / mL, and even more preferably 400 to 600 U / mL.

[0019] Regarding the activity of protein deamidase, the amount of enzyme that liberates 1 μmol of ammonia per minute using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as a substrate is defined as 1 unit (1 U).

[0020] [1-2. (B) Protease] The liquid enzyme preparation of the present invention contains a protease as component (B). In the present invention, the protease is not particularly limited in terms of origin, and examples thereof include bacterial proteases.

[0021] Examples of bacterial proteases include proteases derived from the genera Chryseobacterium, Bacillus, and Geobacillus.

[0022] The protease derived from the genus Chryseobacterium is not particularly limited as long as it can achieve the desired effects of the present invention. Specific examples of proteases derived from the genus Chryseobacterium include Chryseobacterium nematophagum, Chryseobacterium cucumeris, Chryseobacterium lactis, Chryseobacterium rhizoplane, Chryseobacterium joostei, Chryseobacterium shigense, Chryseobacterium proteolyticum, Chryseobacterium gleum, and Chryseobacterium soil. and the like. These proteases derived from the genus Chryseobacterium may be used singly or in combination of two or more. Among these proteases derived from the genus Chryseobacterium, proteases derived from the species Chryseobacterium proteolyticum are preferred from the viewpoint of further enhancing the effect of stabilizing the activity of protein deamidase.

[0023] The protease derived from the genus Chryseobacterium can be prepared by known methods. For example, it can be easily prepared by culturing bacteria of the genus Chryseobacterium and isolating the protease using known means, or by a method using genetic recombination technology.

[0024] Specific examples of proteases derived from the genus Bacillus (or Geobacillus) include Bacillus amyloliquefaciens, Bacillus cereus, Bacillus clausii, Bacillus intermedius, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus, and Bacillus subtilis. Examples of proteases that can be used include proteases derived from Bacillus genus (or Geobacillus genus), Bacillus subtilis, and Bacillus thermoproteolyticus, and these proteases derived from the genus Geobacillus. In the present invention, the above-mentioned proteases may be used alone or in combination of two or more types. Among these proteases derived from the genus Bacillus (or Geobacillus genus), from the viewpoint of further enhancing the effect of stabilizing the activity of protein deamidase, preferred are proteases derived from Bacillus licheniformis, proteases derived from Bacillus amyloliquefaciens, and these proteases derived from the genus Geobacillus.

[0025] The content of component (B) in the liquid enzyme preparation of the present invention is not particularly limited as long as it does not impair the effects of the present invention, and examples thereof include 0.01 to 10,000 U / mL or 0.01 to 5,000 U / mL.

[0026] From the viewpoint of further enhancing the activity stabilizing effect of protein deamidase, the content of component (B) in the liquid enzyme preparation of the present invention is preferably 0.01 to 3,500 U / mL, more preferably 0.01 to 3,000 U / mL or 0.01 to 2,500 U / mL, still more preferably 0.01 to 2,000 U / mL, 0.01 to 1,800 U / mL, 0.01 to 1,650 U / mL, 0.01 to 1,500 U / mL, 0.01 to 1,400 U / mL, 0.01 to 1,300 U / mL, 0.01 to 1,200 U / mL, 0.01 to 1,100 U / mL, 0.01 to 1,000 U / mL, 0.01 to 900 U / mL, 0.01 to 8 ...1,200 U / mL, 0.01 to 1,300 U / mL, 0.01 to 1,400 U / mL, 700 U / mL, 0.01 to 600 U / mL, 0.01 to 500 U / mL, 0.01 to 400 U / mL, 0.01 to 300 U / mL, 0.01 to 200 U / mL, or 0.01 to 150 U / mL, more preferably 0.01 to 100 U / mL, 0.01 to 80 U / mL, 0.01 to 75 U / mL, 0.01 to 70 U / mL, 0. Examples of the protease include 0.01 to 65 U / mL, 0.01 to 40 U / mL, 0.01 to 20 U / mL, 0.01 to 10 U / mL, or 0.01 to 5 U / mL (when the protease is derived from the genus Bacillus (or Geobacillus), it is even more preferably 0.01 to 20 U / mL, 0.01 to 10 U / mL, or 0.01 to 5 U / mL).

[0027] From the viewpoint of effectively obtaining the activity stabilizing effect of protein deamidase, the content of component (B) in the liquid enzyme preparation of the present invention is 0.1 to 2,000 U / mL, 0.5 to 2,000 U / mL, 1 to 2,000 U / mL, 5 to 2,000 U / mL, 10 to 2,000 U / mL, 15 to 2,000 U / mL, 20 to 2,000 U / mL, 30 to 2,000 U / mL, mL, 40-2,000 U / mL, 50-2,000 U / mL, 60-2,000 U / mL, 70-2,000 U / mL, 80-2,000 U / mL, 90-2,000 U / mL, 100-2,000 U / mL, 150-2,000 U / mL, 200-2,000 U / mL, 250-2,000 U / mL, or 300-2,000 U / mL.

[0028] Furthermore, from the viewpoint of enhancing the activity stabilizing effect of protein deamidase by incorporating the component (C), the content of the component (B) in the liquid enzyme preparation of the present invention is preferably 100 to 2,000 U / mL, 150 to 2,000 U / mL, 200 to 2,000 U / mL, 250 to 2,000 U / mL, or 300 to 2,000 U / mL.

[0029] The content of component (B) per 100 U of component (A) in the liquid enzyme preparation of the present invention is not particularly limited as long as it does not impair the effects of the present invention, but examples include 0.002 to 2,000 U or 0.002 to 1,000 U.

[0030] From the viewpoint of further enhancing the activity stabilizing effect of protein deamidase, the content of the component (B) per 100 U of the component (A) in the liquid enzyme preparation of the present invention is preferably 0.002 to 700 U, more preferably 0.002 to 600 U, or 0.002 to 500 U, still more preferably 0.002 to 400 U, 0.002 to 360 U, 0.002 to 330 U, 0.002 to 300 U, 0.002 to 280 U, 0.002 to 260 U, 0.002 to 240 U, ​​0.002 to 220 U, 0.002 to 200 U, 0.002 to 180 U, 0.002 to 160 U,

[0023] The amount of protease may be 0.002 to 140 U, ​​0.002 to 120 U, 0.002 to 100 U, 0.002 to 80 U, 0.002 to 60 U, 0.002 to 40 U, ​​or 0.002 to 30 U, and more preferably 0.002 to 20 U, 0.002 to 16 U, 0.002 to 15 U, 0.002 to 14 U, 0.002 to 13 U, 0.002 to 8 U, 0.002 to 4 U, 0.002 to 2 U, or 0.002 to 1 U (when the protease is derived from the genus Bacillus (or Geobacillus), the amount is even more preferably 0.002 to 4 U, 0.002 to 2 U, or 0.002 to 1 U).

[0031] From the viewpoint of effectively obtaining the activity stabilizing effect of protein deamidase, the content of the component (B) per 100 U of the component (A) in the liquid enzyme preparation of the present invention may be 0.02 to 400 U, 0.1 to 400 U, 0.2 to 400 U, 1 to 400 U, 2 to 400 U, 3 to 400 U, 4 to 400 U, 6 to 400 U, 8 to 400 U, 10 to 400 U, 12 to 400 U, 14 to 400 U, 16 to 400 U, 18 to 400 U, 20 to 400 U, 30 to 400 U, 40 to 400 U, 50 to 400 U, or 60 to 400 U.

[0032] Furthermore, from the viewpoint of enhancing the activity stabilizing effect of protein deamidase by incorporating the component (C), the content of the component (B) per 100 U of the component (A) in the liquid enzyme preparation of the present invention is preferably 20 to 400 U, 30 to 400 U, 40 to 400 U, 50 to 400 U, or 60 to 400 U.

[0033] Protease activity is measured by the Folin method using casein as a substrate. That is, protease activity is measured by carrying out an enzymatic reaction using casein as a substrate at a pH determined by a standard method according to the optimum pH of the protease to be measured, and one unit (1 U) is defined as the amount of enzyme that causes an increase in the Folin test solution color substance equivalent to 1 μg of tyrosine per minute.

[0034] [1-3. (C) Sorbitol] The liquid enzyme preparation of the present invention may contain sorbitol as component (C). There are no particular limitations on when component (C) may be blended into the liquid enzyme preparation of the present invention.

[0035] Component (C) is particularly preferred when the content of component (B) in the liquid enzyme preparation of the present invention is 100 to 2,000 U / mL, 150 to 2,000 U / mL, 200 to 2,000 U / mL, 250 to 2,000 U / mL, or 300 to 2,000 U / mL, or when the content of component (B) per 100 U of component (A) is 20 to 400 U, 30 to 400 U, 40 to 400 U, 50 to 400 U, or 60 to 400 U, in that it can further improve the stability of the liquid enzyme preparation.

[0036] The content of component (C) in the liquid enzyme preparation of the present invention is, for example, 10 w / v% or more, or 15 w / v% or more, preferably 20 w / v% or more, or 25 w / v% or more, more preferably 30 w / v% or more, even more preferably 35 w / v% or more, and even more preferably 38 w / v% or more. In this case, the upper limit of the content range of component (C) is not particularly limited, and examples include 80 w / v% or less, 75 w / v% or less, 65 w / v% or less, 55 w / v% or less, 50 w / v% or less, 45 w / v% or less, or 42 w / v% or less. Specific ranges for the content of component (C) include, for example, 10 to 80 w / v%, or 15 to 75 w / v%, preferably 20 to 65 w / v%, or 25 to 55 w / v%, more preferably 30 to 50 w / v%, even more preferably 35 to 45 w / v%, and even more preferably 38 to 42 w / v%.

[0037] The content of component (C) per 1 U of component (A) in the liquid enzyme preparation of the present invention is, for example, 0.15 mg or more, 0.2 mg or more, 0.3 mg or more, 0.4 mg or more, or 0.45 mg or more, preferably 0.5 mg or more, 0.6 mg or more, and more preferably 0.75 mg or more. The upper limit of the content range of component (C) per 1 U of component (A) in the liquid enzyme preparation of the present invention is not particularly limited, and examples include 1.2 mg or less, 1.1 mg or less, 1 mg or less, 0.85 mg or less, or 0.83 mg or less. Specific ranges of the content of component (C) per 1 U of component (A) include, for example, 0.15 to 1.2 mg, 0.2 to 1.1 mg, 0.3 to 1 mg, 0.4 to 1 mg, or 0.45 to 1 mg, preferably 0.5 to 0.85 mg, or 0.6 to 0.85 mg, and more preferably 0.75 to 0.83 mg.

[0038] [1-4. (D) Citrate] The liquid enzyme preparation of the present invention may contain a citrate as component (D). Specific examples of citrate include alkali metal salts such as potassium salts and sodium salts, and more preferably sodium salts.

[0039] There are no particular limitations on when component (D) may be added to the liquid enzyme preparation of the present invention. Component (D) can be used as a pH buffer. While component (D) suppresses pH fluctuations, it tends to reduce the stability of the liquid enzyme preparation of the present invention. However, it is particularly preferable that the content of component (B) in the liquid enzyme preparation of the present invention is 80 to 2,000 U / mL, 90 to 2,000 U / mL, 100 to 2,000 U / mL, 150 to 2,000 U / mL, 200 to 2,000 U / mL, 250 to 2,000 U / mL, or 300 to 2,000 U / mL, or that the content of component (B) per 100 U of component (A) is 16 to 400 U, 18 to 400 U, 20 to 400 U, 30 to 400 U, 40 to 400 U, 50 to 400 U, or 60 to 400 U, in terms of being able to suppress a decrease in the stability of the liquid enzyme preparation.

[0040] The content of component (D) in the liquid enzyme preparation of the present invention is, for example, 0.5 w / v% or more, preferably 0.8 w / v% or more, more preferably 1 w / v% or more, and even more preferably 1.5 w / v% or more, 2 w / v% or more, 2.5 w / v% or more, or 2.8 w / v% or more. In this case, the upper limit of the content range of component (D) is not particularly limited, and examples include 20 w / v% or less, 18 w / v% or less, 16 w / v% or less, 13 w / v% or less, 11 w / v% or less, 9 w / v% or less, 7 w / v% or less, 5 w / v% or less, 4 w / v% or less, or 3.5 w / v% or less. Specific ranges for the content of component (D) include, for example, 0.5 to 20 w / v%, or 0.5 to 18 w / v%, preferably 0.8 to 16 w / v%, or 0.8 to 13 w / v%, more preferably 1 to 11 w / v%, or 1 to 9 w / v%, and even more preferably 1.5 to 7 w / v%, 2 to 5 w / v%, 2.5 to 4 w / v%, or 2.8 to 3.5 w / v%.

[0041] The content of the (D) component per 1 U of the (A) component in the liquid enzyme preparation of the present invention is, for example, 0.0075 mg or more, preferably 0.012 mg or more, more preferably 0.02 mg or more, and even more preferably 0.025 mg or more, 0.03 mg or more, 0.035 mg or more, 0.04 mg or more, 0.045 mg or more, or 0.05 mg or more. The upper limit of the content range of the (D) component per 1 U of the (A) component in the liquid enzyme preparation of the present invention is not particularly limited, and examples thereof include 0.3 mg or less, 0.27 mg or less, 0.24 mg or less, 0.2 mg or less, 0.16 mg or less, 0.14 mg or less, 0.1 mg or less, 0.09 mg or less, 0.08 mg or less, and 0.07 mg or less. Specific ranges for the amount of component (D) per 1 U of component (A) include 0.0075 to 0.3 mg, preferably 0.012 to 0.27 mg, more preferably 0.02 to 0.24 mg, and even more preferably 0.025 to 0.2 mg, 0.03 to 0.16 mg, 0.035 to 0.14 mg, 0.04 to 0.1 mg, 0.045 to 0.09 mg, 0.045 to 0.08 mg, or 0.05 to 0.07 mg.

[0042] [1-5. Other Components] The liquid enzyme preparation of the present invention contains water as a base, in addition to the above-mentioned components (A) and (B), and the optionally blended components (C) and / or (D). Furthermore, the liquid enzyme preparation of the present invention may or may not contain other components in addition to the components (A), (B), and water, and the optionally blended components (C) and / or (D), to the extent that the effects of the present invention are not affected. Examples of such other components include enzymes other than components (A) and (B), and additives other than components (C) and (D).

[0043] Examples of other enzymes include amylases (α-amylase, β-amylase, glucoamylase), glucosidases (α-glucosidase, β-glucosidase), galactosidases (α-galactosidase, β-galactosidase), peptidases (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, pullulanase, etc. One of these other enzymes may be contained alone, or multiple types may be contained in combination.

[0044] Examples of other additives include buffers, suspending agents, and physiological saline. Examples of buffers include phosphate buffers and acetate buffers. These other additives may be contained alone or in combination.

[0045] [1-6. Formulation, etc.] The liquid enzyme preparation of the present invention is a liquid enzyme composition. The liquid enzyme preparation of the present invention can be prepared by adding predetermined amounts of protein deamidase, protease, predetermined additives, and other components to water, and further sterilizing the mixture as needed.

[0046] The pH (referring to the pH at 25°C; the same applies hereinafter) of the liquid enzyme preparation of the present invention is not particularly limited, but may be, for example, 5.5 or higher, preferably 5.8 or higher. The upper limit of the pH range of the liquid enzyme preparation of the present invention is not particularly limited, but is preferably 8.2 or lower, more preferably 7.5 or lower, even more preferably 7 or lower, and even more preferably 6.5 or lower. Specific pH ranges include, for example, 5.5 to 8.2, preferably 5.5 to 7.5, more preferably 5.8 to 7, and even more preferably 5.8 to 6.5.

[0047] [1-7. Amount of Dissolved Oxygen] The amount of dissolved oxygen contained in the liquid enzyme preparation of the present invention is not particularly limited. In particular, because the liquid enzyme preparation of the present invention exhibits excellent activity stability of protein deamidase, even if the amount of dissolved oxygen contained in the liquid enzyme preparation is not actively reduced, an effective activity stabilization effect can be achieved. From this perspective, suitable examples of the amount of dissolved oxygen in the liquid enzyme preparation include more than 3 mg / L, preferably more than 4 mg / L, more preferably 4.5 mg / L or more, and even more preferably 5 mg / L or more. The upper limit of the range of the amount of dissolved oxygen in the liquid enzyme preparation is not particularly limited, but includes, for example, 8 mg / L or less, preferably 7 mg / L or less, more preferably 6 mg / L or less, and even more preferably 5.5 mg / L or less. Specific ranges of the amount of dissolved oxygen include more than 3 mg / L and less than 8 mg / L, preferably more than 4 mg / L and less than 7 mg / L, more preferably 4.5 to 6 mg / L, and even more preferably 5 to 5.5 mg / L. Note that the liquid enzyme preparation of the present invention does not exclude cases where the amount of dissolved oxygen is 3 mg / L or less.

[0048] [1-8. Placement in a Container] The liquid enzyme preparation of the present invention is appropriately placed in a container capable of holding a liquid.

[0049] Preferably, the liquid enzyme preparation of the present invention is contained in a container whose internal space is purged with an inert gas. The inert gas is not particularly limited, and examples thereof include nitrogen gas and rare gases (helium gas, argon gas, etc.). The method for purging the internal space of the container with an inert gas is not particularly limited, and examples thereof include a method in which a gaseous inert gas or a liquid inert gas is directly added to the container.

[0050] The liquid enzyme preparation of the present invention can improve the activity stability of protein deamidase by being contained in a container whose internal space is purged with an inert gas, even without bubbling an inert gas into the liquid enzyme preparation. Therefore, in a preferred embodiment of the liquid enzyme preparation of the present invention, an inert gas is not bubbling. However, when the liquid enzyme preparation of the present invention is contained in a container whose internal space is purged with an inert gas, a form in which an inert gas is bubbling is not excluded.

[0051] In the liquid enzyme preparation of the present invention housed in the container, the oxygen concentration in the container (i.e., in the gas phase in the container) is, for example, 5 v / v % or less, preferably 3 v / v % or less, more preferably 2 v / v % or less, 1.5 v / v % or less, 1.3 v / v % or less, or 1 v / v % or less. The oxygen concentration range in the container is not particularly limited in its lower limit, and examples include 0.01 v / v % or more, 0.05 v / v % or more, 0.1 v / v % or more, 0.4 v / v % or more, 0.6 v / v % or more, 0.8 v / v % or more, or 1 v / v % or more.

[0052] [1-9. Uses] The liquid enzyme preparation of the present invention can be used in any application that utilizes at least protein deamidase activity. For example, the liquid enzyme preparation of the present invention can be used for the purpose of protein modification. The specific mode of protein modification is not particularly limited, and any modification can be used that utilizes changes in protein properties brought about by the generation of carboxyl groups through deamidation of γ-amide groups and β-amide groups in glutamine residues and asparagine residues in the protein. Specific examples of protein modification include increased protein solubility, increased water dispersibility, improved emulsifying power, and improved emulsion stability.

[0053] Therefore, the liquid enzyme preparation of the present invention can be widely used in the food industry, specifically for applications such as improving the solubility, dispersibility, emulsifying ability, etc. of animal proteins and / or vegetable proteins in a weakly acidic environment, which is the pH range of ordinary foods (for example, for the production of acidic drinks such as coffee whitener, juice, dressings, mayonnaise, and cream); increasing the solubility and dispersibility of poorly soluble vegetable proteins (for example, for the production of tempura flour using wheat gluten); modifying dough in bread and confectionery (for example, for the production of crackers, biscuits, cookies, pizza or pie crusts); and allergen detection of allergenic proteins in foods. Examples of uses include removing or reducing glutamic acid (for example, in the production of foods for patients with wheat allergies); reducing protein mineral sensitivity, increasing the soluble mineral content in liquids containing proteins and minerals, and increasing the absorbability of minerals into the human body (for example, in the production of mineral-rich (for example, calcium) beverages and mineral (for example, calcium) absorption enhancers); reducing bitterness and / or increasing the glutamic acid content (for example, in the production of amino acid-based seasonings (hydrolyzed animal protein (HAP), hydrolyzed vegetable protein (HVP)), miso and soy sauce).

[0054] Furthermore, the liquid enzyme preparation of the present invention can be used in any application that utilizes both the protein deamidase activity and the protease activity.

[0055] The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[0056] [(A) Protein deamidase] Insoluble components such as contaminating polymers were removed from the culture medium of Chryseobacterium proteolyticum, a protein deamidase (protein glutaminase)-producing strain, and the resulting product was purified by ion exchange chromatography. Protein deamidase activity was measured by the following method.

[0057] To 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, 0.1 mL of the sample solution containing protein deamidase was added, and the mixture was left to stand at 37°C for 10 minutes, after which 1 mL of 0.4 M TCA solution was added to terminate 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 the sample solution containing protein deamidase was further added, followed by standing at 37°C for 10 minutes.

[0058] The amount of ammonia generated in the reaction solution was measured using an Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.). The ammonia concentration in the reaction solution was determined from a calibration curve showing the relationship between ammonia concentration and absorbance (630 nm) prepared using an ammonia standard solution (ammonium chloride).

[0059] The activity of protein deamidase was calculated using the following formula, where 1 unit (1 U) is the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the volume of the reaction solution is 2.1, the volume of the enzyme solution is 0.1, Df is the dilution factor of the enzyme solution, and 17.03 is the molecular weight of ammonia.

[0060]

[0061] [(B) Protease] The proteases used in the following test examples are as follows: Chryseobacterium-derived protease: Chryseobacterium proteolyticum-derived protease Bacillus licheniformis-derived protease Bacillus amyloliquefaciens-derived protease

[0062] Protease activity was measured as follows. 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was heated at 37°C for 10 minutes, after which 1 mL of a sample solution containing protease was added and immediately shaken. This solution was then allowed to stand at 37°C for 10 minutes, followed by the addition of 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid), shaking, and again at 37°C for 30 minutes. The first 3 mL of filtrate was discarded, and 2 mL of the next filtrate was measured. 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin's test solution (1→3) were added, shaken well, and allowed to stand at 37°C for 30 minutes. The absorbance (AT) of this solution (enzyme reaction solution) at a wavelength of 660 nm was measured, using water as a control.

[0063] Separately, 1 mL of a sample solution containing protease was measured, 5 mL of trichloroacetic acid test solution (containing 1.8% (w / v) trichloroacetic acid, 1.8% (w / v) sodium acetate, and 0.33 mol / L acetic acid) was added, and the mixture was shaken. 5 mL of 0.6% (w / v) casein solution (0.05 mol / L sodium hydrogen phosphate, pH 8.0) was then added, the mixture was immediately shaken, and the mixture was allowed to stand at 37°C for 30 minutes. This mixture (blank) was treated in the same manner as the enzyme reaction solution described above, except that the absorbance AB was measured.

[0064] 1 mL, 2 mL, 3 mL, and 4 mL of a 1 mg / mL tyrosine standard stock solution (0.2 mol / L hydrochloric acid) were measured, and 0.2 mol / L hydrochloric acid test solution was added to each to make a 100 mL solution. 2 mL of each solution was measured, and 5 mL of 0.55 mol / L sodium carbonate test solution and 1 mL of Folin test solution (1 → 3) were added. The mixture was immediately shaken and left at 37°C for 30 minutes. For each of these solutions, 2 mL of 0.2 mol / L hydrochloric acid test solution was measured and the resulting solution was used as a control. Absorbances A1, A2, A3, and A4 at a wavelength of 660 nm were measured. A calibration curve was created, plotting absorbances A1, A2, A3, and A4 on the vertical axis and the amount of tyrosine (μg) in 2 mL of each solution on the horizontal axis, and the amount of tyrosine (μg) per 2 mL of each solution was calculated. The amount of enzyme that causes an increase in the colored substance of Folin's test solution equivalent to 1 μg of tyrosine per minute was defined as 1 unit (1 U).

[0065]

[0066] Test Example 1 Protein glutaminase (PG, component (A)) purified by ion exchange chromatography was mixed with protease (component (B)), sorbitol (component (C)), or citric acid (component (D)) in the amounts shown in Tables 1 to 3. The pH was adjusted to 6.0 (25°C) using 80% acetic acid or 6N aqueous sodium hydroxide as a pH adjuster, and liquid enzyme preparations with PG concentrations as shown in Tables 1 to 3 were prepared. The prepared liquid enzyme preparation was placed in a container, and nitrogen was injected from a nitrogen cylinder into the gas phase of the storage container (the space above the liquid phase consisting of the liquid enzyme preparation). This was repeated 20 times to almost completely replace the oxygen in the container with nitrogen (i.e., the oxygen in the gas phase was replaced with nitrogen without bubbling nitrogen into the liquid enzyme preparation), and the container was sealed to obtain a container-packed liquid enzyme preparation.

[0067] The containered liquid enzyme preparation thus obtained was allowed to stand at 40°C for one month. The protein deamidase activity after standing was calculated as a relative value (%), assuming that the protein deamidase activity before standing was 100%, and this was taken as the residual activity. The results are shown in Tables 1 to 3. Furthermore, Fig. 1 shows the correlation between the added activity of the blended protease and the residual activity of protein deamidase.

[0068]

[0069]

[0070]

[0071] As shown in Tables 1 to 3, a comparison between Examples 1 to 27 and Comparative Examples 2 to 6 revealed that the activity stabilizing effect of protein deamidase could be enhanced by setting the blending amount of component (B) to about 2,000 U / mL or less (the blending amount of component (B) to 100 U of component (A) is about 400 U or less).

[0072] Furthermore, comparisons between Example 1 and Example 6, between Example 2 and Example 7, between Example 3 and Example 8, between Example 4 and Example 9, between Example 5 and Example 10, and between Example 18 and Example 24 show that when the amount of component (B) is relatively small, the residual activity tends to decrease by adding component (C). However, comparisons between Example 19 and Example 21 and between Example 20 and Example 25 show that when the amount of component (B) is relatively large (specifically, when the amount of component (B) is about 100 U / mL or more (the amount of component (B) is about 20 U or more per 100 U of component (A))), the residual activity improves by adding component (C). Comparing Example 21 with Example 24 and Example 22 with Example 26, it was found that the addition of component (D) tended to slightly decrease the residual activity. However, when the amount of component (B) was relatively large, the liquid enzyme preparation to which component (C) had been added was highly stable. Therefore, even when component (D) was added, the decrease in residual activity could be suppressed, and high stability was effectively achieved.

[0073] The activity stabilization effect of protein deamidase shown in Tables 1 to 3 was also excellent in that it was improved compared to the case where oxygen in the container was not replaced with nitrogen. Such an excellent stabilization effect was obtained even when the amount of dissolved oxygen in the liquid enzyme preparation shown in Tables 1 to 3 exceeded 3 mg / L (specifically, more than 3 mg / L and not more than 8 mg / L), which is shown in Patent Document 3 to result in a decrease in the stability of useful proteins. Therefore, it was completely unexpected that such a stabilization effect could be obtained simply by replacing the internal space with an inert gas regardless of the amount of dissolved oxygen in the liquid enzyme preparation.

Claims

1. A liquid enzyme preparation comprising (A) a protein deamidase and (B) a protease.

2. The liquid enzyme preparation according to claim 1, wherein the liquid enzyme preparation is contained in a container whose internal space is replaced with an inert gas.

3. The liquid enzyme preparation according to claim 1, further comprising (C) sorbitol.

4. The liquid enzyme preparation according to claim 1, further comprising (D) a citrate salt.

5. A liquid enzyme preparation according to claim 3, wherein the content of component (C) is 30 w / v % or more.

6. A liquid enzyme preparation according to claim 4, wherein the content of component (D) is 1 w / v % or more.

7. A liquid enzyme preparation as described in claim 1, having a pH of 5.5 or higher.

8. The liquid enzyme preparation according to claim 1, having a pH of 8.2 or less.

9. The liquid enzyme preparation according to claim 1, wherein the content of component (A) is 0.1 to 10,000 U / ml.

10. A liquid enzyme preparation according to claim 1, wherein the component (A) is a protein deamidase derived from Chryseobacterium proteolyticum.

11. The liquid enzyme preparation according to claim 1, wherein the content of component (B) is 0.01 to 2,000 U / mL.

12. A liquid enzyme preparation according to claim 1, wherein the component (B) is a bacterial protease.

13. The liquid enzyme preparation according to claim 12, wherein the bacterial protease is a protease derived from the genus Chryseobacterium.

14. The liquid enzyme preparation according to claim 12, wherein the bacterial protease is a protease derived from the genus Bacillus and / or Geobacillus.

15. A liquid enzyme preparation according to claim 1, wherein the content of said component (B) per 100 U of said component (A) is 0.002 to 400 U.