Liquid enzyme preparation

Abstract

This invention provides a liquid enzyme formulation of a protein deamidation enzyme with excellent activity stability. [Solution] A liquid enzyme preparation containing a protein deamidation enzyme and 30% w / v or more of sorbitol, with a pH of 5.5 or higher.

Description

Technical Field

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

Background Art

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

[0003] Regarding enzyme preparations, generally, considering dusting and handling, a liquid form is more desirable than a powder form. For this reason, technologies for formulating enzyme preparations in a liquid form have been studied for various enzymes. For example, Patent Document 2 proposes a liquid enzyme preparation characterized by containing a polyol - water suspension containing 25% - 100% (w / w) of polyol and having a pH value within the range of 4.4 - 5.1 and containing at least one milk protein crosslinked and / or modified enzyme. Specifically, in Patent Document 2, it was found that the stability of transglutaminase at pH 5.2 is low, and based on this finding, it is disclosed that the stability of a transglutaminase preparation in a glycerol - water suspension at pH 4.4 - 4.8 and a transglutaminase preparation in a sorbitol - water suspension at pH 4.6 is improved. Along with this, 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 are also disclosed.

Prior Art Documents

Patent Documents

[0004] [Patent Document 1] International Publication No. 2010 / 029685 [Patent Document 2] International Publication No. 2013 / 064736 [Overview of the project] [Problems that the invention aims to solve]

[0005] While Patent Document 2 examines the stability of liquid enzyme preparations containing transglutaminase, it only discloses the formulation of liquid enzyme preparations containing protein deamidase, and does not adequately examine the stability of this enzyme.

[0006] Therefore, the object of the present invention is to provide a liquid enzyme formulation of a protein deamidation enzyme with excellent activity stability. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have discovered that the activity stability of protein deamidase is improved by a new formulation that adjusts the pH to 5.5 or higher and incorporates 30% w / v or more of sorbitol. The present invention was completed based on this finding. The present invention includes the following inventions.

[0008] Item 1. A liquid enzyme preparation containing a protein deamide enzyme and 30% w / v or more of sorbitol, with a pH of 5.5 or higher. Item 2. The liquid enzyme preparation according to Item 1, wherein the sorbitol content is 65 w / v% or less. Item 3. A liquid enzyme preparation as described in Item 1 or 2, having a pH of 8.2 or less. Item 4. A liquid enzyme preparation according to any one of claims 1 to 3, further comprising a sulfite, a thiosulfate, and / or a pyrosulfate. Item 5. A liquid enzyme preparation according to any of items 1 to 4, having a protein deamide enzyme activity of 0.1 to 10,000 U / ml. Item 6. A liquid enzyme preparation according to any one of items 1 to 5, wherein the protein deamide enzyme is derived from Chryseobacterium proteoricum. Item 7. An enzyme activity stabilizer for liquid enzyme preparations of protein deamide enzymes, containing sorbitol as an active ingredient and used at a concentration of 30 w / v% or higher in liquid enzyme preparations of protein deamide enzymes with a pH of 5.5 or higher. Item 8. Further comprising sulfites, thiosulfates, and / or pyrosulfates, as enzyme activity stabilizers for liquid enzyme preparations of protein deamidases as described in Item 7. Item 9. A precipitation inhibitor for liquid enzyme preparations of protein deamide enzymes, containing sorbitol as an active ingredient and used at a concentration of 30 w / v% or higher in liquid enzyme preparations of protein deamide enzymes with a pH of 5.5 or higher. [Effects of the Invention]

[0009] According to the present invention, a liquid enzyme preparation of a protein deamidation enzyme with excellent activity stability is provided. [Brief explanation of the drawing]

[0010] [Figure 1] This shows the results of the activity stability test of the liquid enzyme preparation of protein deamidation enzyme (protein glutaminase: PG) obtained in Test Example 2. [Modes for carrying out the invention]

[0011] [1. Liquid enzyme preparations] The liquid enzyme preparation of the present invention is characterized by containing a protein deamidation enzyme and 30% w / v or more of sorbitol, and having a pH of 5.5 or higher. The liquid enzyme preparation of the present invention will be described in detail below.

[0012] [1-1. Protein Deamidases] Protein deamidases are enzymes that degrade amide group-containing side chains of proteins without cleaving peptide bonds or crosslinking proteins, and their type and origin are not particularly limited. Furthermore, as long as the above-mentioned action is the primary activity, they may also have the ability to degrade amide group-containing side chains of proteins with peptide bond cleavage and crosslinking proteins. Examples of protein deamidases 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 deamidation enzymes include those from the genera Chryseobacterium, Flavobacterium, Empedobacter, Sphingobacterium, and Aureobacterium, as disclosed in JP2000-50887A, JP2001-218590A, and WO2006 / 075772A1. Examples include protein deamide enzymes derived from the genera *Terium* or *Myroides*, protein deamide enzymes derived from the genera *Luteimicrobium*, *Agromyces*, *Microbacterium*, or *Leifsonia* as disclosed in WO2015 / 133590, and commercially available protein glutaminases derived from the genera *Chryseobacterium*. These protein deamide enzymes may be used individually or in combination of multiple types.

[0013] Among these protein deamide enzymes, from the viewpoint of providing a liquid enzyme preparation with even better activity stability, protein deamide enzymes derived from the genus Chryseobacterium are preferred, more preferably protein glutaminases derived from the genus Chryseobacterium, and even more preferably protein glutaminases derived from the species Chryseobacterium proteoricum.

[0014] The protein deamidase can be prepared from the culture broth of the microorganism from which the above protein deamidase is derived. Specific preparation methods include methods for recovering the protein deamidase from the culture broth or cells of the above microorganism. For example, when using a protein deamidase-secreting microorganism, after recovering the cells from the culture broth by filtration, centrifugation, etc. as necessary, the enzyme can be separated and / or purified. Also, when using a non-protein deamidase-secreting microorganism, after recovering the cells from the culture broth as necessary, the cells are disrupted by pressure treatment, ultrasonic treatment, etc. to expose the enzyme, and then the enzyme can be separated and / or purified. As the method for separating and / or purifying the enzyme, known protein separation and / or purification methods can be used without particular limitation, and examples include centrifugation, UF concentration, salting out, and various chromatography methods using ion exchange resins, etc.

[0015] The content of the protein deamidase in the liquid enzyme preparation of the present invention is not particularly limited, but for example, it can be 0.1 to 10,000 U / mL, 1 to 5,000 U / mL, 10 to 2,500 U / ml, 50 to 1,000 U / ml, preferably 100 to 800 U / ml, more preferably 150 to 700 U / ml, still more preferably 250 to 600 U / ml, 250 to 500 U / ml, and even more preferably 300 to 400 U / ml.

[0016] Regarding the activity of the protein deamidase, using benzyloxycarbonyl-L-glutaminylglycine (Z-Gln-Gly) as the substrate, the amount of enzyme that liberates 1 μmol of ammonia per minute is defined as 1 unit (1 U).

[0017] [1-2. Sorbitol] The liquid enzyme preparation of the present invention contains sorbitol. Sorbitol is formulated for the purpose of stabilizing the activity of the protein deamidase.

[0018] The content of sorbitol in the liquid enzyme preparation of the present invention is 30 w / v% or more from the viewpoint of obtaining the desired activity stability. In the liquid enzyme preparation of the present invention, the amount of sorbitol used per 1 U of protein deamidase is determined according to the respective contents of the protein deamidase and sorbitol, but from the viewpoint of obtaining the desired activity stability, preferably 0.8 mg or more can be mentioned.

[0019] From the viewpoint of expressing more excellent activity stability, the content of sorbitol in the liquid enzyme preparation of the present invention may preferably be 35 w / v% or more or 38 w / v% or more. Similarly, from the viewpoint of expressing more excellent activity stability, the amount of sorbitol used per 1 U of protein deamidase in the liquid enzyme preparation of the present invention may preferably be 0.97 mg or more or 1 mg or more. Examples corresponding to these cases include those where the pH of the liquid enzyme preparation of the present invention (pH at 25°C; the same applies hereinafter) is 5.8 to 8.2, preferably 6.0 to 8.0.

[0020] Furthermore, from the viewpoint of expressing even more excellent activity stability, the content of sorbitol in the liquid enzyme preparation of the present invention may preferably be 45 w / v% or more or 48 w / v% or more. Similarly, from the viewpoint of expressing even more excellent activity stability, the amount of sorbitol used per 1 U of protein deamidase in the liquid enzyme preparation of the present invention may preferably be 1.2 mg or more or 1.3 mg or more. Examples corresponding to these cases include those where the pH of the liquid enzyme preparation of the present invention is 6.5 to 7.5, preferably 6.8 to 7.2, and more preferably 6.9 to 7.1.

[0021] While there is no particular upper limit to the sorbitol content range in the liquid enzyme preparation of the present invention, from the viewpoint of exhibiting better activity stability and / or ease of preparation, for example, 65 w / v% or less, preferably 60 w / v% or less, more preferably 57 w / v% or less, even more preferably 55 w / v% or less, and even more preferably 52 w / v% or less. Similarly, while there is no particular upper limit to the amount of sorbitol used per 1U of protein deamidase in the liquid enzyme preparation of the present invention, from the viewpoint of exhibiting better activity stability and / or ease of preparation, for example, 1.8 mg or less, preferably 1.67 mg or less, more preferably 1.59 mg or less, even more preferably 1.53 mg or less, and even more preferably 1.4 mg or less.

[0022] From the viewpoint of exhibiting even better activity stability, the upper limit of the sorbitol content range in the liquid enzyme preparation of the present invention may be preferably 50 w / v% or less, more preferably 47 w / v% or less, even more preferably 45 w / v% or less, and even more preferably 42 w / v% or less. Similarly, from the viewpoint of exhibiting even better activity stability, the upper limit of the amount of sorbitol used per 1U of protein deamidase in the liquid enzyme preparation of the present invention may be preferably 1.4 mg or less, more preferably 1.3 mg or less, even more preferably 1.25 mg or less, and even more preferably 1.16 mg or less. Examples of these cases include when the pH of the liquid enzyme preparation of the present invention is 5.5 to 8.0, preferably 5.5 to 7.2, more preferably 5.8 or more and less than 6.5, even more preferably 5.9 to 6.3, and even more preferably 6.0 to 6.2.

[0023] In certain embodiments of the present invention, sorbitol of 30 w / v% or more in the liquid enzyme preparation may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the sorbitol content in the liquid enzyme preparation of the present invention is preferably 38 w / v% or more or 45 w / v% or more, more preferably 48 w / v% or more or 55 w / v% or more, and even more preferably 58 w / v% or more. Similarly, in certain embodiments of the present invention, sorbitol of 0.8 mg or more per 1 U of protein deamidase in the liquid enzyme preparation may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the amount of sorbitol used per 1 U of protein deamidase in the liquid enzyme preparation of the present invention is preferably 1 mg or more or 1.25 mg or more, more preferably 1.3 mg or more or 1.5 mg or more, and even more preferably 1.6 mg or more. Examples of these embodiments include cases where the pH of the liquid enzyme preparation of the present invention is 5.5 or more and less than 6.75, more preferably 5.5 to 6.5.

[0024] Furthermore, in another specific embodiment of the present invention, sorbitol of 38 w / v% or more, or 45 w / v% or more, in the liquid enzyme preparation of the present invention may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the sorbitol content in the liquid enzyme preparation of the present invention is preferably 48 w / v% or more, or 55 w / v% or more, more preferably 58 w / v% or more. Similarly, in another specific embodiment of the present invention, sorbitol of 1 mg or more, or 1.25 mg or more per 1 U of protein deamidase in the liquid enzyme preparation of the present invention may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the amount of sorbitol used per 1 U of protein deamidase in the liquid enzyme preparation of the present invention is preferably 1.3 mg or more, or 1.5 mg or more, more preferably 1.6 mg or more. Examples of these embodiments include cases where the pH of the liquid enzyme preparation of the present invention is 6.75 or more and less than 7.5, more preferably 6.8 to 7.2.

[0025] Furthermore, in yet another specific embodiment of the present invention, sorbitol of 48 w / v% or more or 55 w / v% or more in the liquid enzyme preparation of the present invention may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the sorbitol content in the liquid enzyme preparation of the present invention is preferably 58 w / v% or more. Similarly, in another specific embodiment of the present invention, sorbitol of 1.3 mg or more or 1.5 mg or more per 1 U of protein deamidase in the liquid enzyme preparation of the present invention may be added for the purpose of suppressing sedimentation in addition to stabilizing activity. In this embodiment, from the viewpoint of enhancing the sedimentation suppression effect, the amount of sorbitol used per 1 U of protein deamidase in the liquid enzyme preparation of the present invention is preferably 1.6 mg or more. An example of these embodiments is when the pH of the liquid enzyme preparation of the present invention is 7.5 or higher.

[0026] [1-3. Sulfites, thiosulfates, and pyrosulfates] From the viewpoint of exhibiting even better activity stability, the liquid enzyme preparation of the present invention preferably further contains sulfite, thiosulfate, and / or pyrosulfate as predetermined salts. Specifically, these predetermined salts include metal salts of alkali metals (potassium salt, sodium salt, etc.) and alkaline earth metals (calcium salt, etc.). These predetermined salts may be used individually or in combination of multiple types.

[0027] Among these, from the viewpoint of exhibiting even better activity stability, sulfites and pyrosulfates are more preferred, sulfites are even more preferred, or alkali metal salts are even more preferred, and sodium salts are even more preferred. Furthermore, among these, from the viewpoint of exhibiting particularly excellent activity stability, alkali metal sulfites are especially preferred, and sodium sulfite is the most preferred.

[0028] When the liquid enzyme preparation of the present invention contains the above-mentioned predetermined salt, the amount of the predetermined salt is not particularly limited and can be set appropriately according to the effect of improving activity stabilization to be imparted. For example, 0.01 to 2 w / v%, preferably 0.03 to 1 w / v%, preferably 0.04 to 0.8 w / v%, more preferably 0.06 to 0.6 w / v% or 0.08 to 0.6 w / v%, even more preferably 0.15 to 0.55 w / v%, 0.2 to 0.55 w / v%, 0.3 to 0.55 w / v%, or 0.4 to 0.55 w / v% can be found. Alternatively, the amount of the predetermined salt per 1U of protein deamidase can be, for example, 0.00025 to 0.05 mg, preferably 0.0008 to 0.026 mg, preferably 0.001 to 0.023 mg, more preferably 0.0015 to 0.017 mg or 0.002 to 0.017 mg, even more preferably 0.004 to 0.015 mg, 0.005 to 0.015 mg, 0.008 to 0.015 mg, or 0.01 to 0.015 mg.

[0029] [1-4. Other ingredients] The liquid enzyme preparation of the present invention may contain other components in addition to the protein deamidase and sorbitol described above, to an extent that does not affect the effects of the present invention. Examples of other components include other enzymes other than protein deamidase, additives, etc.

[0030] Other enzymes include, for example, amylase (α-amylase, β-amylase, glucoamylase), glucosidase (α-glucosidase, β-glucosidase), galactosidase (α-galactosidase, β-galactosidase), protease (acid protease, neutral protease, alkaline protease), peptidase (leucine peptidase, aminopeptidase), lipase, esterase, cellulase, phosphatase (acid phosphatase, alkaline phosphatase), nuclease, deaminase, oxidase, dehydrogenase, glutaminase, pectinase, catalase, dextranase, transglutaminase, pullulanase, etc. These other enzymes may be included individually or in combination of multiple types.

[0031] Examples of additives include excipients, buffers, suspending agents, stabilizers, preservatives, antimicrobials, physiological saline, polyhydric alcohols (referring to polyhydric alcohols other than sorbitol as described above; the same applies hereinafter), pH adjusters, etc. These additives may be included individually or in combination of multiple types.

[0032] Among these additives, from the viewpoint of further improving the activity stability of the liquid enzyme preparation of the present invention, it is preferable that the liquid enzyme preparation of the present invention substantially contains no polyhydric alcohols. Polyhydric alcohols include non-sugar polyhydric alcohols such as glycerin and propylene glycol, and sugar alcohols other than sorbitol. It should be noted that substantially free of polyhydric alcohols means that the liquid enzyme preparation of the present invention may contain polyhydric alcohols to an extent that does not impair its activity stability. Specifically, the polyhydric alcohol content is 1 w / v% or less, preferably 0.1 w / v% or less, more preferably 0.01 w / v% or less, and most preferably 0 w / v%.

[0033] [1-5. Formulation, etc.] The liquid enzyme preparation of the present invention is a liquid preparation. The liquid enzyme preparation of the present invention can be prepared by adding predetermined amounts of each component to water and dissolving them, or by separating and / or purifying unwanted components such as contaminating polymers from the culture medium of a protein deamide enzyme producing strain, adding an aqueous sorbitol solution, then adding a pH adjuster as appropriate to adjust to a predetermined pH, and / or adding sulfites, thiosulfates, and / or pyrosulfates as needed, and further sterilizing as necessary.

[0034] The pH (at 25°C) of the liquid enzyme preparation of the present invention is 5.5 or higher from the viewpoint of obtaining the desired activity stability. The upper limit of the pH range of the liquid enzyme preparation of the present invention is not particularly limited, but from the viewpoint of exhibiting even better activity stability, values ​​such as 8.2 or lower, preferably 8.0 or lower, more preferably 7.6 or lower, even more preferably 7.2 or lower, even more preferably 7.0 or lower, 6.8 or lower, 6.5 or lower, 6.2 or lower, 6.0 or lower, or 5.7 or lower are recommended.

[0035] [1-6.Application] The liquid enzyme preparation of the present invention can be used for any application that utilizes protein deamidation enzyme activity. For example, the liquid enzyme preparation of the present invention can be used for the purpose of modifying proteins. The specific form of protein modification is not particularly limited and can be any method that utilizes the change in protein properties brought about by the generation of carboxyl groups through the deamidation of γ-amide and β-amide groups of glutamine and asparagine residues of proteins. Specifically, protein modification can include increased protein solubility, increased water dispersibility, improved emulsifying power, and improved emulsion stability.

[0036] Therefore, the protein deamide enzyme liquid enzyme preparation of the present invention can be widely used in the food industry. Specifically, it can be used to improve the solubility, dispersibility, and emulsification of animal proteins and / or plant proteins in a weakly acidic environment, which is within the pH range of normal foods (for example, in the production of acidic beverages such as coffee whiteners and juices, dressings, mayonnaise, and cream); to increase the solubility and dispersibility of poorly soluble plant proteins (for example, in the production of tempura flour using wheat gluten); to modify dough in bread and confectionery (for example, in the production of crackers, biscuits, cookies, pizza or pie crusts); and to remove or reduce allergens in allergenic proteins in food (e.g. Examples include: the manufacture of food for wheat allergy sufferers; applications to reduce the mineral sensitivity of proteins, increase the soluble mineral content in liquids containing proteins and minerals, and improve the absorption of minerals into the human body (e.g., applications in the manufacture of high-mineral (e.g., calcium) beverages and mineral (e.g., calcium) absorption enhancers); applications to reduce bitterness, improve the protein degradation rate of proteases, and / or enhance glutamic acid content (e.g., applications in the manufacture of amino acid-based seasonings (hydrolyzed animal protein (HAP), hydrolyzed plant protein (HVP)), and miso and soy sauce).

[0037] [2. Enzyme activity stabilizers for liquid enzyme preparations of protein deamidases and sediment inhibitors for liquid enzyme preparations of protein deamidases] As described above, in liquid enzyme preparations containing protein deamidase with a pH of 5.5 or higher, sorbitol added at a concentration of 30 w / v% or higher improves the activity stability of the liquid enzyme preparation, i.e., the stability of protein deamidase activity. Accordingly, the present invention also provides an enzyme activity stabilizer for liquid enzyme preparations of protein deamidase, which contains sorbitol as an active ingredient and is used at a concentration of 30 w / v% or higher in liquid enzyme preparations of protein deamidase with a pH of 5.5 or higher. From the viewpoint of exhibiting even better activity stability, the enzyme activity stabilizer for liquid enzyme preparations of protein deamidase of the present invention preferably further contains, in addition to sorbitol, a predetermined salt such as sulfite, thiosulfate, and / or pyrosulfate.

[0038] Furthermore, as described above, sorbitol added at a concentration of 30 w / v% or higher in a liquid enzyme preparation containing a protein deamidase with a pH of 5.5 or higher can suppress sedimentation of the liquid enzyme preparation. Accordingly, the present invention also provides a sedimentation inhibitor for liquid enzyme preparations of protein deamidase, which contains sorbitol as an active ingredient and is used at a concentration of 30 w / v% or higher in a liquid enzyme preparation of protein deamidase with a pH of 5.5 or higher.

[0039] More preferably, the sediment inhibitor of the present invention is used at a concentration of 30 w / v% or more in a liquid enzyme preparation of protein deamide enzyme with a pH of 5.5 or higher and less than 6.75, at a concentration of 38 w / v% or more in a liquid enzyme preparation of protein deamide enzyme with a pH of 6.75 or higher and less than 7.5, or at a concentration of 48 w / v% or more in a liquid enzyme preparation of protein deamide enzyme with a pH of 7.5 or higher.

[0040] In the enzyme activity stabilizer and precipitation inhibitor for the liquid enzyme preparation of protein deamide enzyme of the present invention, the types and amounts of each component are as shown in the "1. Liquid Enzyme Preparation" section above. [Examples]

[0041] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0042] [Test Example 1] Unwanted components such as contaminating polymers were removed from the culture medium of the protein glutaminase-producing strain Chryseobacterium proteoricum, and aqueous sorbitol and / or glycerin were added. Subsequently, the pH was adjusted to 5.0 or 6.0 using 1N hydrochloric acid or 1N sodium hydroxide aqueous solution to prepare a liquid enzyme preparation with the concentrations of protein glutaminase (PG), sorbitol, and glycerin as shown in Table 1.

[0043] The resulting liquid enzyme preparation was left at 60°C for 72 hours. The protein deamidase activity levels before and after standing were measured by the following method.

[0044] To 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly, 0.1 mL of sample solution containing protein deamidase was added and left to stand at 37°C for 10 minutes. Then, 1 mL of 0.4 M TCA solution was added to stop the reaction. As a blank, 1 mL of 0.2 M phosphate buffer (pH 6.5) containing 30 mM Z-Gln-Gly was added to 1 mL of 0.4 M TCA reagent, and then 0.1 mL of sample solution containing protein deamidase was added and left to stand at 37°C for 10 minutes.

[0045] The amount of ammonia produced in the reaction solution was measured using the Ammonia Test Wako (Fujifilm Wako Pure Chemical Industries) for the solution obtained as described above. 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 ammonia standard solution (ammonium chloride).

[0046] The activity of the protein deamide enzyme was calculated using the following formula, where 1 unit (1U) is defined as the amount of enzyme that produces 1 μmol of ammonia per minute. In the formula, the reaction volume is 2.1, the enzyme solution volume is 0.1, and Df is the dilution ratio of the enzyme solution. Also, 17.03 is the molecular weight of ammonia.

[0047]

number

[0048] The residual activity was calculated as the relative value (%) of the protein deamidase activity after standing, with the protein deamidase activity before standing set to 100%. The results are shown in Table 1.

[0049] [Table 1]

[0050] As shown in Table 1, when glycerin was added to the liquid enzyme preparation of PG (Comparative Examples 2-6), when 20 w / v% sorbitol was added (Comparative Example 7), and when 30 w / v% sorbitol was added and the pH was adjusted to 5.0 (Comparative Example 8), the activity stability did not improve significantly compared to Comparative Example 1. However, when 30 w / v% sorbitol was added and the pH was adjusted to 6.0 (Example 1), a remarkable improvement in activity stability was observed.

[0051] [Test Example 2] Liquid enzyme preparations containing protein glutaminase (PG) as a protein deamidation enzyme and sorbitol (without glycerin) were prepared in the same manner as in Test Example 1, except that the pH was adjusted to 5.5, 6.0, 6.5, 7.0, or 8.0, and the amount of sorbitol was set to 0 w / v%, 20 w / v%, 30 w / v%, 40 w / v%, 50 w / v%, or 60 w / v%. The PG content in the liquid enzyme preparation was 360 U / ml. The residual activity of the obtained liquid enzyme preparation was measured after standing at 60°C for 72 hours in the same manner as in Test Example 1. The results are shown in Figure 1.

[0052] As shown in Figure 1, a liquid enzyme preparation containing a protein deamidase and 30% or more w / v sorbitol, with a pH of 5.5 or higher, showed a significant improvement in activity stability.

[0053] [Test Example 3] The liquid enzyme preparation obtained in Test Example 2 was dispensed into 1 ml microcentrifuge tubes (2.0 ml capacity) and left to stand at 60°C for 72 hours. The appearance was observed, and the presence and extent of sediment were evaluated based on the following criteria. Table 2 shows representative images of the appearance corresponding to each criterion. The results are shown in Table 3.

[0054] -: No sediment was observed, and the liquid enzyme preparation was clear. +: A very small amount of sediment was observed. ++: A small amount of sediment was observed. +++: A large amount of sediment was observed.

[0055] [Table 2]

[0056] [Table 3]

[0057] As shown in Table 3, a large amount of sediment was generated when sorbitol was not included, but sedimentation was suppressed by adding 30 w / v% or more of sorbitol. In particular, when the pH of the liquid enzyme preparation was 5.5, 6.0, and 6.5, adding 30 w / v% or more of sorbitol showed excellent sedimentation suppression, when the pH of the liquid enzyme preparation was 7.0, adding 40 w / v% or more of sorbitol showed excellent sedimentation suppression, and when the pH of the liquid enzyme preparation was 8.0, adding 50 w / v% or more of sorbitol showed excellent sedimentation suppression.

[0058] [Test Example 4] Liquid enzyme preparations were prepared using the liquid enzyme preparation of Example 1 prepared in Test Example 1, and also by further adding sodium sulfite, sodium thiosulfate, sodium pyrosulfate, magnesium sulfate, disodium ethylenediaminetetraacetate, or potassium chloride in the amounts shown in Table 4 to the liquid enzyme preparation of Example 1. The residual activity of these liquid enzyme preparations was measured in the same manner as in Test Example 1, except that the standing conditions were 40°C for 2 weeks. The results are shown in Table 4.

[0059] [Table 4]

[0060] As is clear from Table 4, even when various salts were added to a pH 6.0 liquid enzyme preparation containing protein glutaminase and 30 w / v% sorbitol, an effective enzyme activity stabilization effect was obtained. In particular, when sodium sulfite, sodium thiosulfate, or sodium pyrosulfate were added, an even higher enzyme activity stabilization effect was obtained, and this trend was especially pronounced when sodium sulfite and sodium pyrosulfate were added.

Claims

1. A liquid enzyme preparation containing a protein deamidation enzyme and 30% w / v or more sorbitol, with a pH of 5.5 or higher.

2. The liquid enzyme preparation according to claim 1, wherein the sorbitol content is 65 w / v% or less.

3. A liquid enzyme preparation according to claim 1 or 2, wherein the pH is 8.2 or lower.

4. Furthermore, the liquid enzyme preparation according to any one of claims 1 to 3, comprising sulfites, thiosulfates, and / or pyrosulfates.

5. A liquid enzyme preparation according to any one of claims 1 to 4, wherein the protein deamidation enzyme activity is 0.1 to 10,000 U / ml.

6. The liquid enzyme preparation according to any one of claims 1 to 5, wherein the protein deamidase is derived from Chryseobacterium proteoricum.

7. A stabilizer for the enzyme activity of liquid enzyme preparations of protein deamidase, containing sorbitol as the active ingredient and used at a concentration of 30 w / v% or higher in liquid enzyme preparations of protein deamidase with a pH of 5.5 or higher.

8. Furthermore, an enzyme activity stabilizer for a liquid enzyme preparation of a protein deamidase according to claim 7, comprising a sulfite, a thiosulfate, and / or a pyrosulfate.

9. A precipitation inhibitor for liquid enzyme preparations of protein deamidase, containing sorbitol as an active ingredient and used at a concentration of 30 w / v% or higher in liquid enzyme preparations of protein deamidase with a pH of 5.5 or higher.

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