Protein glutamine liquid enzyme

Abstract

The present invention relates to a stabilizer composition for protein glutaminase and a liquid enzyme formulation comprising said composition. The stabilizer composition comprises the following components: polyol, saccharides, edible colloid, a sulfur-containing antioxidant, a buffer solution, an optional chloride and the balance of water. The liquid enzyme preparation comprises a protein glutaminase obtained by fermentation with Chryseobacterium prion and centrifugation and ultrafiltration treatment, and the stabilizer composition of the invention. The invention also relates to a method for preparing the stabilizer composition and the liquid enzyme preparation. After the liquid enzyme preparation is stored at normal temperature for 365 days, the enzyme activity is still kept at 85% or above, and the liquid enzyme preparation is uniform in texture, convenient to use and wide in application scene.

Description

Technical Field

[0001] This invention belongs to the field of biological enzyme preparations, and more specifically relates to a stabilizer composition for protein glutaminase and a liquid preparation of protein glutaminase comprising said composition. Background Technology

[0002] Protein-glutaminase (EC 3.5.1.44), abbreviated as PG, can remove the amide group from various proteins, polypeptides, or short peptides. It can convert L-β-glutamine into L-glutamic acid and ammonia, and it only acts on the glutamine group of proteins or peptides, without affecting asparagine residues or free glutamine. At the same time, it does not cause protein cross-linking or hydrolysis, thereby improving the solubility and emulsifying properties of proteins. It has broad application prospects in the food industry (especially in the dairy and beverage industries).

[0003] Currently, protein glutaminase products on the domestic and international markets are mainly solid powders. Powders have problems such as causing dust, poor solubility and dispersibility during application. Since the application scenarios of protein glutaminase are mainly concentrated in the dairy and beverage industries, liquid enzyme preparations have more advantages in practical applications.

[0004] In summary, there is an urgent need in the art to develop a stabilizer composition for protein glutaminase and a liquid formulation of protein glutaminase that exhibits excellent stability and can be stored at room temperature for extended periods while essentially maintaining its activity. Therefore, this invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to develop a stabilizer composition for protein glutaminase and a liquid formulation of protein glutaminase containing the composition, wherein the enzyme formulation has excellent storage stability, for example, the enzyme activity after storage at room temperature (25°C) for 180-365 days is still more than 90% of the initial enzyme activity, preferably more than 95%.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] 1. A stabilizer composition for protein glutaminase, wherein the composition comprises or consists of the following components by weight percentage:

[0008] (a) 20-75% polyols;

[0009] (b) 20-75% carbohydrates;

[0010] (c) 0.5-8% edible gums;

[0011] (d) 0.05-1% sulfur-containing antioxidants;

[0012] (e) 1-20% 25-75 mM buffer;

[0013] (f) 0-5% chloride; and

[0014] (g) The remaining amount of water.

[0015] 2. The composition according to claim 1, wherein the composition comprises or consists of the following components by weight percentage:

[0016] (a) 20-60% polyols;

[0017] (b) 20-60% carbohydrates;

[0018] (c) 0.5-5% edible gums;

[0019] (d) 0.05-0.5% sulfur-containing antioxidants;

[0020] (e) 1-15% 20-60 mM buffer;

[0021] (f) 0-1% chloride; and

[0022] (g) The remaining amount of water.

[0023] 3. The composition according to claim 1 or 2, wherein the composition comprises or consists of the following components by weight percentage:

[0024] (a) 30-55% polyols;

[0025] (b) 30-55% carbohydrates;

[0026] (c) 0.5-3% edible gums;

[0027] (d) 0.05-0.3% sulfur-containing antioxidants;

[0028] (e) 1-12% 30-50 mM buffer;

[0029] (f) 0-0.5% chloride; and

[0030] (g) The remaining amount of water.

[0031] 4. The composition according to any one of items 1-3, wherein the polyol is selected from the group consisting of glycerol, sorbitol, xylitol, propylene glycol, and neopentyl glycol.

[0032] 5. The composition according to any one of items 1-4, wherein the sugar is selected from the group consisting of trehalose, sorbitol, glucose, sucrose and fructose.

[0033] 6. The composition according to any one of items 1-5, wherein the edible gum is selected from the group consisting of gelatin, pectin, guar gum, carrageenan, locust bean gum, xanthan gum, agar, sodium alginate and konjac gum.

[0034] 7. The composition according to any one of items 1-6, wherein the sulfur-containing antioxidant is selected from the group consisting of glutathione, sodium sulfite and ergothioneine.

[0035] 8. The composition according to any one of items 1-7, wherein the buffer is selected from the group consisting of phosphate buffer, citrate buffer and Tris-hydrochloric acid buffer.

[0036] 9. The composition according to any one of claims 1-8, wherein the chloride is a chloride selected from the group consisting of calcium chloride, magnesium chloride, ferric chloride, sodium chloride, and potassium chloride.

[0037] 10. A method for preparing a composition of any one of items 1-9, wherein the components are added in the following order by mass percentage: polyol, sugar, edible gum, antioxidant, optional chloride, buffer solution and water.

[0038] 11. A liquid formulation of a protein glutaminase, comprising:

[0039] (i) 1-10000 U / ml protein glutaminase solution; and

[0040] (ii) A stabilizer composition comprising, by weight, 50-90% of any one of items 1-9 of the formulation.

[0041] 12. The formulation according to item 11, wherein the protein glutaminase solution is obtained by fermentation using bacteria capable of expressing protein glutaminase and centrifugation and ultrafiltration of the fermentation broth.

[0042] 13. The formulation according to claim 12, wherein the bacteria are selected from the group consisting of Chryseobacterium, Streptomyces and Bacillus, preferably Chryseobacterium proteolyticum or Bacillus subtilis.

[0043] 14. The formulation according to item 12 or 13, wherein the centrifugation is performed under the following conditions:

[0044] (i) Centrifugation speed is 5000-15000 RPM;

[0045] (ii) The centrifugation temperature is 0-30℃;

[0046] (iii) Centrifugation time is 15 minutes to 3 hours; and / or

[0047] (iv) The pH during centrifugation is 6.5-8.0.

[0048] 15. The formulation according to any one of claims 12-14, wherein the ultrafiltration membrane has a molecular weight cutoff of 3-100 kDa.

[0049] 16. The formulation according to any one of claims 12-15, wherein the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3-50 times.

[0050] 17. The formulation according to any one of items 11-16, wherein the protein glutaminase solution is sterilized by filtration, preferably through a filter membrane of 0.22-0.45 μm.

[0051] 18. The formulation according to any one of items 11-17, wherein the stabilizer composition is sterilized at 90-115°C for 5-20 minutes.

[0052] 19. The formulation according to any one of claims 11-18, wherein the pH of the formulation is 5.0-8.5.

[0053] 20. The formulation according to any one of items 11-19, wherein the enzyme activity of the formulation remains at more than 85%, preferably more than 95%, of the initial enzyme activity after being placed at room temperature for 365 days.

[0054] 21. A method for preparing the liquid formulation described in any one of 11-19, comprising:

[0055] (i) Provide protein glutaminase solution at 1-10000 U / ml;

[0056] (ii) Provide a stabilizer composition as described in any one of items 1-9; and

[0057] (iii) The protein glutaminase solution is mixed with the stabilizer composition such that the stabilizer composition accounts for 50-90% of the liquid formulation by weight.

[0058] 22. The method according to item 21, wherein the protein glutaminase solution is obtained by fermentation using bacteria capable of expressing protein glutaminase and centrifugation and ultrafiltration of the fermentation broth.

[0059] 23. The method according to claim 22, wherein the bacteria are selected from the group consisting of Chryseobacterium, Streptomyces and Bacillus, preferably Chryseobacterium proteolyticum or Bacillus subtilis.

[0060] 24. The method according to item 22 or 23, wherein the centrifugation is performed under the following conditions:

[0061] (i) Centrifugation speed is 5000-15000 RPM;

[0062] (ii) The centrifugation temperature is 0-30℃;

[0063] (iii) Centrifugation time is 15 minutes to 3 hours; and / or

[0064] (iv) The pH during centrifugation is 6.5-8.0.

[0065] 25. The method according to any one of items 22-24, wherein the ultrafiltration membrane has a molecular weight cutoff of 3-100 kDa.

[0066] 26. The method according to any one of items 22-25, wherein the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3-50 times.

[0067] 27. The method according to any one of items 21-26, wherein the protein glutaminase solution is sterilized by filtration, preferably through a filter membrane of 0.22-0.45 μm.

[0068] 28. The method according to any one of items 21-27, wherein the stabilizer composition has been sterilized at 90-115°C for 5-20 minutes.

[0069] 29. The method according to any one of items 21-28, wherein the pH of the liquid formulation is 5.0-8.5.

[0070] 30. The method according to any one of items 21-29, wherein the enzyme activity of protein glutaminase remains at more than 85%, preferably more than 95%, of the initial enzyme activity after the liquid preparation is placed at room temperature for 365 days.

[0071] To make the technical solution of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. Invention Details

[0073] 1. The stabilizer composition of the present invention

[0074] In one aspect, the present invention provides a stabilizer composition for protein glutaminase. In one embodiment, the composition comprises, or consists of, the following components by weight percentage: (a) 20-75% polyol; (b) 20-75% sugar; (c) 0.5-8% edible gum; (d) 0.05-1% sulfur-containing antioxidant; (e) 1-20% 50 mM buffer; (f) 0-5% chloride; and (g) the balance being water. In one embodiment, the composition comprises, or consists of, the following components by weight percentage: (a) 20-60% polyol; (b) 20-60% sugars; (c) 0.5-5% edible gums; (d) 0.05-0.5% sulfur-containing antioxidants; (e) 1-15% 50 mM buffer solution; (f) 0-1% chloride; and (g) the balance water. In another embodiment, the composition comprises, or consists of, the following components by weight percentage: (a) 30-55% polyol; (b) 30-55% sugars; (c) 0.5-3% edible gums; (d) 0.05-0.3% sulfur-containing antioxidants; (e) 1-12% 50 mM buffer solution; (f) 0-0.5% chloride; and (g) the balance water.

[0075] In one embodiment, the composition comprises, by weight percentage, 20-75%, 25-75%, 30-75%, 35-75%, 40-75%, 45-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 20-70%, 25-70%, 30-70%, 35-70%, 40-70%, 45-70%, 50-70%, 55-70%, 60-70%, 65-70%, 20-65%, 25-65%, 30-65%, 35-65%, 40-65%, 45-65%, 50-65%, 55-65%, 60-65%, 20-60%, 25-6% Polyols of 0%, 30-60%, 35-60%, 40-60%, 45-60%, 50-60%, 55-60%, 20-55%, 25-55%, 30-55%, 35-55%, 40-55%, 45-55%, 50-55%, 20-50%, 25-50%, 30-50%, 35-50%, 40-50%, 45-50%, 20-45%, 25-45%, 30-45%, 35-45%, 40-45%, 20-40%, 25-40%, 30-40%, 35-40%, 20-35%, 25-35%, 30-35%, 20-30%, 25-30%, or 20-25%. In one embodiment, the polyol is used to increase the conformational rigidity of the protein glutaminase through hydrogen bonding, stabilizing the nonpolar surface of the protein glutaminase and preventing protein aggregation. In one embodiment, the polyol is selected from the group consisting of glycerol, sorbitol, xylitol, propylene glycol, and neopentyl glycol. In one embodiment, the composition contains 20-75%, 20-60%, or 30-55% glycerol and / or sorbitol by weight percentage.

[0076] In one embodiment, the composition comprises, by weight percentage, 20-75%, 25-75%, 30-75%, 35-75%, 40-75%, 45-75%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 20-70%, 25-70%, 30-70%, 35-70%, 40-70%, 45-70%, 50-70%, 55-70%, 60-70%, 65-70%, 20-65%, 25-65%, 30-65%, 35-65%, 40-65%, 45-65%, 50-65%, 55-65%, 60-65%, 20-60%, 25-6% Sugars in the range of 0%, 30-60%, 35-60%, 40-60%, 45-60%, 50-60%, 55-60%, 20-55%, 25-55%, 30-55%, 35-55%, 40-55%, 45-55%, 50-55%, 20-50%, 25-50%, 30-50%, 35-50%, 40-50%, 45-50%, 20-45%, 25-45%, 30-45%, 35-45%, 40-45%, 20-40%, 25-40%, 30-40%, 35-40%, 20-35%, 25-35%, 30-35%, 20-30%, 25-30%, or 20-25%. In one embodiment, the sugars are used to maintain the conformation and water-holding capacity of the protein glutaminase active site, protecting and stabilizing enzyme activity. In one embodiment, the sugar is selected from the group consisting of trehalose, sorbitol, glucose, sucrose, and fructose. In one embodiment, the composition comprises a total of 20-75%, 20-60%, or 30-55% trehalose, glucose, and / or sucrose by weight percentage.

[0077] In one embodiment, the composition comprises, by weight percentage: 0.5-8%, 1-8%, 1.5-8%, 2-8%, 2.5-8%, 3-8%, 3.5-8%, 4-8%, 4.5-8%, 5-8%, 5.5-8%, 6-8%, 6.5-8%, 7-8%, 7.5-8%, 0.5-7.5%, 1-7.5%, 1.5-7.5%, 2-7.5%, 2.5-7.5%, 3-7.5%, 3.5-7.5%, 4-7.5%, 4.5-7.5%, 5-7.5%, 5.5-7.5%, 6-7.5%, 6.5-7.5%. %, 7-7.5%, 0.5-7%, 1-7%, 1.5-7%, 2-7%, 2.5-7%, 3-7%, 3.5-7%, 4-7%, 4.5-7%, 5-7%, 5.5-7%, 6-7%, 6.5-7%, 0.5-6.5%, 1-6.5%, 1.5-6.5%, 2-6.5%, 2.5-6.5%, 3-6.5%, 3.5-6.5%, 4-6.5%, 4.5-6.5%, 5-6.5%, 5.5-6.5%, 6-6.5%, 0.5-6%, 1-6%, 1.5-6%, 2-6%, 2.5-6%, 3- 6%, 3.5-6%, 4-6%, 4.5-6%, 5-6%, 5.5-6%, 0.5-5.5%, 1-5.5%, 1.5-5.5%, 2-5.5%, 2.5-5.5%, 3-5.5%, 3.5-5.5%, 4-5.5%, 4.5-5.5%, 5-5.5%, 0.5-5%, 1-5%, 1.5-5%, 2-5%, 2.5-5%, 3-5%, 3.5-5%, 4-5%, 4.5-5%, 0.5-4.5%, 1-4.5%, 1.5-4.5%, 2-4.5%, 2.5-4.5%, 3-4.5%. Edible gums at concentrations of 3.5-4.5%, 4-4.5%, 0.5-4%, 1-4%, 1.5-4%, 2-4%, 2.5-4%, 3-4%, 3.5-4%, 0.5-3.5%, 1-3.5%, 1.5-3.5%, 2-3.5%, 2.5-3.5%, 3-3.5%, 0.5-3%, 1-3%, 1.5-3%, 2-3%, 2.5-3%, 0.5-2.5%, 1-2.5%, 1.5-2.5%, 2-2.5%, 0.5-2%, 1-2%, 1.5-2%, 0.5-1.5%, 1-1.5%, or 0.5-1%. In one embodiment, the edible gums synergistically form a micronetwork structure with polyols and sugars, fixing the conformation of the protein glutaminase and preventing mechanical vibration or freeze-thaw damage. In one embodiment, the edible gum is selected from the group consisting of gelatin, pectin, guar gum, carrageenan, locust bean gum, xanthan gum, agar, sodium alginate, and konjac gum.In one embodiment, the edible gum is selected from the group consisting of gelatin, guar gum, and xanthan gum. In one embodiment, the composition comprises a total of 0.5-8%, 0.5-5%, or 0.5-3% gelatin, guar gum, and / or xanthan gum by weight percentage.

[0078] In one embodiment, the composition comprises, by weight percentage, 0.05-1%, 0.08-1%, 0.1-1%, 0.15-1%, 0.2-1%, 0.3-1%, 0.4-1%, 0.5-1%, 0.6-1%, 0.7-1%, 0.8-1%, 0.9-1%, 0.05-0.9%, 0.08-0.9%, 0.1-0.9%, 0.15-0.9%, 0.2-0.9%, 0.3-0.9%, 0.4-0.9%, 0.5-0.9%, 0 0.6-0.9%, 0.7-0.9%, 0.8-0.9%, 0.05-0.8%, 0.08-0.8%, 0.1-0.8%, 0.15-0.8%, 0.2-0.8%, 0.3-0.8%, 0.4-0.8%, 0.5-0.8%, 0.6-0.8%, 0.7-0.8%, 0.05-0.7%, 0.08-0.7%, 0.1-0.7%, 0.15-0.7%, 0.2-0.7%, 0.3-0.7%, 0.4-0.7%, 0 0.5-0.7%, 0.6-0.7%, 0.05-0.6%, 0.08-0.6%, 0.1-0.6%, 0.15-0.6%, 0.2-0.6%, 0.3-0.6%, 0.4-0.6%, 0.5-0.6%, 0.05-0.5%, 0.08-0.5%, 0.1-0.5%, 0.15-0.5%, 0.2-0.5%, 0.3-0.5%, 0.4-0.5%, 0.05-0.4%, 0.08-0.4%, 0.1-0.4% Sulfur-containing antioxidants at concentrations of 0.15-0.4%, 0.2-0.4%, 0.3-0.4%, 0.05-0.3%, 0.08-0.3%, 0.1-0.3%, 0.15-0.3%, 0.2-0.3%, 0.05-0.2%, 0.08-0.2%, 0.1-0.2%, 0.15-0.2%, 0.05-0.15%, 0.08-0.15%, 0.1-0.15%, 0.05-0.1%, 0.08-0.1%, or 0.05-0.08%. In one embodiment, the sulfur-containing antioxidant is used to neutralize free radicals to protect the active site of protein glutaminase, prevent protein oxidation, and synergize with edible gums to further enhance the conformational rigidity of the protein glutaminase molecule and prolong its half-life. In one embodiment, the sulfur-containing antioxidant is selected from the group consisting of glutathione, sodium sulfite, and ergothioneine. In one embodiment, the composition comprises a total of 0.05-1%, 0.05-0.5%, or 0.05-0.3% glutathione (GSH) and / or sodium sulfite by weight percentage.

[0079] In one embodiment, the composition comprises, by weight percentage, 1-20%, 2-20%, 5-20%, 8-20%, 10-20%, 12-20%, 15-20%, 18-20%, 1-18%, 2-18%, 5-18%, 8-18%, 10-18%, 12-18%, 15-18%, 1-15%, 2-15%, 5-15%, 8-15%, 10-15%, 12-15%, 1-12%, 2-12%, 5-12%, 8-12%, 10-12%, 1-10%, 2-10%, 5-10%, 8-10%, 1-8%, 2-8%, 5-8%, 1-5%, 2-5%, or 1-2% buffer solution. In one embodiment, the buffer solution is 25-75 mM, 30-75 mM, 35-75 mM, 40-75 mM, 45-75 mM, 50-75 mM, 55-75 mM, 60-75 mM, 65-75 mM, 70-75 mM, 25-70 mM, 30-70 mM, 35-70 mM, 40-70 mM, 45-70 mM, 50-70 mM, 55-70 mM, 60-70 mM, 65-70 mM, 25-65 mM, 30-65 mM, 35-65 mM, 40-65 mM, 45-65 mM, 50-65 mM, 55-65 mM, 60-65 mM, 25-60 mM, 30-60 mM, 3 ... Buffer solutions of mM, 40-60 mM, 45-60 mM, 50-60 mM, 55-60 mM, 25-55 mM, 30-55 mM, 35-55 mM, 40-55 mM, 45-55 mM, 50-55 mM, 25-50 mM, 30-50 mM, 35-50 mM, 40-50 mM, 45-50 mM, 25-45 mM, 30-45 mM, 35-40 mM, 40-45 mM, 25-40 mM, 30-40 mM, 35-40 mM, 25-35 mM, 30-35 mM, or 30-35 mM. In one embodiment, the buffer solution is used to maintain the constancy of the storage environment for protein glutaminase. In one embodiment, the buffer is selected from the group consisting of phosphate buffer, citrate buffer, and Tris-hydrochloric acid buffer. In one embodiment, the composition comprises, by weight percentage, a total of 1-20%, 1-15%, or 1-12% of 50 mM phosphate buffer and / or 50 mM citrate buffer.

[0080] In one embodiment, the composition comprises, by weight percentage, 0-5%, 0.5-5%, 1-5%, 1.5-5%, 2-5%, 2.5-5%, 3-5%, 3.5-5%, 4-5%, 4.5-5%, 0-4.5%, 0.5-4.5%, 1-4.5%, 1.5-4.5%, 2-4.5%, 2.5-4.5%, 3-4.5%, 3.5-4.5%, 4-4.5%, 0-4%, 0.5-4%, 1-4%, 1.5-4%, 2-4%, 2.5-4%, 3-4%, 3.5-4%. The chlorides are 0-3.5%, 0.5-3.5%, 1-3.5%, 1.5-3.5%, 2-3.5%, 2.5-3.5%, 3-3.5%, 0-3%, 0.5-3%, 1-3%, 1.5-3%, 2-3%, 2.5-3%, 0-2.5%, 0.5-2.5%, 1-2.5%, 1.5-2.5%, 2-2.5%, 0-2%, 0.5-2%, 1-2%, 1.5-2%, 0-1.5%, 0.5-1.5%, 1-1.5%, 0-1%, 0.5-1%, or 0-0.5%. In one embodiment, the metal ions in the chloride act as cofactors for the enzyme, maintaining and promoting the function of protein glutaminase. In one embodiment, the chloride is selected from the group consisting of calcium chloride, magnesium chloride, ferric chloride, sodium chloride, and potassium chloride. In one embodiment, the composition comprises, by weight percentage, a total of 0-5%, 0-1%, or 0-0.5% calcium chloride, magnesium chloride, and / or ferric chloride.

[0081] In one embodiment, the components of the composition work synergistically to maintain the activity, thermal stability, and storage stability of the protein glutaminase and to expand the application scenarios of the enzyme.

[0082] 2. A method for preparing the stabilizer composition of the present invention.

[0083] In one aspect, a method is provided for preparing the stabilizer composition of the present invention, comprising mixing the components in the stated mass percentages to prepare the stabilizer composition. In one embodiment, the method comprises uniformly mixing the components in the stated mass percentages to prepare the stabilizer composition. In one embodiment, the method comprises adding the components in the stated mass percentages in the following order: polyol, sugar, edible gum, antioxidant, optional chloride, buffer solution, and water.

[0084] 3. The liquid formulation of the protein glutaminase of the present invention.

[0085] In one aspect, a liquid formulation of protein glutaminase is provided, comprising (a) a protein glutaminase solution of 1-10000 U / ml; and (b) a stabilizer composition of the present invention, by weight, comprising 50-90% of the formulation. In one embodiment, the liquid formulation comprises (a) a protein glutaminase solution of 250-7500 U / ml; and (b) a stabilizer composition of the present invention, by weight, comprising 50-80% of the formulation. In one embodiment, the liquid formulation comprises (a) a protein glutaminase solution of 500-5000 U / ml; and (b) a stabilizer composition of the present invention, by weight, comprising 50-60% of the formulation.

[0086] In one embodiment, the liquid formulation comprises 1-10000 U / ml, 50-10000 U / ml, 100-10000 U / ml, 250-10000 U / ml, 500-10000 U / ml, 750-10000 U / ml, 1000-10000 U / ml, 2500-10000 U / ml, 5000-10000 U / ml, 7500-10000 U / ml, 1-7500 U / ml, 50-7500 U / ml, 100-7500 U / ml, 250-7500 U / ml, 500-7500 U / ml, 750-7500 U / ml, 1000-7500 U / ml, 2500-7500 U / ml, 500-7500 U / ml, 750-7500 U / ml, 1000-7500 U / ml, 2500-7500 U / ml, 5000-7500 U / ml. U / ml, 1-5000 U / ml, 50-5000 U / ml, 100-5000 U / ml, 250-5000 U / ml, 500-5000 U / ml, 750-5000 U / ml, 1000-5000 U / ml, 2500-5000 U / ml, 1-2500 U / ml, 50-2500 U / ml, 100-2500U / ml, 250-2500 U / ml, 500-2500 U / ml, 750-2500 U / ml, 1000-2500 U / ml, 1-1000 U / ml, 50-1000 U / ml, 100-1000 U / ml, 250-1000 U / ml, 500-1000 U / ml, 750-1000 Protein glutaminase solutions with concentrations of 1-750 U / ml, 50-750 U / ml, 100-750 U / ml, 250-750 U / ml, 500-750 U / ml, 1-500 U / ml, 50-500 U / ml, 100-500 U / ml, 250-500 U / ml, 1-250 U / ml, 50-250 U / ml, 100-250 U / ml, 1-100 U / ml, 50-100 U / ml, or 1-50 U / ml. In one embodiment, the method includes providing a protein glutaminase solution with a concentration of 100-5000 U / ml.

[0087] In one embodiment, the protein glutaminase solution is obtained by fermentation using bacteria capable of expressing protein glutaminase, followed by centrifugation and ultrafiltration of the fermentation broth. In one embodiment, the bacteria capable of expressing protein glutaminase are selected from the group consisting of *Chryseobacterium*, *Streptomyces*, and *Bacillus*. In one embodiment, the bacteria are *Chryseobacterium proteolyticum* or *Bacillus subtilis*. In one embodiment, the protein glutaminase solution is obtained by fermentation using *Chryseobacterium proteolyticum*, followed by centrifugation and ultrafiltration of the fermentation broth.

[0088] In one embodiment, the centrifugation is carried out under the following conditions: (i) a centrifugation speed of 5000-15000 RPM; (ii) a centrifugation temperature of 0-30°C; (iii) a centrifugation time of 15 minutes to 3 hours; and / or (iv) a pH of 6.5-8.0 during the centrifugation process.In one embodiment, the centrifugation speed is 5000-15000 RPM, 6000-15000 RPM, 7000-15000 RPM, 8000-15000 RPM, 9000-15000 RPM, 10000-15000 RPM, 11000-15000 RPM, 12000-15000 RPM, 13000-15000 RPM, 14000-15000 RPM, 5000-14000 RPM, 6000-14000 RPM, 7000-14000 RPM, 8000-14000 RPM, 9000-14000 RPM, 10000-14000 RPM, 11000-14000 RPM, 12000-14000 RPM. RPM, 13000-14000 RPM, 5000-13000 RPM, 6000-13000 RPM, 7000-13000RPM, 8000-13000 RPM, 9000-13000 RPM, 10000-13000 RPM, 11000-13000 RPM, 12000-13000RPM, 5000-12000 RPM, 6000-12000 RPM, 7000-12000 RPM, 8000-12000 RPM, 9000-12000RPM, 10000-12000 RPM, 11000-12000 RPM, 5000-11000 RPM, 6000-11000 RPM, 7000-11000RPM, 8000-11000 RPM, 9000-11000 RPM, 10000-11000 RPM, 5000-10000 RPM, 6000-10000RPM, 7000-10000 RPM, 8000-10000 RPM, 9000-10000 RPM, 5000-9000 RPM, 6000-9000 RPM, 7000-9000 RPM, 8000-9000 RPM, 5000-8000 RPM, 6000-8000 RPM, 7000-8000 RPM, 5000-7000 RPM, 6000-7000 RPM or 5000-6000 In one embodiment, the centrifugation temperature is 0-30°C, 5-30°C, 10-30°C, 15-30°C, 20-30°C, 25-30°C, 0-25°C, 5-25°C, 10-25°C, 15-25°C, 20-25°C, 0-20°C, 5-20°C, 10-20°C, 15-20°C, 0-15°C, 5-15°C, 10-15°C, 0-10°C, 5-10°C, or 0-5°C.In one embodiment, the duration of the centrifugation is 15 minutes to 3 hours, 30 minutes to 3 hours, 45 minutes to 3 hours, 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, 2.5 to 3 hours, 15 minutes to 2.5 hours, 30 minutes to 2.5 hours, 45 minutes to 2.5 hours, 1 to 2.5 hours, 1.5 to 2.5 hours, 2 to 2.5 hours, 15 minutes to 2 hours, 30 minutes to 2 hours, 45 minutes to 2 hours, 1 to 2 hours, 1.5 to 2 hours, 15 minutes to 1.5 hours, 30 minutes to 1.5 hours, 45 minutes to 1.5 hours, 1 to 1.5 hours, 15 minutes to 1 hour, 30 minutes to 1 hour, 45 minutes to 1 hour, 15 to 45 minutes, 30 to 45 minutes, or 15 to 30 minutes. In one embodiment, the pH during the centrifugation process is 6.5-8.0, 6.6-8.0, 6.8-8.0, 7.0-8.0, 7.2-8.0, 7.4-8.0, 7.6-8.0, 7.8-8.0, 6.5-7.8, 6.6-7.8, 6.8-7.8, 7.0-7.8, 7.2-7.8, 7.4-7.8, 7.6-7.8, 6.5-7.6, or 6.6-7. 6, 6.8-7.6, 7.0-7.6, 7.2-7.6, 7.4-7.6, 6.5-7.4, 6.6-7.4, 6.8-7.4, 7.0-7.4, 7.2-7.4, 6.5-7.2, 6.6-7.2, 6.8-7.2, 7.0-7.2, 6.5-7.0, 6.6-7.0, 6.8-7.0, 6.5-6.8, 6.6-6.8, or 6.5-6.6. In one embodiment, the supernatant after centrifugation of the *Aureobacillus prionatus* fermentation broth includes impurities selected from the group consisting of multiple proteases and small molecule impurities. In one embodiment, the multiple proteases include multiple metalloproteinases. In one embodiment, the small molecule impurities are selected from the group consisting of monosaccharides, organic acids, inorganic acids, and inorganic salts. In one embodiment, the impurities affect the stability of protein glutaminase.

[0089] In one embodiment, the supernatant after centrifugation is subjected to ultrafiltration membrane treatment. In one embodiment, the ultrafiltration membrane used for the ultrafiltration membrane treatment has a molecular weight cutoff of 3-100 kDa, 5-100 kDa, 8-100 kDa, 10-100 kDa, 25-100 kDa, 50-100 kDa, 75-100 kDa, 3-75 kDa, 5-75 kDa, 8-75 kDa, 10-75 kDa, 25-75 kDa, 50-75 kDa, 3-50 kDa, 5-50 kDa, 8-50 kDa, 10-50 kDa, 25-50 kDa, 3-25 kDa, 5-25 kDa, 8-25 kDa, 10-25 kDa, 3-10 kDa, 5-10 kDa, 8-10 kDa, 3-8 kDa, 5-8 kDa, or 3-5 kDa. In one embodiment, the ultrafiltration membrane used in the ultrafiltration membrane treatment has a molecular weight cutoff of 3 kDa, 5 kDa, 8 kDa, 10 kDa, 25 kDa, 50 kDa, 75 kDa, or 100 kDa. In another embodiment, the ultrafiltration membrane used in the ultrafiltration membrane treatment has a molecular weight cutoff of 10 kDa. In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3-50 times, 5-50 times, 8-50 times, 10-50 times, 15-50 times, 20-50 times, 25-50 times, 30-50 times, 35-50 times, 40-50 times, 45-50 times, 3-45 times, 5-45 times, 8-45 times, 10-45 times, 15-45 times, 20-45 times, 25-45 times, 30-45 times, 35-45 times, 40-45 times, 3-40 times, 5-40 times, 8-40 times, 10-40 times, 15-40 times, 20-40 times, 25-40 times, 30-40 times, 35- 40x, 3-35x, 5-35x, 8-35x, 10-35x, 15-35x, 20-35x, 25-35x, 30-35x, 3-30x, 5-30x, 8-30x, 10-30x, 15-30x, 20-30x, 25-30x, 3-25x, 5-25x, 8-25x, 10-25x, 15-25x, 20-25x, 3-20x, 5-20x, 8-20x, 10-20x, 15-20x, 3-15x, 5-15x, 8-15x, 10-15x, 3-10x, 5-10x, 8-10x, 3-8x, 5-8x or 3-5x. In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 10 times. In another embodiment, the ultrafiltration membrane treatment removes some of the protease and most of the small molecule impurities while concentrating the fermentation broth supernatant, thereby greatly improving the stability of the protein glutaminase solution.

[0090] In one embodiment, the protein glutaminase solution is sterilized by filtration. In another embodiment, the protein glutaminase solution is sterilized by passing it through a filter membrane with a diameter of 0.22-0.45 μm, 0.25-0.45 μm, 0.3-0.45 μm, 0.35-0.45 μm, 0.4-0.45 μm, 0.22-0.4 μm, 0.25-0.4 μm, 0.3-0.4 μm, 0.35-0.4 μm, 0.22-0.35 μm, 0.25-0.35 μm, 0.3-0.35 μm, 0.22-0.3 μm, 0.25-0.3 μm, or 0.22-0.25 μm. In yet another embodiment, the protein glutaminase solution is sterilized by passing it through a 0.22 μm sterile filter membrane.

[0091] In one embodiment, the liquid formulation comprises, by weight, 50-90%, 55-90%, 60-90%, 65-90%, 70-90%, 75-90%, 80-90%, 85-90%, 50-85%, 55-85%, 60-85%, 65-85%, 70-85%, 75-85%, 80-85%, 50-80%, 55-80%, 60-80%, 65-80%, 70-80%, 75-80%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 50-70%, 55-70%, 60-70%, 65-70%, 50-65%, 55-65%, 60-65%, 50-60%, 55-60%, or 50-55% of the stabilizer composition of the present invention. In one embodiment, the stabilizer composition is subjected to high-temperature sterilization. In another embodiment, the stabilizer composition is sterilized at 90-115°C, 92-115°C, 95-115°C, 98-115°C, 100-115°C, 102-115°C, 105-115°C, 108-115°C, 110-115°C, 112-115°C, 90-112°C, 92-112°C, 95-112°C, 98-112°C, 100-112°C, 10... 2-112℃, 105-112℃, 108-112℃, 110-112℃, 90-110℃, 92-110℃, 95-110℃, 98-110℃, 100-110℃, 102-110℃, 105-110℃, 108-110℃, 90-108℃, 92-108℃, 95-108℃, 98-108℃, 100-108℃, 102-1 08℃, 105-108℃, 90-105℃, 92-105℃, 95-105℃, 98-105℃, 100-105℃, 102-105℃, 90-102℃, 92-102℃, 95-102℃, 98-102℃, 100-102℃, 90-100℃, 92-100℃, 95-100℃, 98-100℃, 90-98℃, 92-98℃ Heat treatment at ℃, 95-98℃, 90-95℃, 92-95℃, 90-92℃ for 5-20, 8-20, 10-20, 12-20, 15-20, 18-20, 5-18, 8-18, 10-18, 12-18, 15-18, 5-15, 8-15, 10-15, 12-15, 5-12, 8-12, 10-12, 5-10, 8-10 or 5-8 minutes.

[0092] In one embodiment, the pH of the liquid formulation is 5.0-8.5, 5.5-8.5, 6.0-8.5, 6.5-8.5, 7.0-8.5, 7.5-8.5, 8.0-8.5, 5.0-8.0, 5.5-8.0, 6.0-8.0, 6.5-8.0, 7.0-8.0, 7.5-8.0, 5.0-7.5, 5.5-7.5, 6.0-7.5, 6.5-7.5, 7.0-7.5, 5.0-7.0, 5.5-7.0, 6.0-7.0, 6.5-7.0, 5.0-6.5, 5.5-6.5, 6.0-6.5, 5.0-6.0, 5.5-6.0, or 5.0-5.5. In one embodiment, the enzyme activity of the liquid formulation remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of its initial enzyme activity after being stored at room temperature for 30 days. In another embodiment, the enzyme activity of the liquid formulation remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of its initial enzyme activity after being stored at room temperature for 180 days. In one embodiment, the enzyme activity of the liquid formulation remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of its initial enzyme activity after being stored at room temperature for 365 days. In one embodiment, the room temperature is 20-25°C.

[0093] 4. A method for preparing the liquid formulation of the present invention.

[0094] In one aspect, a method for preparing a liquid formulation of the protein glutaminase of the present invention is provided, comprising: (i) providing a protein glutaminase solution of 1-10000 U / ml and a stabilizer composition of the present invention; and (ii) mixing the protein glutaminase solution with the stabilizer composition of the present invention such that the stabilizer composition of the present invention accounts for 50-90% by weight of the liquid formulation. In one embodiment, the method comprises: (i) providing a protein glutaminase solution of 250-7500 U / ml and a stabilizer composition of the present invention; and (ii) mixing the protein glutaminase solution with the stabilizer composition of the present invention such that the stabilizer composition of the present invention accounts for 50-80% by weight of the liquid formulation. In one embodiment, the method comprises: (i) providing a protein glutaminase solution of 500-5000 U / ml and a stabilizer composition of the present invention; and (ii) mixing the protein glutaminase solution with the stabilizer composition of the present invention such that the stabilizer composition of the present invention accounts for 50-60% by weight of the liquid formulation.

[0095] In one embodiment, the method includes providing 1-10000 U / ml, 50-10000 U / ml, 100-10000 U / ml, 250-10000 U / ml, 500-10000 U / ml, 750-10000 U / ml, 1000-10000 U / ml, 2500-10000 U / ml, 5000-10000 U / ml, 7500-10000 U / ml, 1-7500 U / ml, 50-7500 U / ml, 100-7500 U / ml, 250-7500 U / ml, 500-7500 U / ml, 750-7500 U / ml, 1000-7500 U / ml, 2500-7500 U / ml, 500-7500 U / ml, 1000-7500 U / ml, 2500-7500 U / ml, 5000-7500 U / ml. U / ml, 1-5000 U / ml, 50-5000 U / ml, 100-5000 U / ml, 250-5000 U / ml, 500-5000 U / ml, 750-5000 U / ml, 1000-5000 U / ml, 2500-5000 U / ml, 1-2500 U / ml, 50-2500 U / ml, 100-2500U / ml, 250-2500 U / ml, 500-2500 U / ml, 750-2500 U / ml, 1000-2500 U / ml, 1-1000 U / ml, 50-1000 U / ml, 100-1000 U / ml, 250-1000 U / ml, 500-1000 U / ml, 750-1000 Protein glutaminase solutions with concentrations of 1-750 U / ml, 50-750 U / ml, 100-750 U / ml, 250-750 U / ml, 500-750 U / ml, 1-500 U / ml, 50-500 U / ml, 100-500 U / ml, 250-500 U / ml, 1-250 U / ml, 50-250 U / ml, 100-250 U / ml, 1-100 U / ml, 50-100 U / ml, or 1-50 U / ml. In one embodiment, the method includes providing a protein glutaminase solution with a concentration of 100-5000 U / ml.

[0096] In one embodiment, the protein glutaminase solution is obtained by fermentation using bacteria capable of expressing protein glutaminase and subsequent solid-liquid separation of the fermentation broth. In one embodiment, the bacteria capable of expressing protein glutaminase are selected from the group consisting of *Chryseobacterium*, *Streptomyces*, and *Bacillus*. In one embodiment, the bacteria are *Chryseobacterium proteolyticum* or *Bacillus subtilis*. In one embodiment, the solid-liquid separation includes centrifugation and ultrafiltration. In one embodiment, the protein glutaminase solution is obtained by fermentation using *Chryseobacterium proteolyticum* and subsequent centrifugation and ultrafiltration of the fermentation broth.

[0097] In one embodiment, the centrifugation is carried out under the following conditions: (i) a centrifugation speed of 5000-15000 RPM; (ii) a centrifugation temperature of 0-30°C; (iii) a centrifugation time of 15 minutes to 3 hours; and / or (iv) a pH of 6.5-8.0 during the centrifugation process.In one embodiment, the centrifugation speed is 5000-15000 RPM, 6000-15000 RPM, 7000-15000 RPM, 8000-15000 RPM, 9000-15000 RPM, 10000-15000 RPM, 11000-15000 RPM, 12000-15000 RPM, 13000-15000 RPM, 14000-15000 RPM, 5000-14000 RPM, 6000-14000 RPM, 7000-14000 RPM, 8000-14000 RPM, 9000-14000 RPM, 10000-14000 RPM, 11000-14000 RPM, 12000-14000 RPM. RPM, 13000-14000 RPM, 5000-13000 RPM, 6000-13000 RPM, 7000-13000RPM, 8000-13000 RPM, 9000-13000 RPM, 10000-13000 RPM, 11000-13000 RPM, 12000-13000RPM, 5000-12000 RPM, 6000-12000 RPM, 7000-12000 RPM, 8000-12000 RPM, 9000-12000RPM, 10000-12000 RPM, 11000-12000 RPM, 5000-11000 RPM, 6000-11000 RPM, 7000-11000RPM, 8000-11000 RPM, 9000-11000 RPM, 10000-11000 RPM, 5000-10000 RPM, 6000-10000RPM, 7000-10000 RPM, 8000-10000 RPM, 9000-10000 RPM, 5000-9000 RPM, 6000-9000 RPM, 7000-9000 RPM, 8000-9000 RPM, 5000-8000 RPM, 6000-8000 RPM, 7000-8000 RPM, 5000-7000 RPM, 6000-7000 RPM or 5000-6000 In one embodiment, the centrifugation temperature is 0-30°C, 5-30°C, 10-30°C, 15-30°C, 20-30°C, 25-30°C, 0-25°C, 5-25°C, 10-25°C, 15-25°C, 20-25°C, 0-20°C, 5-20°C, 10-20°C, 15-20°C, 0-15°C, 5-15°C, 10-15°C, 0-10°C, 5-10°C, or 0-5°C.In one embodiment, the duration of the centrifugation is 15 minutes to 3 hours, 30 minutes to 3 hours, 45 minutes to 3 hours, 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, 2.5 to 3 hours, 15 minutes to 2.5 hours, 30 minutes to 2.5 hours, 45 minutes to 2.5 hours, 1 to 2.5 hours, 1.5 to 2.5 hours, 2 to 2.5 hours, 15 minutes to 2 hours, 30 minutes to 2 hours, 45 minutes to 2 hours, 1 to 2 hours, 1.5 to 2 hours, 15 minutes to 1.5 hours, 30 minutes to 1.5 hours, 45 minutes to 1.5 hours, 1 to 1.5 hours, 15 minutes to 1 hour, 30 minutes to 1 hour, 45 minutes to 1 hour, 15 to 45 minutes, 30 to 45 minutes, or 15 to 30 minutes. In one embodiment, the pH during the centrifugation process is 6.5-8.0, 6.6-8.0, 6.8-8.0, 7.0-8.0, 7.2-8.0, 7.4-8.0, 7.6-8.0, 7.8-8.0, 6.5-7.8, 6.6-7.8, 6.8-7.8, 7.0-7.8, 7.2-7.8, 7.4-7.8, 7.6-7.8, 6.5-7.6, or 6.6-7. 6, 6.8-7.6, 7.0-7.6, 7.2-7.6, 7.4-7.6, 6.5-7.4, 6.6-7.4, 6.8-7.4, 7.0-7.4, 7.2-7.4, 6.5-7.2, 6.6-7.2, 6.8-7.2, 7.0-7.2, 6.5-7.0, 6.6-7.0, 6.8-7.0, 6.5-6.8, 6.6-6.8, or 6.5-6.6. In one embodiment, the supernatant after centrifugation of the *Aureobacillus prionatus* fermentation broth includes impurities selected from the group consisting of multiple proteases and small molecule impurities. In one embodiment, the multiple proteases include multiple metalloproteinases. In one embodiment, the small molecule impurities are selected from the group consisting of monosaccharides, organic acids, inorganic acids, and inorganic salts. In one embodiment, the impurities affect the stability of protein glutaminase.

[0098] In one embodiment, the supernatant after centrifugation is subjected to ultrafiltration membrane treatment. In one embodiment, the ultrafiltration membrane used for the ultrafiltration membrane treatment has a molecular weight cutoff of 3-100 kDa, 5-100 kDa, 8-100 kDa, 10-100 kDa, 25-100 kDa, 50-100 kDa, 75-100 kDa, 3-75 kDa, 5-75 kDa, 8-75 kDa, 10-75 kDa, 25-75 kDa, 50-75 kDa, 3-50 kDa, 5-50 kDa, 8-50 kDa, 10-50 kDa, 25-50 kDa, 3-25 kDa, 5-25 kDa, 8-25 kDa, 10-25 kDa, 3-10 kDa, 5-10 kDa, 8-10 kDa, 3-8 kDa, 5-8 kDa, or 3-5 kDa. In one embodiment, the ultrafiltration membrane used in the ultrafiltration membrane treatment has a molecular weight cutoff of 3 kDa, 5 kDa, 8 kDa, 10 kDa, 25 kDa, 50 kDa, 75 kDa, or 100 kDa. In another embodiment, the ultrafiltration membrane used in the ultrafiltration membrane treatment has a molecular weight cutoff of 10 kDa. In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3-50 times, 5-50 times, 8-50 times, 10-50 times, 15-50 times, 20-50 times, 25-50 times, 30-50 times, 35-50 times, 40-50 times, 45-50 times, 3-45 times, 5-45 times, 8-45 times, 10-45 times, 15-45 times, 20-45 times, 25-45 times, 30-45 times, 35-45 times, 40-45 times, 3-40 times, 5-40 times, 8-40 times, 10-40 times, 15-40 times, 20-40 times, 25-40 times, 30-40 times, 35- 40x, 3-35x, 5-35x, 8-35x, 10-35x, 15-35x, 20-35x, 25-35x, 30-35x, 3-30x, 5-30x, 8-30x, 10-30x, 15-30x, 20-30x, 25-30x, 3-25x, 5-25x, 8-25x, 10-25x, 15-25x, 20-25x, 3-20x, 5-20x, 8-20x, 10-20x, 15-20x, 3-15x, 5-15x, 8-15x, 10-15x, 3-10x, 5-10x, 8-10x, 3-8x, 5-8x or 3-5x. In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 3, 5, 8, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times.In one embodiment, the ultrafiltration membrane treatment concentrates the protein glutaminase in the fermentation broth by 10 times. In another embodiment, the ultrafiltration membrane treatment removes some of the protease and most of the small molecule impurities while concentrating the fermentation broth supernatant, thereby greatly improving the stability of the protein glutaminase solution.

[0099] In one embodiment, the protein glutaminase solution is sterilized by filtration. In another embodiment, the protein glutaminase solution is sterilized by passing it through a filter membrane with a diameter of 0.22-0.45 μm, 0.25-0.45 μm, 0.3-0.45 μm, 0.35-0.45 μm, 0.4-0.45 μm, 0.22-0.4 μm, 0.25-0.4 μm, 0.3-0.4 μm, 0.35-0.4 μm, 0.22-0.35 μm, 0.25-0.35 μm, 0.3-0.35 μm, 0.22-0.3 μm, 0.25-0.3 μm, or 0.22-0.25 μm. In yet another embodiment, the protein glutaminase solution is sterilized by passing it through a 0.22 μm sterile filter membrane.

[0100] In one embodiment, the method includes providing the stabilizer composition of the present invention, and when mixing it with the protein glutaminase solution, the stabilizer composition of the present invention comprises, by weight, 50-90%, 55-90%, 60-90%, 65-90%, 70-90%, 75-90%, 80-90%, 85-90%, 50-85%, 55-85%, 60-85%, 65-85%, 70-85% of the liquid formulation. 75-85%, 80-85%, 50-80%, 55-80%, 60-80%, 65-80%, 70-80%, 75-80%, 50-75%, 55-75%, 60-75%, 65-75%, 70-75%, 50-70%, 55-70%, 60-70%, 65-70%, 50-65%, 55-65%, 60-65%, 50-60%, 55-60%, or 50-55%. In one embodiment, the stabilizer composition is subjected to high-temperature sterilization. In one embodiment, the stabilizer composition is used at temperatures of 90-115°C, 92-115°C, 95-115°C, 98-115°C, 100-115°C, 102-115°C, 105-115°C, 108-115°C, 110-115°C, 112-115°C, 90-112°C, 92-112°C, 95-112°C, 98-112°C, 100-112°C, and 10... 2-112℃, 105-112℃, 108-112℃, 110-112℃, 90-110℃, 92-110℃, 95-110℃, 98-110℃, 100-110℃, 102-110℃, 105-110℃, 108-110℃, 90-108℃, 92-108℃, 95-108℃, 98-108℃, 100-108℃, 102-1 08℃, 105-108℃, 90-105℃, 92-105℃, 95-105℃, 98-105℃, 100-105℃, 102-105℃, 90-102℃, 92-102℃, 95-102℃, 98-102℃, 100-102℃, 90-100℃, 92-100℃, 95-100℃, 98-100℃, 90-98℃, 92-98℃ Heat treatment at ℃, 95-98℃, 90-95℃, 92-95℃, 90-92℃ for 5-20, 8-20, 10-20, 12-20, 15-20, 18-20, 5-18, 8-18, 10-18, 12-18, 15-18, 5-15, 8-15, 10-15, 12-15, 5-12, 8-12, 10-12, 5-10, 8-10 or 5-8 minutes.

[0101] In one embodiment, the pH of the liquid formulation prepared by the method is 5.0-8.5, 5.5-8.5, 6.0-8.5, 6.5-8.5, 7.0-8.5, 7.5-8.5, 8.0-8.5, 5.0-8.0, 5.5-8.0, 6.0-8.0, 6.5-8.0, 7.0-8.0, 7.5-8.0, 5.0-7.5, 5.5-7.5, 6.0-7.5, 6.5-7.5, 7.0-7.5, 5.0-7.0, 5.5-7.0, 6.0-7.0, 6.5-7.0, 5.0-6.5, 5.5-6.5, 6.0-6.5, 5.0-6.0, 5.5-6.0, or 5.0-5.5. In one embodiment, the enzyme activity of the liquid formulation remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of the initial enzyme activity after being stored at room temperature for 30 days. In another embodiment, the enzyme activity of the liquid formulation prepared by the method remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of the initial enzyme activity after being stored at room temperature for 180 days. In one embodiment, the enzyme activity of the liquid formulation remains at 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% of its initial enzyme activity after being stored at room temperature for 365 days. In one embodiment, the room temperature is 20-25°C.

[0102] 5. Technical advantages and beneficial effects of the present invention

[0103] Compared with the prior art, the present invention has at least the following technical advantages and beneficial effects:

[0104] (i) The present invention provides a stabilizer composition for protein glutaminase, wherein the sugars and polyols in the composition work together to form a stable structure to protect enzyme activity, the edible gums work together with the polyols and sugars to form a micro-network structure to fix the conformation of protein glutaminase and prevent mechanical vibration or freeze-thaw damage, and the sulfur-containing antioxidants work together with the edible gums to further enhance the conformational rigidity of protein glutaminase molecules and prolong the half-life, and the metal ions act as cofactors of the enzyme to maintain and promote the functionality of protein glutaminase;

[0105] (ii) By centrifuging and ultrafiltration, a large number of inorganic impurities and proteases that affect the stability of protein glutaminase in the fermentation broth are removed, which significantly improves the overall stability of the liquid enzyme preparation. The liquid preparation can be stored at room temperature (20-25℃) for 365 days with an enzyme activity retention rate of more than 85%, preferably more than 95%. Detailed Implementation

[0106] The present invention will be further described below with reference to specific embodiments, but these specific embodiments should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make various changes or modifications to these specific embodiments without departing from the scope of the technical solution of the present invention, and the changed and modified implementation schemes still fall within the protection scope of the present invention.

[0107] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0108] Example 1. Production of protein glutaminase (PG enzyme) by fermentation using *Cyclophorus glutamate*.

[0109] This embodiment relates to a fermentation method for the efficient production of PG enzyme using *Cryptobrachium utilis*, comprising the following steps:

[0110] 1.1 Preparation of seed culture

[0111] Take 2 ml of glycerol tubes of *Aureobacterium utilis* that have been verified to have stable characteristics, and inoculate them into seed culture medium. The seed culture medium consists of the following components: 15-25 g / L (e.g., 17 g / L) soybean peptone; 2-5 g / L (e.g., 2.5 g / L) anhydrous magnesium sulfate; 1.5-5 g / L (e.g., 2 g / L) yeast extract, and adjust the pH to 5.5-6.5 (e.g., 6.0). Place the medium in a constant temperature shaker and incubate overnight for 8-12 hours (e.g., 12 hours) at 29-32℃ (e.g., 30℃) and 200-250 r / min (e.g., 200 rpm). Harvest the seed culture.

[0112] 1.2 Vaccination

[0113] The overnight seed culture was inoculated into a fermenter containing 1-10 L (e.g., 4 L) of fermentation medium, at an inoculation rate of 5-10% (e.g., 8%). The fermentation medium consisted of: 22-32 g / L (e.g., 25 g / L) soybean peptone; 1-5 g / L (e.g., 2-4 g / L) sodium dihydrogen phosphate dihydrate; 0.2-0.8 g / L (e.g., 0.3 g / L) magnesium sulfate heptahydrate; 0.1-0.5 g / L (e.g., 0.1 g / L) ferrous sulfate heptahydrate; 0.2-0.5 g / L (e.g., 0.3 g / L) potassium chloride; and 0.6-1.5 g / L (e.g., 0.8 g / L) anhydrous nickel sulfate. The pH was adjusted to 5.0-6.0 (e.g., 5.5).

[0114] 1.3 Fermentation

[0115] Set the fermentation temperature to 30-32℃ (e.g., 30℃), the aeration rate to 0.3-0.6 vvm (e.g., 0.5 vvm), control the dissolved oxygen value of the fermentation system to be no less than 20% (e.g., no less than 30%), and the agitator speed to 300-700 r / min. When fermentation has been going on for 4-6 hours (e.g., 5 hours), start adding the nutrient composition, which consists of the following components: 45-65 g / L (e.g., 60 g / L) soybean peptone; 5-10 g / L (e.g., 6 g / L) sodium dihydrogen phosphate dihydrate; 1-5 g / L (e.g., 2 g / L) magnesium sulfate heptahydrate; 0.3-0.8 g / L (e.g., 0.4 g / L) ferrous sulfate heptahydrate; and 0.3-0.8 g / L (e.g., 0.4 g / L) potassium chloride. Control the flow rate at 120-160 ml / h (e.g., 150 ml / h). Add 1-2 g / L (e.g., 1 g / L) of polyoxypropylene ethylene glycerol ether and 1-2 g / L (e.g., 1.5 g / L) of betaine after 18-22 hours (e.g., 20 hours) of fermentation. Take samples every 2 hours at 8, 12, 16, and 20 hours of fermentation to measure PG enzyme activity.

[0116] 1.4 PG enzyme activity assay method

[0117] 10 μL of fermentation supernatant was diluted 5-fold and added to a 96-well plate. 100 μL of 6.74 g / L Cbz-Gln-Gly solution was added. The reaction was incubated at 37℃ for 30 minutes, then 100 μL of TCA solution was added to terminate the reaction. The ammonia content of the solution after the reaction was determined by the phenol method, and the absorbance was measured at 630 nm (A1). Another 10 μL of fermentation supernatant was diluted 5-fold and added to a 96-well plate. 100 μL of TCA solution was added, and the reaction was incubated for 30 minutes. 100 μL of 6.74 g / L Cbz-Gln-Gly solution was added, and the absorbance was measured at 630 nm using the phenol method (A2). One unit of enzyme activity (U) is defined as the amount of enzyme required to produce 1 μmol of ammonia per minute. The enzyme activity calculation formula is as follows:

[0118] Enzyme activity (U / ml) = (A1-A2) × 21 × 5 / 17.03 / 30 / K.

[0119] Where A1: A of the experimental sample 630 Absorbance value; A2: Blank A 630 Absorbance value; K: Slope of the standard curve for ammonia determined by phenol hypochlorite spectrophotometry.

[0120] Example 2. Post-treatment of *Lactobacillus prionogenae* fermentation broth

[0121] This embodiment provides a method for post-processing the fermentation broth of *Aureobacillus prionatus*, including the following steps:

[0122] 2.1 Centrifugation

[0123] The fermentation broth of *Chlorella vulgaris* was separated using a solid-liquid separation device to remove solid impurities and obtain a supernatant containing a large amount of protein glutaminase.

[0124] The fermentation broth of *Aureobacillus prionatus* was subjected to solid-liquid separation using a centrifuge. The centrifugation speed was controlled at 5000-15000 rpm, the centrifugation time at 15 minutes to 3 hours, and the centrifugation temperature at 0-30℃. Preferably, the centrifugation speed was 8000-12000 rpm, the centrifugation time at 30 minutes to 1 hour, and the centrifugation temperature at 0-20℃.

[0125] In one specific implementation, the centrifugation speed is controlled at 10,000 rpm, the centrifugation time is 30 minutes, and the centrifugation temperature is 4°C.

[0126] 2.2 Ultrafiltration Membrane Treatment

[0127] The supernatant of the centrifuged *Gynostemma pentaphyllum* fermentation broth was processed through a membrane separation device. An ultrafiltration membrane with a suitable pore size was selected to remove some impurities, proteases, small sugars, and inorganic salts, resulting in a concentrated solution of protein glutaminase with high enzyme activity.

[0128] The supernatant of the fermentation broth of *Aureobacterium utilis* was further separated into solid and liquid components by ultrafiltration membrane equipment. The ultrafiltration membrane size was selected as 5-30 kD. The fermentation broth was concentrated to 1 / 5 to 1 / 20 of the initial volume. The ultrafiltration membrane retentate was recovered and aseptically filtered using a 0.22-0.45 μm filter membrane.

[0129] In one specific embodiment, an ultrafiltration membrane with a size of 10 kD is selected, the fermentation broth is concentrated to 1 / 10 of its initial volume, the ultrafiltration membrane retentate is recovered, and the filtrate is treated with aseptic filtration using a 0.22 μm filter membrane.

[0130] Example 3. Stabilizer composition 1 of the present invention

[0131] Prepare stabilizer composition 1 of the present invention according to Table 1 and sterilize it at 100°C for 10 minutes.

[0132] Table 1. Stabilizer composition 1 of the present invention

[0133]

[0134] The fermentation broth of *Aureobacterium utilis* obtained in Example 1 and the post-treated fermentation broth of *Aureobacterium utilis* obtained in Example 2 were respectively mixed with the stabilizer composition 1 of the present invention to obtain liquid enzyme preparations 1-2 of the present invention. The fermentation broth of Example 1 without the addition of the stabilizer composition 1 of the present invention (comparative enzyme preparation 1) was used as a control.

[0135] The stability of the liquid enzyme preparations of the present invention was measured: the enzyme activity A1 of protein glutaminase in enzyme preparation 1 and liquid enzyme preparations 1-2 of the present invention was measured as the original enzyme activity. After the preparations were kept at room temperature (20-25℃) for 1 month (30 days), the enzyme activity A2 was measured again. The residual percentage of protein glutaminase activity calculated according to the following formula was used as the stability indicator:

[0136] Stability = A2 / A1 * 100%

[0137] The stability results are shown in Table 2. The stability of the liquid enzyme preparations 1-2 of the present invention, with the addition of 80% of the stabilizer composition of the present invention, was significantly improved (3.4-5.1 times that of the control) compared to the untreated case. Furthermore, the stability-enhancing effect of the stabilizer composition of the present invention on post-treated fermentation broth was more significant than that on untreated case (1.5 times that of untreated case with post-treatment).

[0138] Table 2. Stability of liquid formulations of different protein glutaminases

[0139]

[0140] Example 4. Stabilizer compositions 2-7 of the present invention and comparative compositions 2-5

[0141] The fermentation broth of *Aureobacterium utilis* was collected and centrifuged to separate the solids and liquids. The centrifugation speed was controlled at 9000 rpm, the centrifugation time at 35 min, and the centrifugation temperature at 4℃. The centrifuged solids were discarded, and the fermentation supernatant of *Aureobacterium utilis* was retained. 10 L of the fermentation supernatant was collected and concentrated to 1 L using an ultrafiltration membrane with a size of 10 kD. The ultrafiltration membrane retentate was recovered and aseptically filtered through a 0.22 μm filter membrane to obtain a protein glutaminase solution of 500 U / mL.

[0142] Comparative compositions 2-5 and stabilizer compositions 3-8 of the present invention were prepared according to Table 3 and sterilized at 100 °C for 10 minutes respectively.

[0143] Table 3. Components and contents (wt%) of comparative compositions 2-5 and stabilizer compositions 2-7 of the present invention

[0144]

[0145] The protein glutaminase solution of 500 U / mL obtained above was mixed with comparative compositions 2-5 and stabilizer compositions 3-8 of the present invention in different proportions, and the stability was determined after storage at room temperature (20-25℃) for 12 months (365 days). The results are shown in Table 4.

[0146] Table 4. Stability of liquid formulations of different protein glutaminases

[0147]

[0148] As shown in Table 4, when the stabilizer compositions of the present invention are added at a content of 50-90%, the enzyme activity residual rate of the post-treated protein glutaminase (PG enzyme) solution reaches more than 85% after storage at room temperature (20-25°C) for 12 months (365 days). Among them, stabilizer composition 5 of the present invention can achieve an enzyme activity residual rate of up to 96% after storage at room temperature for 365 days, showing excellent stability. Compared with the results of other examples and comparative examples, it can be seen that the significant improvement in the stability of the liquid formulation of protein glutaminase of the present invention is the result of the synergistic effect between the polyols, sugars, edible gums, and sulfur-containing antioxidants in the stabilizer compositions of the present invention, as well as their combined effect with buffer solution and optional metal chloride components.

[0149] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any way. Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and the scope of protection of the present invention is defined by the appended specifications. Those skilled in the art can make various changes or modifications to the above-disclosed technical content without departing from the scope of the present invention, and all such changes and modifications fall within the scope of protection of the present invention.

Claims

1. A stabilizer composition for protein glutaminase, wherein the composition comprises or consists of the following components by weight percentage: (a) 20-75% polyols; (b) 20-75% carbohydrates; (c) 0.5-8% edible gums; (d) 0.05-1% sulfur-containing antioxidants; (e) 1-20% 25-75 mM buffer; (f) 0-5% chloride; and (g) The remaining amount of water.

2. The composition according to claim 1, wherein the composition comprises or consists of the following components by weight percentage: (a) 20-60% polyols; (b) 20-60% carbohydrates; (c) 0.5-5% edible gums; (d) 0.05-0.5% sulfur-containing antioxidants; (e) 1-15% 20-60 mM buffer; (f) 0-1% chloride; and (g) The remaining amount of water.

3. The composition according to claim 1 or 2, wherein the composition comprises or is composed of the following components by weight percentage: (a) 30-55% polyols; (b) 30-55% carbohydrates; (c) 0.5-3% edible gums; (d) 0.05-0.3% sulfur-containing antioxidants; (e) 1-12% 30-50 mM buffer; (f) 0-0.5% chloride; and (g) The remaining amount of water.

4. The composition according to any one of claims 1-3, wherein the polyol is selected from the group consisting of glycerol, sorbitol, xylitol, propylene glycol, and neopentyl glycol.

5. The composition according to any one of claims 1-4, wherein the sugar is selected from the group consisting of trehalose, sorbitol, glucose, sucrose, and fructose.

6. The composition according to any one of claims 1-5, wherein the edible gum is selected from the group consisting of gelatin, pectin, guar gum, carrageenan, locust bean gum, xanthan gum, agar, sodium alginate and konjac gum.

7. The composition according to any one of claims 1-6, wherein the sulfur-containing antioxidant is selected from the group consisting of glutathione, sodium sulfite, and ergothioneine.

8. The composition according to any one of claims 1-7, wherein the buffer is selected from the group consisting of phosphate buffer, citrate buffer and Tris-hydrochloric acid buffer.

9. The composition according to any one of claims 1-8, wherein the chloride is a chloride selected from the group consisting of calcium chloride, magnesium chloride, ferric chloride, sodium chloride, and potassium chloride.

10. A method for preparing a composition according to any one of claims 1-9, wherein the components are added in the following order by weight percentage: polyol, sugar, edible gum, antioxidant, optional chloride, buffer solution and water.

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