Method for rapidly and efficiently producing protein glutaminase

Abstract

The present invention relates to a method for the production of protein glutaminase (protein-glutaminase; the EC is 3.5. 1.44; the present invention relates to a seed medium composition and a fermentation medium composition for a PG enzyme (e.g., PG enzyme). The invention relates to a method for producing PG enzyme through fermentation. The method comprises the following steps: fermenting for 3-8 hours by adopting a fermentation medium composition, and feeding a nutrient composition, and supplementing the surfactant composition when the fermentation is carried out for 13-18 hours. According to the method, the fermentation period can be shortened by 29%, the fermentation enzyme activity is improved, and the enzyme activity can reach 12.3 U / ml after 20-hour fermentation.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for rapidly and efficiently producing protein glutaminase (PG enzyme) and / or improving the enzymatic activity efficiency of PG enzyme production. Background Art

[0002] Protein-glutaminase (EC 3.5.1.44; PG enzyme) is a new type of protein deamidase that can specifically catalyze the deamidation of glutamine residues in substrate proteins or polypeptides to form glutamate, while releasing ammonia. Due to the specificity of its catalytic function, it can modify proteins without changing the protein structure, such as solubility, emulsification, foaming and gelation. In addition, it can avoid excessive hydrolysis caused by chemical and other protease treatments that damage the original properties of proteins, and is an enzyme with great application prospects in the food processing industry. The enzymatic deamination of PG enzyme can improve the physical and chemical properties of plant proteins, thereby providing a new method for transforming them into plant protein foods comparable to animal proteins.

[0003] PG enzyme was first discovered and isolated from the bacterial Chryseobacterium proteolyticum strain 9670T by Yamaguchi et al. in 2000. PG enzyme derived from Chryseobacterium proteolyticum was approved by the National Health Commission as a new enzyme preparation for the food industry in 2020.

[0004] The production optimization of PG enzyme in Chryseobacterium utilis is currently achieved through two main approaches: strain selection and culture process optimization. For example, by screening new Chryseobacterium utilis, the fermentation enzyme activity can reach 2.15U / mL (patent publication number: CN114149934A); secondly, selecting an appropriate culture medium or culture process is usually an important means of production optimization. According to existing reports, there are many culture optimization methods that can improve the fermentation enzyme activity of PG: by adding zeolite as a pH stabilizer to the culture medium, the PG fermentation enzyme activity of Chryseobacterium utilis can be increased to 2.0U / mL (patent publication number: CN107325977A); by adding glycine and food-grade Tween-80, the PG yield can be increased by 244% (patent publication number: CN116463324). In order to reduce the production cost of PG enzyme, in addition to improving enzyme activity, improving the production efficiency of PG by shortening the fermentation cycle is a very effective strategy.

[0005] In summary, the prior art still lacks a method for rapidly and efficiently producing PG enzyme using Chryseobacterium utilis. In view of this, the present invention is proposed. Summary of the invention

[0006] The object of the present invention is to provide a method for producing PG enzyme using Chryseobacterium utilis, which can not only produce PG enzyme quickly and efficiently, but also greatly shorten the fermentation cycle and improve the fermentation enzyme activity. The specific technical scheme of the present invention is as follows:

[0007] 1. A method for producing protein glutaminase by fermentation, comprising:

[0008] (a) using the seed culture medium composition to perform seed culture on Chryseobacterium utilis to obtain a seed culture;

[0009] (b) fermenting the seed culture using a fermentation medium composition;

[0010] (c) adding the nutrient composition 3-8 hours into the fermentation; and

[0011] (d) optionally adding a surfactant composition after the fermentation has been carried out for 13-18 hours.

[0012] 2. The method according to claim 1, wherein the seed culture medium composition comprises:

[0013] 15-25 g / L, preferably 17-20 g / L of soy peptone,

[0014] 2-5 g / L, preferably 2.5-4 g / L, of anhydrous magnesium sulfate, and

[0015] 1.5-5g / L, preferably 2-4g / L yeast powder;

[0016] The pH of the seed culture medium composition is 5.5-6.5, preferably 5.8-6.2.

[0017] 3. The method according to item 1 or 2, wherein the seed culture is carried out under the following conditions:

[0018] (1) The culture temperature is 29-32°C;

[0019] (2) culturing under shaking conditions of 200-250 r / min; and / or

[0020] (3) The culture is carried out for 8-12 hours.

[0021] 4. The method according to any one of items 1 to 3, wherein the fermentation medium composition comprises:

[0022] 22-32 g / L, preferably 25-30 g / L of soy peptone,

[0023] 1-5 g / L, preferably 2-4 g / L of sodium dihydrogen phosphate dihydrate,

[0024] 0.2-0.8 g / L, preferably 0.3-0.7 g / L of magnesium sulfate heptahydrate,

[0025] 0.1-0.5 g / L, preferably 0.1-0.3 g / L of ferrous sulfate heptahydrate,

[0026] 0.2-0.5 g / L, preferably 0.3-0.5 g / L potassium chloride, and

[0027] 0.6-1.5 g / L, preferably 0.8-1.2 g / L, anhydrous nickel sulfate;

[0028] The pH of the fermentation medium composition is 5.0-6.0, preferably 5.2-5.6.

[0029] 5. The method according to any one of items 1 to 4, wherein the fermentation is carried out under the following conditions:

[0030] (1) Temperature of 30-32°C;

[0031] (2) Stirring at 300-700 r / min;

[0032] (3) The dissolved oxygen value of the system is not less than 30%; and / or

[0033] (4) Fermentation is carried out for 24-36 hours.

[0034] 6. The method according to any one of items 1 to 5, wherein in step (c), a nutrient composition is added after the fermentation is carried out for 4 to 6 hours, preferably 5 hours, and the nutrient composition comprises:

[0035] 45-65g / L, preferably 50-60g / L soy peptone,

[0036] 5-10 g / L, preferably 6-8 g / L of sodium dihydrogen phosphate dihydrate,

[0037] 1-5 g / L, preferably 2-4 g / L of magnesium sulfate heptahydrate,

[0038] 0.3-0.8 g / L, preferably 0.4-0.6 g / L of ferrous sulfate heptahydrate, and

[0039] 0.3-0.8 g / L, preferably 0.4-0.6 g / L potassium chloride.

[0040] 7. The method according to any one of items 1 to 6, wherein the nutrient composition in step (c) is added to the fermentation system at a rate of 120-160 ml / h, preferably 150 ml / h.

[0041] 8. The method according to any one of items 1 to 7, wherein in step (d), a surfactant composition is added after the fermentation is carried out for 14 to 17 hours, preferably 15 to 16 hours, and the surfactant composition comprises:

[0042] 1-2 g / L of polyoxypropylene oxyethylene glyceryl ether, preferably 1-1.5 g / L of polyoxypropylene oxyethylene glyceryl ether; and

[0043] 1-2 g / L betaine, preferably 1.5-2 g / L betaine.

[0044] In order to make the technical solution of the present invention clearer, the present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The growth curves (OD) of the bacterial cells obtained by fermentation using the method of the present invention and the control method without the feeding strategy of step (c) of the present invention are shown in FIG. 600 ) comparison.

[0046] Figure 2 Comparison of the enzyme activity levels (U / mL) of PG enzymes produced by the method of the present invention and the control method that does not adopt the feeding strategy of step (c) of the present invention;

[0047] Figure 3 A comparison of the enzyme activity levels (U / mL) of PG enzymes produced by the method of the present invention and the control method which does not adopt the surfactant addition strategy of step (d) of the present invention; and

[0048] Figure 4 The enzymatic activity levels (U / mL) of PG enzymes produced by the method of the present invention and the control method which does not adopt the feeding strategy of step (c) and the surfactant addition strategy of step (d) of the present invention are compared. DETAILED DESCRIPTION OF THE INVENTION

[0050] 1. The method for producing PG enzyme (PG) of the present invention

[0051] In one aspect, the present invention relates to a method for fermenting and producing PG enzyme and / or improving the production efficiency of PG produced by Chryseobacterium utilis, the method comprising the following steps: (a) using a seed culture medium composition to carry out seed culture of Chryseobacterium utilis to obtain a seed culture; (b) using a fermentation medium composition to ferment the seed culture; (c) adding a nutrient solution composition after 3-8 hours of fermentation; and (d) optionally adding a surfactant composition after 15-25 hours of fermentation.

[0052] In one embodiment, the seed culture medium composition comprises or consists of the following components: 15-25 g / L soy peptone, 2-5 g / L anhydrous magnesium sulfate, and 1.5-5 g / L yeast powder. In one embodiment, the pH of the seed culture medium composition is 5.5-6.5. In one embodiment, the seed culture medium composition comprises or consists of the following components: 17-20 g / L soy peptone, 2.5-4 g / L anhydrous magnesium sulfate, and 2-4 g / L yeast powder. In one embodiment, the pH of the seed culture medium composition is 5.8-6.2, preferably 6.0. In one embodiment, the seed culture is carried out under the following conditions: (1) the culture temperature is 29-32°C; (2) the culture is carried out under shaking conditions of 200-250 r / min; and / or (3) the culture is carried out for 8-12 hours. In one embodiment, a seed culture is obtained after the seed culture. In one embodiment, the seed culture is inoculated into a fermentation tank filled with a fermentation medium for fermentation culture. In one embodiment, the inoculation amount is 5%-11%, preferably 6%-11%, more preferably 8%-10%.

[0053] In one embodiment, the fermentation medium composition comprises or consists of the following components: 22-32 g / L soy peptone, 1-5 g / L sodium dihydrogen phosphate dihydrate, 0.2-0.8 g / L magnesium sulfate heptahydrate, 0.1-0.5 g / L ferrous sulfate heptahydrate, 0.2-0.5 g / L potassium chloride, and 0.6-1.5 g / L anhydrous nickel sulfate. In one embodiment, the pH of the fermentation medium composition is 5.0-6.0. In one embodiment, the fermentation medium composition comprises or consists of the following components: 25-30 g / L soy peptone, 2-4 g / L sodium dihydrogen phosphate dihydrate, 0.3-0.7 g / L magnesium sulfate heptahydrate, 0.1-0.3 g / L ferrous sulfate heptahydrate, 0.3-0.5 g / L potassium chloride, and 0.8-1.2 g / L anhydrous nickel sulfate. In one embodiment, the pH of the fermentation medium composition is 5.2-5.6, preferably 5.5. In one embodiment, the fermentation is carried out under the following conditions: (1) the temperature is 30-32°C; (2) under stirring at 300-700 r / min; (3) the dissolved oxygen value of the system is not less than 30%; and / or (4) the fermentation is carried out for 24-36 hours. In one embodiment, the seed culture is obtained after the seed culture.

[0054] In one embodiment, in step (c), the nutritional composition is supplemented after fermentation for 4-6 hours, 4.5-5.5 hours, 4.7-5.3 hours, 4.8-5.2 hours or 5 hours. In one embodiment, the nutritional composition comprises: 45-65 g / L soy peptone, 5-10 g / L sodium dihydrogen phosphate dihydrate, 1-5 g / L magnesium sulfate heptahydrate, 0.3-0.8 g / L ferrous sulfate heptahydrate, and 0.3-0.8 g / L potassium chloride. In one embodiment, the nutritional composition comprises: 50-60 g / L soy peptone, 6-8 g / L sodium dihydrogen phosphate dihydrate, 2-4 g / L magnesium sulfate heptahydrate, 0.4-0.6 g / L ferrous sulfate heptahydrate, and 0.4-0.6 g / L potassium chloride. In one embodiment, in step (c), the nutrient composition is added to the fermentation system at a flow rate of 120-160 ml / h, preferably 150 ml / h.

[0055] In one embodiment, the surfactant composition is added after fermentation for 13-18 hours, 14-17.5 hours, 15-17 hours, 15.5-16.5 hours, 15-16 hours or 16 hours. In one embodiment, the surfactant composition comprises polyoxypropylene oxyethylene glycerol ether and betaine. In one embodiment, the surfactant composition comprises: 1-2 g / L of polyoxypropylene oxyethylene glycerol ether and 1-2 g / L of betaine. In one embodiment, the surfactant composition comprises: 1-1.5 g / L of polyoxypropylene oxyethylene glycerol ether and 1.5-2 g / L of betaine.

[0056] 2. Technical advantages and beneficial effects of the present invention

[0057] Compared with the prior art, the present invention has at least the following technical advantages and beneficial effects:

[0058] (1) In a liquid submerged culture system, the cell passage cycle of Chryseobacterium utilis is short and the PG expression level is low, which is not conducive to the continuous and efficient production of PG. The present invention creatively proposes an excellent feeding strategy (including feeding time, feeding nutrient composition, and feeding method) that can extend the cell cycle in the system so that it can continuously and efficiently produce PG; and

[0059] (2) The conventional fermentation method of Chryseobacterium utilis has a low efficiency in producing PG, which is not conducive to industrial production. The present invention creatively proposes to add a preferred surfactant in the late stage of fermentation to change the cell membrane permeability, thereby shortening the fermentation cycle by 29% and increasing the enzyme activity to above 12.3 U / ml, thereby significantly improving the production efficiency of PG. DETAILED DESCRIPTION

[0060] The present invention will be further described below in conjunction with specific embodiments, but these specific embodiments cannot be understood as limiting the scope of protection of the present invention. Those skilled in the art may 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 embodiments still fall within the scope of protection of the present invention.

[0061] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. The materials and reagents used in the following embodiments, unless otherwise specified, can be obtained from commercial sources. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0062] Example 1. Fermentation production method of PG enzyme of the present invention

[0063] A fermentation method for efficiently producing PG enzyme using Chryseobacterium utilis comprises the following steps:

[0064] 1.1 Preparation of seed culture

[0065] Take 2 ml of a glycerol tube of Chryseobacterium utilis with stable properties after verification, and inoculate it into a seed culture medium. The components of the seed culture medium are as follows: 15-25 g / L (e.g., 17 g / L) soy 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 powder, adjust the pH to 5.5-6.5 (e.g., 6.0), place in a constant temperature shaker, and culture overnight at 29-32°C (e.g., 30°C) and 200-250 r / min (e.g., 200 rpm) for 8-12 hours (e.g., 12 hours), and harvest the seed culture.

[0066] 1.2 Inoculation

[0067] The above-mentioned overnight cultured seed culture is inoculated into a fermentation tank filled with 1-10 L (e.g. 4 L) of fermentation medium, and the inoculation amount is 5-10% (e.g. 8%). The fermentation medium components are as follows: 22-32 g / L (e.g. 25 g / L) soy 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; 0.6-1.5 g / L (e.g. 0.8 g / L) anhydrous nickel sulfate. The pH is adjusted to 5.0-6.0 (e.g. 5.5).

[0068] 1.3 Fermentation

[0069] The fermentation temperature is set to 30-32°C (e.g. 30°C), the ventilation volume is set to 0.3-0.6vvm (e.g. 0.5vvm), the dissolved oxygen value of the fermentation system is controlled to be not less than 20% (e.g. not less than 30%), the stirring paddle speed is 300-700r / min, and the nutrient composition is started to be added when the fermentation is carried out for 4-6 hours (e.g. 5 hours). The components of the nutrient composition are as follows: 45-65g / L (e.g. 60g / L) soy peptone; 5-10g / L (e.g. 6g / L) sodium dihydrogen phosphate dihydrate; 1-5g / L (e.g. 2g / L) magnesium sulfate heptahydrate; 0.3-0.8g / L (e.g. 0.4g / L) ferrous sulfate heptahydrate; 0.3-0.8g / L (e.g. 0.4g / L) potassium chloride, and the flow acceleration is controlled at 120-160ml / h (e.g. 150ml / h). When the fermentation is carried out for 18-22 hours (for example, 20 hours), 1-2 g / L (for example, 1 g / L) of polyoxypropylene oxyethylene glycerol ether and 1-2 g / L (for example, 1.5 g / L) of betaine are added. Samples are taken every 2 hours to measure the PG enzyme activity after 8 hours, 12 hours, 16 hours, and 20 hours of fermentation.

[0070] 1.4PG enzyme activity determination method

[0071] Take 10 μL of fermentation supernatant and dilute it 5 times and add it to a 96-well plate, add 100 μL of 6.74 g / L Cbz-Gln-Gly solution to it. React at 37°C for 30 minutes, then add 100 μL of TCA solution to terminate the reaction. The ammonia content of the solution after the reaction was determined by the phenol method, and the absorbance value at 630 nm was A1. Take another 10 μL of fermentation supernatant and dilute it 5 times and add it to a 96-well plate. React with 100 μL of TCA solution for 30 minutes, then add 100 μL of 6.74 g / L Cbz-Gln-Gly solution, and also use the phenol method to determine the absorbance value at 630 nm as A2. One enzyme activity unit (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:

[0072] Enzyme activity (U / ml) = (A1-A2)×21×5 / 17.03 / 30 / K.

[0073] Where A1: A of the experimental sample 630 Absorbance value; A2: blank A 630 Absorbance value; K: slope of the standard curve of ammonia determined by phenol hypochlorite spectrophotometry.

[0074] Example 2. Growth curves (OD) of the bacterial cells obtained by fermentation by the method of the present invention and the control method without the feeding strategy of step (c) of the present invention 600 )

[0075] 2.1 Preparation of seed culture

[0076] Take 2 ml of a glycerol tube of Chryseobacterium utilis with stable properties after verification, and inoculate it into a seed culture medium. The components of the seed culture medium are as follows: 15 g / L soy peptone; 2.0 g / L anhydrous magnesium sulfate; 3 g / L yeast powder. Adjust the pH to 6.2, place in a constant temperature shaker, and culture overnight at 30.5°C and 220 rpm for 12 hours, and harvest the seed culture.

[0077] 2.2 Inoculation

[0078] The above overnight cultured seed culture was inoculated into a fermentation tank filled with 5L fermentation medium at an inoculation rate of 7%. The fermentation medium composition is as follows: 25g / L soy peptone; 3g / L sodium dihydrogen phosphate dihydrate; 0.25g / L magnesium sulfate heptahydrate; 0.2g / L ferrous sulfate heptahydrate; 0.4g / L potassium chloride; 1.2g / L anhydrous nickel sulfate. The pH was adjusted to 5.5.

[0079] 2.3 Fermentation

[0080] The fermentation temperature was set to 30°C, the ventilation volume was set to 0.5vvm, the dissolved oxygen value of the fermentation system was controlled to be not less than 30%, the stirring paddle speed was 600r / min, and the working group began to add the nutrient composition after 5.2 hours of fermentation. The components of the nutrient composition are as follows: 55g / L soy peptone; 6g / L sodium dihydrogen phosphate dihydrate; 2g / L magnesium sulfate heptahydrate; 0.5g / L ferrous sulfate heptahydrate; 0.5g / L potassium chloride, and the flow acceleration was controlled at 150ml / h. A control group was also set up. The fermentation method of the control group was different from the above fermentation method only in that the nutrient composition was not added, and the other conditions were the same. Samples were taken at 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, and 30 hours of fermentation to monitor the growth status of the bacteria (OD 600 ).

[0081] like Figure 1 As shown in FIG. 1 , the bacterial growth of the working group and the control group in the initial period (about 7 hours) after the nutrient composition was added was basically similar, and both of them reached an OD of about 18 after about 12 hours of fermentation. 600 However, after the bacterial growth reaches the maximum value, the OD 600 It remained basically unchanged for about 18 hours thereafter (less than 10% lower than the maximum value); while the OD 600 It decreases to about 40% of the maximum value in about 18 hours thereafter.

[0082] Example 3. Comparative experiment of the enzyme activity level (U / mL) of PG enzyme produced by the method of the present invention and the control method without the feeding strategy of step (c) of the present invention

[0083] 3.1 Preparation of seed culture

[0084] Take 2 ml of the glycerol tube of Chryseobacterium utilis with stable properties after verification, and inoculate it into the seed culture medium. The components of the seed culture medium are as follows: 18 g / L soy peptone; 2.5 g / L anhydrous magnesium sulfate; 2.5 g / L yeast powder. Adjust the pH to 6.0, place it in a constant temperature shaker, and culture it overnight at 30°C and 200 rpm for 12.5 hours, and harvest the seed culture.

[0085] 3.2 Inoculation

[0086] The above overnight cultured seed culture was inoculated into a fermentation tank filled with 4 L fermentation medium, with an inoculum amount of 8%. The fermentation medium composition is as follows: 30 g / L soy peptone; 3.5 g / L sodium dihydrogen phosphate dihydrate; 0.3 g / L magnesium sulfate heptahydrate; 0.2 g / L ferrous sulfate heptahydrate; 0.4 g / L potassium chloride; 1.0 g / L anhydrous nickel sulfate. The pH was adjusted to 5.5.

[0087] 3.3 Fermentation

[0088] The fermentation temperature was set to 30°C, the ventilation was set to 0.6vvm, the dissolved oxygen value of the fermentation system was controlled to be not less than 25%, the stirring paddle speed was 700r / min, and the working group began to add the nutrient composition after 5.0 hours of fermentation. The components of the nutrient composition were as follows: 60g / L soy peptone; 7g / L sodium dihydrogen phosphate dihydrate; 3g / L magnesium sulfate heptahydrate; 0.6g / L ferrous sulfate heptahydrate; 0.6g / L potassium chloride, and the flow acceleration was controlled at 140ml / h. A control group was also set up. The fermentation method of the control group differed from the above fermentation method only in that the nutrient composition was not added, and the other conditions were the same. Samples were taken at 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, and 30 hours of fermentation to determine the PG enzyme activity (U / ml).

[0089] like Figure 2 As shown, the PG enzyme activity of the working group in the early stage of starting the feeding of the nutrient composition is basically similar to that of the control group. The growth rate of the enzyme activity of the working group of the feeding nutrient composition is better than that of the control group after 8 hours. The control group reaches the maximum enzyme activity of 6.4U / ml at 28 hours of fermentation, while the working group reaches the maximum enzyme activity of 8.5U / ml at 24 hours of fermentation. Not only the fermentation time is shortened by 4 hours, but the enzyme activity is about 1.3 times the highest enzyme activity of the control group.

[0090] Example 4. Comparative experiment of the enzyme activity level (U / mL) of PG enzyme produced by the method of the present invention and the control method without the surfactant addition strategy of step (c) and / or (d) of the present invention

[0091] 4.1 Preparation of seed culture

[0092] Take 2 ml of the glycerol tube of Chryseobacterium utilis with stable properties after verification, and inoculate it into the seed culture medium. The components of the seed culture medium are as follows: 20 g / L soy peptone; 3.5 g / L anhydrous magnesium sulfate; 3 g / L yeast powder. The pH is adjusted to 6.5, placed in a constant temperature shaker, and cultured overnight at 30°C and 220 rpm for 12.0 hours, and the seed culture is harvested.

[0093] 4.2 Inoculation

[0094] The above overnight cultured seed culture was inoculated into a fermentation tank filled with 6L fermentation medium, with an inoculum amount of 6%. The fermentation medium composition is as follows: 26g / L soy peptone; 3g / L sodium dihydrogen phosphate dihydrate; 0.2g / L magnesium sulfate heptahydrate; 0.3g / L ferrous sulfate heptahydrate; 0.3g / L potassium chloride; 1.2g / L anhydrous nickel sulfate. The pH was adjusted to 5.5.

[0095] 4.3 Fermentation

[0096] The fermentation temperature was set to 30°C, the ventilation was set to 0.5vvm, the dissolved oxygen value of the fermentation system was controlled to be not less than 30%, the stirring paddle speed was 500r / min, and the nutrient composition was added after 5 hours of fermentation. The components of the nutrient composition were as follows: 60g / L soy peptone; 7g / L sodium dihydrogen phosphate dihydrate; 3g / L magnesium sulfate heptahydrate; 0.6g / L ferrous sulfate heptahydrate; 0.6g / L potassium chloride, and the flow acceleration was controlled at 120ml / h. The working group added a surfactant composition (1g / L polyoxypropylene oxyethylene glycerol ether and 1.5g / L betaine) when the fermentation was carried out for 16 hours. Control groups I and II were also set up. The difference between the fermentation method of control group I and the above-mentioned fermentation method of the working group was that the surfactant composition was not added, and the other conditions were the same. The difference between the fermentation method of control group II and the above-mentioned fermentation method of the working group was that neither the nutrient composition nor the surfactant composition was added, and the other conditions were the same. Samples were taken at 8, 12, 16, 20, 24, 28 and 30 hours of fermentation to determine PG enzyme activity (U / ml).

[0097] like Figure 3As shown, since the working group and the control group I in this embodiment both added the nutrient composition, the PG enzyme activity of the two was basically similar before the addition of the surfactant composition. After the surfactant composition was added for 16 hours of fermentation, the enzyme activity increased significantly, and the highest enzyme activity was reached at 20 hours of fermentation, which was about 12.3U / ml, which was 45% higher than that of the control group I, and the fermentation time was shortened by 4 hours, indicating that the addition of the surfactant composition has a significant promoting effect on the secretion expression of the PG enzyme.

[0098] like Figure 4 As shown, by comparing the working group in this embodiment with the control group II, the fermentation regulation method of adding the nutrient composition and the surfactant composition not only increased the enzyme activity by 92%, but also shortened the fermentation cycle from the original 28 hours to 20 hours, shortened by 29%, which can significantly improve the production efficiency of PG enzyme, thereby reducing production costs, and is a very effective fermentation regulation strategy.

[0099] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that this is only for illustration and the protection scope of the present invention is limited by the attached items. Those skilled in the art may make various changes or modifications to the technical contents disclosed above without departing from the scope of the technical solution of the present invention, and these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A method for producing protein glutaminase by fermentation, comprising: (a) using the seed culture medium composition to perform seed culture on Chryseobacterium utilis to obtain a seed culture; (b) fermenting the seed culture using a fermentation medium composition; (c) adding the nutrient composition 3-8 hours into the fermentation; and (d) optionally adding a surfactant composition after the fermentation has been carried out for 13-18 hours.

2. The method according to claim 1, wherein the seed culture medium composition comprises: 15-25 g / L, preferably 17-20 g / L of soy peptone, 2-5 g / L, preferably 2.5-4 g / L, of anhydrous magnesium sulfate, and 1.5-5g / L, preferably 2-4g / L yeast powder; The pH of the seed culture medium composition is 5.5-6.5, preferably 5.8-6.

2.

3. The method according to claim 1 or 2, wherein the seed culture is carried out under the following conditions: (1) The culture temperature is 29-32°C; (2) culturing under shaking conditions of 200-250 r / min; and / or (3) The culture is carried out for 8-12 hours.

4. The method according to any one of claims 1 to 3, wherein the fermentation medium composition comprises: 22-32 g / L, preferably 25-30 g / L of soy peptone, 1-5 g / L, preferably 2-4 g / L of sodium dihydrogen phosphate dihydrate, 0.2-0.8 g / L, preferably 0.3-0.7 g / L of magnesium sulfate heptahydrate, 0.1-0.5 g / L, preferably 0.1-0.3 g / L of ferrous sulfate heptahydrate, 0.2-0.5 g / L, preferably 0.3-0.5 g / L potassium chloride, and 0.6-1.5 g / L, preferably 0.8-1.2 g / L, anhydrous nickel sulfate; The pH of the fermentation medium composition is 5.0-6.0, preferably 5.2-5.

6.

5. The method according to any one of claims 1 to 4, wherein the fermentation is carried out under the following conditions: (1) Temperature of 30-32°C; (2) Stirring at 300-700 r / min; (3) The dissolved oxygen value of the system is not less than 30%; and / or (4) Fermentation is carried out for 24-36 hours.

6. The method according to any one of claims 1 to 5, wherein in step (c), a nutrient composition is added after the fermentation is carried out for 4 to 6 hours, preferably 5 hours, and the nutrient composition comprises: 45-65g / L, preferably 50-60g / L soy peptone, 5-10 g / L, preferably 6-8 g / L of sodium dihydrogen phosphate dihydrate, 1-5 g / L, preferably 2-4 g / L of magnesium sulfate heptahydrate, 0.3-0.8 g / L, preferably 0.4-0.6 g / L of ferrous sulfate heptahydrate, and 0.3-0.8 g / L, preferably 0.4-0.6 g / L potassium chloride.

7. The method according to any one of claims 1 to 6, wherein the nutrient composition in step (c) is added to the fermentation system at a rate of 120-160 ml / h, preferably 150 ml / h.

8. The method according to any one of claims 1 to 7, wherein in step (d), a surfactant composition is added after the fermentation is carried out for 14 to 17 hours, preferably 15 to 16 hours, wherein the surfactant composition comprises 1 to 2 g / L, preferably 1 to 1.5 g / L, of polyoxypropylene oxyethylene glycerol ether and 1 to 2 g / L, preferably 1.5 to 2 g / L of betaine.

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