Total synthetic culture medium for producing protein glutaminase through fermentation of Chryseobacterium prion and preparation method and application of total synthetic culture medium for producing protein glutaminase through fermentation of Chryseobacterium prion

Abstract

The invention discloses a total synthesis culture medium for culturing Chryseobacterium prion to produce protein glutaminase through fermentation, the culture medium comprises a carbon source, a nitrogen source and inorganic salt, and the specific composition and content of the culture medium are as follows: the carbon source is glucose; the nitrogen source is prepared from glycine, proline, leucine, serine, phenylalanine, glutamic acid, cysteine, arginine, asparagine, lysine, isoleucine, valine, tyrosine and glutamine; the inorganic salts comprise K2HPO4, KH2PO4, MgSO4. 7H2O and FeSO4. 7H2O, and the inorganic salts comprise K2HPO4, KH2PO4, MgSO4. 7H2O and FeSO4. 7H2O. The invention also discloses a preparation method of the synthetic culture medium and a method for producing protein glutaminase in the total synthetic culture medium. The fully synthetic culture medium disclosed by the invention can be applied to culture of Chryseobacterium prion and has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a fully synthetic culture medium for the proliferation of Chryseobacterium utilis and fermentation to produce protein glutaminase, as well as a preparation method and application thereof. Background Art

[0002] Protein glutaminase is a new type of hydrolase that can specifically act on the glutamine residues on the side chains of various proteins or polypeptides, causing them to deamidate, converting the amide side chain of the glutamine residues into carboxyl groups and releasing ammonia, thereby improving the solubility, foaming and emulsifying properties of the protein. Protein glutaminase has mild action conditions, high efficiency and no adverse side effects. It will not cause protein hydrolysis and cross-linking, and has good development prospects in the food industry.

[0003] Chryseobacterium utilis is a strain that produces protein glutaminase. Currently, the culture of Chryseobacterium utilis mainly adopts natural culture medium, in which the nitrogen source is a natural component with unclear or unstable composition, such as yeast powder or peptone, which affects the stability of fermentation production and the repeatability of scientific research, and is not conducive to the quantitative analysis and research of cell metabolic characteristics. Since Chryseobacterium utilis does not use inorganic nitrogen sources, its growth is limited in conventional bacterial synthetic culture medium, and it hardly produces protein glutaminase.

[0004] Therefore, there is an urgent need to provide a fully synthetic culture medium for producing protein glutaminase by fermentation of Chryseobacterium proteolyticus. Summary of the invention

[0005] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a fully synthetic culture medium for the proliferation of Chryseobacterium utilis and the fermentation production of protein glutaminase and a preparation method thereof.

[0006] The fully synthetic medium is a medium prepared from substances with fully understood chemical compositions, and has strong repeatability, and is suitable for use in laboratories for research on the nutritional requirements, metabolism, strain selection, and genetic analysis of Chryseobacterium utilis. The preparation of the fully synthetic medium for culturing Chryseobacterium utilis to ferment and produce protein glutaminase mentioned in the present invention follows the basic principles of preparing the medium, and its composition and preparation method are determined from carbon sources, nitrogen sources, and inorganic salts, respectively. Among them, carbohydrates are good carbon sources and energy materials for general microorganisms, and glucose, as a monosaccharide, can be well utilized by Chryseobacterium utilis, so glucose is selected as the carbon source of the synthetic medium mentioned in the present invention.

[0007] The physiological functions of inorganic salts in the body are mainly as components of enzyme active centers, maintaining the stability of biomacromolecules and cell structures, regulating and maintaining the osmotic pressure balance of cells, controlling the redox potential of cells, and serving as energy sources for the growth of certain microorganisms. K2HPO4 and KH2PO4 are commonly used pH buffers in culture media, and MgSO4·7H2O and FeSO4·7H2O are inorganic salt components in natural synthetic culture media currently used to culture Chryseobacterium utilis to ferment and produce protein glutaminase. These four inorganic salts provide most elements such as potassium, phosphorus, sulfur, magnesium, and iron.

[0008] The selection of nitrogen source is the main technical difficulty that needs to be overcome in the present invention. Chryseobacterium utilis does not grow or ferment to produce protein glutaminase in a culture medium with only an inorganic nitrogen source, and most organic nitrogen sources are complex in composition and cannot be used in a fully synthetic culture medium, so only amino acids can be selected as the nitrogen source for the fully synthetic culture medium. There are many types of amino acids. The present invention optimizes the types and concentrations of amino acids, and clarifies the nitrogen source composition of the fully synthetic culture medium for culturing Chryseobacterium utilis to ferment and produce protein glutaminase.

[0009] The present invention provides a fully synthetic culture medium for culturing Chryseobacterium utilis to ferment and produce protein glutaminase. The fully synthetic culture medium comprises a carbon source, a nitrogen source and an inorganic salt, and its specific composition and content are as follows:

[0010] The carbon source: glucose 0.25-2.5 g / L;

[0011] The nitrogen source: glycine 0.5-2g / L, proline 0.5-2g / L, leucine 0.5-2g / L, serine 0.5-2g / L, phenylalanine 0.5-2g / L, glutamic acid 0.5-2g / L, cysteine ​​0.125-1g / L, arginine 0.125-1g / L, asparagine 0.5-2g / L, lysine 0.5-2g / L, isoleucine 0.5-2g / L, valine 0.5-2g / L, tyrosine 0.5-2g / L, glutamine 0.5-2g / L;

[0012] The inorganic salts include: K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 2.5 g / L, and FeSO4·7H2O 0.05 g / L.

[0013] Preferably, the fully synthetic culture medium comprises 0.25-9 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.5 g / L glycine, 0.5 g / L proline, 0.5 g / L leucine, 1.5 g / L serine, 1.5 g / L phenylalanine, 1.5 g / L glutamate, 1 g / L cysteine, 1 g / L arginine, 1 g / L asparagine, 1 g / L lysine, 1 g / L isoleucine, 1 g / L valine, 1 g / L tyrosine, and 1 g / L glutamine.

[0014] The present invention also provides a method for preparing a fully synthetic culture medium, the specific steps of which are as follows:

[0015] (1) After the carbon source and inorganic salt are dissolved in water in proportion, high pressure and high temperature sterilization is performed at a sterilization temperature of 115° C. for 20 min to obtain solution A;

[0016] (2) The nitrogen sources were prepared into 25× mother solutions, and sterilized by filtration using a sterile filter membrane (membrane pore size ≤ 0.2 μm) to obtain nitrogen source stock solutions;

[0017] (3) After the solution A obtained in step (1) is cooled to 25° C., a nitrogen source stock solution is added and mixed to obtain the fully synthetic culture medium;

[0018] In the step (1), the composition and content of the carbon source in the fully synthetic culture medium are: glucose 0.25-2.5 g / L; preferably, 0.25 g / L.

[0019] In the step (1), the composition and content of inorganic salts in the fully synthetic culture medium are: K2HPO4 1g / L, KH2PO4 0.5g / L, MgSO4·7H2O 2.5g / L, FeSO4·7H2O 0.05g / L; preferably, K2HPO4 1g / L, KH2PO4 0.5g / L, MgSO4·7H2O 2.5g / L, FeSO4·7H2O 0.05g / L.

[0020] In the step (3), the composition and content of the nitrogen source in the fully synthetic culture medium are: glycine 0.5-2g / L, proline 0.5-2g / L, leucine 0.5-2g / L, serine 0.5-2g / L, phenylalanine 0.5-2g / L, glutamic acid 0.5-2g / L, cysteine ​​0.125-1g / L, arginine 0.125-1g / L, asparagine 0.5-2g / L, lysine 0.5-2g / L, isoleucine 0.5-2g / L, valine 0.5-2g / L L, tyrosine 0.5-2g / L, glutamine 0.5-2g / L; preferably, the composition and content of the nitrogen source in the fully synthetic culture medium are: glycine 0.5g / L, proline 0.5g / L, leucine 0.5g / L, serine 1.5g / L, phenylalanine 1.5g / L, glutamate 1.5g / L, cysteine ​​1g / L, arginine 1g / L, asparagine 1g / L, lysine 1g / L, isoleucine 1g / L, valine 1g / L, tyrosine 1g / L, glutamine 1g / L.

[0021] The present invention also provides a fully synthetic culture medium prepared by the method described above.

[0022] The present invention also provides an application of the above-mentioned fully synthetic culture medium in culturing Chryseobacterium utilis.

[0023] The present invention also provides a method for producing protein glutaminase in the above-mentioned fully synthetic medium, the method comprising: inserting Chryseobacterium proteolyticum YF810 into the fully synthetic medium for shaking culture for 12h-24h to obtain the fermentation liquid of the protein glutaminase. The strain is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, the deposit time is February 6, 2015, and the deposit number is CGMCC No. 10532.

[0024] In the method, the culture temperature is 26-32°C;

[0025] The fully synthetic culture medium comprises: 0.25-2.5 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.5-2 g / L aminoacid, 0.5-2 g / L proline, 0.5-2 g / L leucine, 0.5-2 g / L serine, 0.5-2 g / L phenylalanine, 0.5-2 g / L glutamic acid, 0.125-1 g / L cysteine, 0.125-1 g / L arginine, 0.5-2 g / L asparagine, 0.5-2 g / L lysine, 0.5-2 g / L isoleucine, 0.5-2 g / L valine, 0.5-2 g / L tyrosine, and 0.5-2 g / L glutamine.

[0026] Preferably, the culture medium comprises: glucose 0.25 g / L, K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 0.05 g / L, glycine 0.5 g / L, proline 0.5 g / L, leucine 0.5 g / L, serine 1.5 g / L, phenylalanine 1.5 g / L, glutamate 1.5 g / L, cysteine ​​1 g / L, arginine 1 g / L, asparagine 1 g / L, lysine 1 g / L, isoleucine 1 g / L, valine 1 g / L, tyrosine 1 g / L, and glutamine 1 g / L.

[0027] In a specific embodiment, the method for using the fully synthetic medium to culture Chryseobacterium utilis to ferment and produce protein glutaminase comprises the following steps:

[0028] (1) Preparation of plate culture medium: In 1 L of distilled water, add 10 g of yeast peptone FP103, 2 g of yeast extract powder FM888, and 2.05 g of MgSO4·7H2O, adjust the pH to 7.0 with NaOH solution, add 18-20 g of agar strips, and sterilize at 121°C for 20 min to prepare plate culture medium;

[0029] (2) Preparation of seed culture medium: Add 10 g of yeast peptone FP103, 2 g of yeast extract powder FM888, and 2.05 g of MgSO4·7H2O to 1 L of distilled water, adjust the pH to 7.0 with NaOH solution, and sterilize at 121°C for 20 min.

[0030] (3) Seed preparation: Chryseobacterium utilis was used as the bacterial species and first inoculated into the plate culture medium prepared in step (1) for culture at a temperature of 30° C. for 16 h. An appropriate amount of bacterial cells was scraped with an inoculation loop and inoculated into the seed culture medium in step (2) for shaking culture at a temperature of 30° C. for 200 rpm for 8 h.

[0031] (4) Fermentation culture: The seed solution cultured in step (3) was inoculated into the fully synthetic culture medium for shaking culture, with an inoculation amount of 7%, a culture temperature of 30° C., a culture speed of 200 rpm, and a culture time of 16 h.

[0032] The present invention takes the bacteria-based fully synthetic medium as the optimization starting point, and optimizes the types and concentrations of the carbon source and nitrogen source of the medium. In the initial medium, the growth of Chryseobacterium utilis is restricted and no protein glutaminase is produced. In the fully synthetic medium, Chryseobacterium utilis proliferates and grows well, the biomass is increased by 311.43% compared with the initial medium, and the protein glutaminase activity increases to 1.71U / mL.

[0033] The fully synthetic culture medium of the present invention has simple ingredients, all of which are common reagents, small dosage, low cost, simple preparation method, and can be used for the normal proliferation and growth of Chryseobacterium utilis and the production of protein glutaminase, providing an important basis for the study of the mechanism of Chryseobacterium utilis fermentation synthesis of protein glutaminase. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 This is the effect of different nitrogen sources on the activity of protein glutaminase in Example 2 of the present invention.

[0036] Figure 2 This is the effect of glucose concentration on protein glutaminase activity in Example 3 of the present invention.

[0037] Figure 3 This is the effect of a single amino acid deficiency on the enzymatic activity of protein glutaminase in Example 4 of the present invention.

[0038] Figure 4 The figure shows the effects of different concentrations of glycine, proline, serine, leucine, valine, phenylalanine and glutamate on the activity of protein glutaminase in Example 5 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is further described in detail with reference to the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0040] The following description of at least one exemplary embodiment is actually only illustrative and is by no means intended to limit the present invention and its application or use. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limiting. Therefore, other examples of the exemplary embodiments may have different values.

[0042] The invention provides a fully synthetic culture medium for culturing Chryseobacterium utilis to ferment and produce protein glutaminase, the culture medium comprising a carbon source, a nitrogen source and an inorganic salt, and the specific composition and content thereof are as follows: the carbon source: 0.25-2.5 g / L of glucose; the nitrogen source: 0.5-2 g / L of glycine, 0.5-2 g / L of proline, 0.5-2 g / L of leucine, 0.5-2 g / L of serine, 0.5-2 g / L of phenylalanine, 0.5-2 g / L of glutamine Acid 0.5-2g / L, cysteine ​​0.125-1g / L, arginine 0.125-1g / L, asparagine 0.5-2g / L, lysine 0.5-2g / L, isoleucine 0.5-2g / L, valine 0.5-2g / L, tyrosine 0.5-2g / L, glutamine 0.5-2g / L; the inorganic salts: K2HPO41g / L, KH2PO40.5g / L, MgSO4·7H2O 2.5g / L, FeSO4·7H2O0.05g / L. The present invention also proposes a method for preparing the synthetic culture medium and a method for producing protein glutaminase in the fully synthetic culture medium. The fully synthetic culture medium of the present invention can be used in culturing Chryseobacterium utilis, and has broad application prospects.

[0043] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. The materials and reagents used in the present examples are commercially available unless otherwise specified. The reagents used in the present examples are all analytical grade reagents and can be purchased from regular channels.

[0044] A protein glutaminase producing strain, Chryseobacterium proteolyticum YF810, is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, the address of which is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 6, 2015, and the deposit number is CGMCC No. 10532.

[0045] Method for determining the activity of protein glutaminase: Protein glutaminase converts the glutamine amide group in Cbz-Gln-Gly into a hydroxyl group accompanied by the release of ammonia. In an alkaline medium, ammonia reacts with phenol with sodium nitrosylferrocyanide as a catalyst and sodium hypochlorite as an oxidant to generate a blue-green indigo blue dye, which has a specific absorption effect on the spectrum with a wavelength of 630nm. The relative activity of PG enzyme can be reflected by measuring OD630.

[0046] The fermentation broth was centrifuged at 10000r / min and 4°C for 5min to obtain the supernatant, which was diluted 2 times. 10μL of the supernatant dilution was added to a 96-well microplate, and then 100μL of the preheated Cbz-Gln-Gly solution (0.674g of dipeptide Cbz-Gln-Gly was weighed and fixed to 100mL with 0.176mol / L PB buffer (pH6.5)) was added thereto, and reacted in an air bath at 37°C for 30min. Then 100μL of trichloroacetic acid solution was added, mixed evenly, and the reaction was terminated. The ammonia content in the solution after the reaction was determined by the phenol method. A630 was determined as the absorbance value A1. The control group was first added with trichloroacetic acid solution and reacted for 30min before adding the substrate solution. The rest of the operations were the same as the experimental group. A630 was determined as A2. The amount of enzyme required to produce 1μmol of ammonia per minute is defined as one enzyme activity unit (IU). The formula for calculating enzyme activity in the fermentation broth is as follows:

[0047] Enzyme activity (U / mL) = (A1-A2) / 17.03 / 30 / a×21×5

[0048] A1: absorbance value of the experimental group; A2: absorbance value of the control group; a: slope of the standard curve of ammonia determined by phenol hypochlorite spectrophotometry.

[0049] The present invention will be further described below by way of examples.

[0050] Example 1 Production of protein glutaminase by culturing Chryseobacterium utilis in a fully synthetic medium

[0051] (1) Preparation of plate culture medium: In 1 L of distilled water, add 10 g of yeast peptone FP103, 2 g of yeast extract powder FM888, and 2.05 g of MgSO4·7H2O, adjust the pH to 7.0 with NaOH solution, add 18-20 g of agar strips, and sterilize at 121°C for 20 min to prepare plate culture medium;

[0052] (2) Preparation of seed culture medium: Add 10 g of yeast peptone FP103, 2 g of yeast extract powder FM888, and 2.05 g of MgSO4·7H2O to 1 L of distilled water, adjust the pH to 7.0 with NaOH solution, and sterilize at 121°C for 20 min.

[0053] (3) Seed preparation: Chryseobacterium utilis was used as the bacterial species and first inoculated into the plate culture medium prepared in step (1) for culture at a temperature of 30° C. for 16 h. An appropriate amount of bacterial cells was scraped with an inoculation loop and inoculated into the seed culture medium in step (2) for shaking culture at a temperature of 30° C. for 200 rpm for 8 h.

[0054] (4) Preparation of synthetic culture medium: In 1L distilled water, add 0.25g glucose, 1g K2HPO4, 0.5g KH2PO4, 2.5g MgSO4·7H2O, 2.5g FeSO4·7H2O 0.05g, high pressure and high temperature sterilization, the sterilization temperature is 115℃, and the sterilization time is 20min; the final concentrations are calculated based on glycine 0.5g / L, proline 0.5g / L, leucine 0.5g / L, serine 1.5g / L, phenylalanine 1.5g / L, glutamic acid 1.5g / L, cysteine ​​1g / L, arginine 1g / L, asparagine 1g / L, lysine 1g / L, isoleucine 1g / L, valine 1g / L, tyrosine 1g / L, and glutamine 1g / L, prepare a nitrogen source solution, filter and sterilize with a sterile filter membrane (filter membrane pore size ≤0.2μm), add to the culture medium aqueous solution and mix evenly to obtain a fully synthetic culture medium;

[0055] (5) Fermentation culture: The seed solution cultured in step (3) was inoculated into a fully synthetic culture medium for shaking culture, with an inoculation amount of 7%, a culture temperature of 30° C., a culture speed of 200 rpm, and a culture time of 16 h;

[0056] (6) Determination of protein glutaminase activity: The enzyme activity was determined using the enzyme activity determination method. The results showed that the enzyme activity of protein glutaminase was 1.71 U / mL.

[0057] Example 2 Effect of different nitrogen sources on protein glutaminase activity

[0058] In this example, 1 g / L of (NH4)2SO4, 1 g / L of 20 kinds of amino acids and a combination of the two were selected as nitrogen sources to explore the optimal nitrogen source for Chryseobacterium utilis to produce protein glutaminase. The concentrations of the remaining components were 2.5 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, and 0.05 g / L FeSO4·7H2O. The components and contents of the seed culture medium and the culture method were the same as those in Example 1 of the present invention.

[0059] The results are as follows Figure 1As shown, when the nitrogen source is (NH4)2SO4, the growth of Chryseobacterium utilis is limited and almost no protein glutaminase is produced. When the nitrogen source is 20 amino acids, the enzyme activity of protein glutaminase is 0.38U / mL.

[0060] Example 3 Effect of glucose concentration on protein glutaminase activity

[0061] In this embodiment, 0.25g / L, 0.5g / L, 1g / L, 1.5g / L, 2g / L and 2.5g / L of glucose are selected as carbon sources to explore the optimal carbon source concentration for the production of protein glutaminase by Chryseobacterium utilis. On the basis of Example 2 of the present invention, 20 kinds of amino acids are selected as nitrogen sources, and the concentration of each amino acid is 1g / L. The concentrations of the remaining components are K2HPO41g / L, KH2PO40.5g / L, MgSO4·7H2O 2.5g / L, and FeSO4·7H2O0.05g / L. The components and contents of the seed culture medium and the culture method are the same as in Example 1.

[0062] The results are as follows Figure 2 As shown, when the glucose concentration was 0.25 g / L, the enzyme activity of protein glutaminase was the highest, which was 0.57 U / mL.

[0063] Example 4 Single deletion experiment of amino acid components

[0064] This embodiment designs a single amino acid deficiency experiment to screen amino acid components that have a significant effect on the growth of Chryseobacterium utilis and the production of protein glutaminase. On the basis of Example 3 of the present invention, 0.25g / L of glucose is selected as the carbon source. The concentrations of the remaining components are K2HPO41g / L, KH2PO40.5g / L, MgSO4·7H2O 2.5g / L, FeSO4·7H2O0.05g / L. The components and contents of the seed culture medium and the culture method are the same as in Example 1. The concentration of each amino acid is 1g / L. During the experiment, one amino acid is deleted each time, and the concentrations of the remaining amino acids remain unchanged.

[0065] The results are as follows Figure 3 As shown in the figure, the 20 amino acids play different roles in the growth of Chryseobacterium utilis and the production of protein glutaminase. Among them, the enzyme activity increased significantly after the single deletion of cysteine, methionine, arginine, and tryptophan, indicating that these amino acids may have an inhibitory effect on the production and accumulation of protein glutaminase. The enzyme activity decreased significantly after the single deletion of glycine, proline, serine, leucine, valine, phenylalanine, and glutamate, indicating that these amino acids may play a relatively important role in the production and accumulation of protein glutaminase.

[0066] Example 5 Concentration gradient experiment of key amino acids

[0067] In this example, based on Example 4 of the present invention, a concentration gradient experiment of some key amino acids was designed to explore the optimal concentrations of these amino acids for the growth of Chryseobacterium utilis and the production of protein glutaminase. Based on Example 3 of the present invention, 0.25 g / L of glucose was selected as the carbon source. The concentrations of the remaining components were K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 2.5 g / L, and FeSO4·7H2O 0.05 g / L. The components, contents, and culture methods of the seed culture medium are the same as those of Example 1 of the present invention. The initial concentration of each amino acid was 1 g / L. During the experiment, the concentration of one amino acid was changed each time, and the concentrations of the remaining amino acids remained unchanged. The concentrations of glycine, proline, serine, leucine, valine, phenylalanine, and glutamic acid were selected as 0 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, and 2 g / L, respectively.

[0068] The results are as follows Figure 4 As shown, the optimal ratio is as follows: glycine 0.5 g / L, proline 0.5 g / L, serine 1.5 g / L, leucine 0.5 g / L, valine 1 g / L, phenylalanine 1.5 g / L, and glutamic acid 1.5 g / L.

[0069] Example 6 Orthogonal design method to optimize amino acid combination

[0070] This example designs an orthogonal test based on Example 4 and Example 5 of the present invention to optimize the composition of amino acids. Four amino acids, cysteine, methionine, arginine, and tryptophan, are determined as the objects of investigation, and other components in the culture medium are fixed, and a four-factor two-level orthogonal test is used. Based on Example 2 of the present invention, 0.25g / L of glucose is selected as the carbon source. The concentrations of the remaining components are K2HPO41g / L, KH2PO40.5g / L, MgSO4·7H2O 2.5g / L, and FeSO4·7H2O 0.05g / L. On the basis of Example 4, 0.5 g / L of glycine, 0.5 g / L of proline, 1.5 g / L of serine, 0.5 g / L of leucine, 1 g / L of valine, 1.5 g / L of phenylalanine, 1.5 g / L of glutamic acid, 1 g / L of glutamine, 1 g / L of lysine, 1 g / L of histidine, 1 g / L of alanine, 1 g / L of isoleucine, 1 g / L of asparagine, 1 g / L of aspartic acid, 1 g / L of tyrosine, and 1 g / L of threonine were selected. The components and contents of the seed culture medium and the culture method were the same as those in Example 1 of the present invention.

[0071] Table 1 Results of orthogonal test with four factors and two levels

[0072]

[0073] The results are shown in Table 1. After analysis, the optimal ratio was as follows: 1 g / L cysteine, 1 g / L arginine, no addition of methionine and tryptophan, and the remaining components remained unchanged.

[0074] Example 7 Orthogonal design method to optimize amino acid combination

[0075] In this embodiment, on the basis of Embodiment 4, Embodiment 5 and Embodiment 6 of the present invention, an orthogonal test is designed to optimize the composition of amino acids. Aspartic acid, asparagine, lysine, isoleucine, alanine, valine, tyrosine, histidine, glutamic acid and threonine wet weight amino acids are determined as the objects of investigation, and other components in the culture medium are fixed, and an orthogonal test with ten factors and two levels is adopted. On the basis of Embodiment 2, 0.25g / L of glucose is selected as the carbon source. The concentrations of the remaining components are K2HPO41g / L, KH2PO40.5g / L, MgSO4·7H2O2.5g / L, and FeSO4·7H2O 0.05g / L. On the basis of Embodiment 4 and Embodiment 5 of the present invention, 0.5g / L of glycine, 0.5g / L of proline, 1.5g / L of serine, 0.5g / L of leucine, 1.5g / L of phenylalanine, 1.5g / L of glutamic acid, 1g / L of cysteine, and 1g / L of arginine are selected. The components and contents of the seed culture medium and the culture method are the same as those in Example 1 of the present invention.

[0076] Table 2 Results of orthogonal test of ten factors and two levels

[0077]

[0078] The results are shown in Table 2. After analysis, the optimal ratio is as follows: asparagine 1g / L, lysine 1g / L, isoleucine 1g / L, valine 1g / L, tyrosine 1g / L, glutamine 1g / L, and the other components remain unchanged.

[0079] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0080] As used in the present invention, the term "comprising" is an open expression, that is, including the contents specified in the present invention but not excluding other contents.

[0081] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0082] Although the embodiments of the present description have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present description, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A fully synthetic culture medium for culturing Chryseobacterium utilis to produce protein glutaminase by fermentation, characterized in that: The culture medium comprises a carbon source, a nitrogen source and an inorganic salt; wherein, The carbon source includes 0.25-2.5 g / L of glucose; The nitrogen source includes glycine 0.5-2g / L, proline 0.5-2g / L, leucine 0.5-2g / L, serine 0.5-2g / L, phenylalanine 0.5-2g / L, glutamic acid 0.5-2g / L, cysteine ​​0.125-1g / L, arginine 0.125-1g / L, asparagine 0.5-2g / L, lysine 0.5-2g / L, isoleucine 0.5-2g / L, valine 0.5-2g / L, tyrosine 0.5-2g / L, and glutamine 0.5-2g / L; The inorganic salts include K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 2.5 g / L, and FeSO4·7H2O 0.05 g / L.

2. The fully synthetic medium according to claim 1, characterized in that The fully synthetic culture medium comprises: 0.25-2.5 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.5 g / L glycine, 0.5 g / L proline, 0.5 g / L leucine, 1.5 g / L serine, 1.5 g / L phenylalanine, 1.5 g / L glutamic acid, 1 g / L cysteine, 1 g / L arginine, 1 g / L asparagine, 1 g / L lysine, 1 g / L isoleucine, 1 g / L valine, 1 g / L tyrosine, and 1 g / L glutamine.

3. A method for preparing a fully synthetic culture medium, characterized in that: The preparation method comprises the following steps: (1) After the carbon source and inorganic salt are dissolved in water in proportion, high pressure and high temperature sterilization is performed at a sterilization temperature of 115° C. for 20 min to obtain solution A; (2) The nitrogen sources were prepared into 25× mother solutions, and sterilized by filtration with a sterile filter membrane to obtain nitrogen source stock solutions; (3) After the solution A obtained in step (1) is cooled to 25° C., a nitrogen source stock solution is added and mixed to obtain the fully synthetic culture medium.

4. The preparation method according to claim 3, characterized in that: In the step (1), the composition and content of the carbon source in the fully synthetic culture medium are: glucose 0.25-2.5 g / L; And / or, the composition and content of inorganic salts in the fully synthetic culture medium are: K2HPO4 1 g / L, KH2PO4 0.5 g / L, MgSO4·7H2O 2.5 g / L, FeSO4·7H2O 0.05 g / L.

5. The preparation method according to claim 3, characterized in that: In the step (3), the composition and content of the nitrogen source in the fully synthetic culture medium are: glycine 0.5-2g / L, proline 0.5-2g / L, leucine 0.5-2g / L, serine 0.5-2g / L, phenylalanine 0.5-2g / L, glutamate 0.5-2g / L, cysteine ​​0.125-1g / L, arginine 0.125-1g / L, asparagine 0.5-2g / L, lysine 0.5-2g / L, isoleucine 0.5-2g / L, valine 0.5-2g / L, tyrosine 0.5-2g / L, and glutamine 0.5-2g / L.

6. A fully synthetic culture medium prepared by the method according to any one of claims 3 to 5.

7. Use of the fully synthetic culture medium as described in any one of claims 1, 2, and 6 in culturing Chryseobacterium utilis.

8. A method for producing protein glutaminase in the fully synthetic medium as claimed in any one of claims 1, 2 and 6, characterized in that: The method comprises: inoculating Chryseobacterium proteolyticumYF810 into a fully synthetic culture medium and shaking and culturing it for 12 hours to 24 hours to obtain a fermentation liquid of the protein glutaminase; The Chryseobacterium proteolyticum YF810 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on February 6, 2015, with the deposit number of CGMCC No.10532.

9. The method according to claim 8, characterized in that In the method, the culture temperature is 26-32°C; and / or, The fully synthetic culture medium comprises: 0.25-2.5 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.5-2 g / L aminoacid, 0.5-2 g / L proline, 0.5-2 g / L leucine, 0.5-2 g / L serine, 0.5-2 g / L phenylalanine, 0.5-2 g / L glutamic acid, 0.125-1 g / L cysteine, 0.125-1 g / L arginine, 0.5-2 g / L asparagine, 0.5-2 g / L lysine, 0.5-2 g / L isoleucine, 0.5-2 g / L valine, 0.5-2 g / L tyrosine, and 0.5-2 g / L glutamine.

10. The method according to claim 9, characterized in that The fully synthetic culture medium comprises: 0.25-2.5 g / L glucose, 1 g / L K2HPO4, 0.5 g / L KH2PO4, 2.5 g / L MgSO4·7H2O, 0.05 g / L FeSO4·7H2O, 0.5 g / L glycine, 0.5 g / L proline, 0.5 g / L leucine, 1.5 g / L serine, 1.5 g / L phenylalanine, 1.5 g / L glutamic acid, 1 g / L cysteine, 1 g / L arginine, 1 g / L asparagine, 1 g / L lysine, 1 g / L isoleucine, 1 g / L valine, 1 g / L tyrosine, and 1 g / L glutamine.

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