Mutant of protein glutaminase and method for activating protein glutaminase in one step

Abstract

The invention relates to a mutant of protein glutaminase and a method for activating the mutant in one step, and belongs to the technical field of biology. According to the invention, a protein glutaminase mutant with improved enzymatic activity is obtained through saturated mutation screening, and fusion protein composed of super-folded green fluorescent protein sfGFP, a protein glutaminase propeptide region, intein Mth RIR1 and a protein glutaminase mature region is expressed in bacillus subtilis. Wherein the intein spontaneously cuts the fusion protein, the activated mature protein glutaminase is obtained in one step, the problem that in the prior art, the activation of protein glutaminase zymogen cannot be realized due to limited protease of bacillus subtilis is solved, and the enzyme activity of the prepared mature protein glutaminase can reach 40.2 U / mL.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a mutant of protein glutaminase and a method for one-step activation thereof. BACKGROUND

[0002] Protein glutaminase (PG, EC 3.5.1.44) is a novel enzyme preparation in the field of food industry, which specifically catalyzes the deamidation reaction of glutamine residues in proteins or polypeptides to generate glutamic acid and ammonia. Compared with traditional deamidation enzymes, protein glutaminase has significant advantages: the action process does not cause protein hydrolysis, can maintain the structural integrity of the substrate, and has high substrate specificity and low allergenic risk. The enzyme has important modification function on food raw materials rich in glutamine (such as wheat, soybean protein), which can significantly improve the solubility and enhance the emulsification, foaming and other food processing properties by exposing the hydrophilic sites of the protein and optimizing the local conformation, and has broad application potential.

[0003] Protein glutaminase is expressed in the form of zymogen (pro-PG), which contains a pro-peptide region (Pro region) and a mature enzyme region (mPG). The enzyme itself is inactive and needs to be activated through a specific activation process (such as protease cleavage) to become active. Bacillus subtilis, as an FDA certified food safety level (GRAS) expression host, has the advantages of universal codon system and high secretion ability, and is widely used in the production of food enzyme preparations. However, studies have shown that Bacillus subtilis WB600, WB800 and other engineering strains have inherent defects when expressing protein glutaminase zymogen: limited by the insufficient activity of endogenous protease, the zymogen cannot be fully activated, and must rely on the auxiliary cleavage of exogenous protease. This not only significantly increases the production cost, but also increases the risk of degradation of the target product due to the introduction of heterologous protease, and the complexity of the subsequent purification process is significantly increased. Therefore, it is necessary to develop a convenient and efficient one-step activation method for protein glutaminase in Bacillus subtilis. SUMMARY

[0004] To solve the above technical problems, the present application first screens a protein glutaminase mutant L115K with improved enzyme activity through saturation mutation, and then expresses a fusion protein containing a super-folded green fluorescent protein sfGFP, a protein glutaminase pro-peptide region, an intein Mth RIR1 and a protein glutaminase mature region in Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168, wherein the sfGFP mediates the transport of the fusion protein to the fermentation broth, and the intein spontaneously cleaves the fusion protein to obtain the activated mature protein glutaminase in one step.

[0005] The first object of the present application is to provide a mutant of protein glutaminase, wherein the leucine at the first position of the parent sequence shown in SEQ ID NO. 1 is mutated into lysine.

[0006] The second object of the present application is to provide a gene encoding the mutant.

[0007] The third object of the present application is to provide a one-step activation method of protein glutaminase, wherein a fusion protein composed of a super-folded green fluorescent protein, a protein glutaminase pro-peptide region, an intein and a protein glutaminase mature region is expressed in Bacillus subtilis in sequence, the intein spontaneously cleaves the fusion protein to obtain the activated protein glutaminase.

[0008] The nucleotide sequence of the protein glutaminase pro-peptide region is shown in SEQ ID NO. 5; the intein is Mth RIR1, and the nucleotide sequence is shown in SEQ ID NO. 6; the parent sequence of the protein glutaminase mature region is shown in SEQ ID NO. 1, and the leucine at the first position is mutated into lysine.

[0009] The intein is a self-catalytic type protein splicing element, which can realize self-cleavage through a four-step nucleophilic reaction mechanism under the condition of no cofactor and energy supply, and connect the two flanking peptides with a new peptide bond to form a functional protein. The C-terminal cleavage type intein mutant Mth RIR1 has a length of 134 amino acids, which is the shortest continuous intein known at present. -1 The G, C1A double mutant retains high C-terminal specific cleavage activity.

[0010] The fusion protein is transported from the intracellular of Bacillus subtilis to the fermentation broth by the non-classical secretion pathway mediated by the super-folded green fluorescent protein sfGFP.

[0011] Further, the expression uses P nprE , P amyQ or P lytR as the promoter.

[0012] In an embodiment of the present application, the expression uses P nprE as the promoter.

[0013] Further, the expression uses pP43NMK as the expression vector.

[0014] Further, the Bacillus subtilis is Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168.

[0015] In an embodiment of the present application, the Bacillus subtilis is Bacillus subtilis WB600.

[0016] A fourth object of the present application is to provide a recombinant expression vector, which sequentially expresses a super-folded green fluorescent protein, a protein glutaminase pro-peptide region, an intein and a protein glutaminase mature region, with pP43NMK as a backbone, P nprE , P amyQ or PlytR as a promoter.

[0017] The nucleotide sequence of the protein glutaminase pro-peptide region is shown in SEQ ID NO. 5; the intein is Mth RIR1, and the nucleotide sequence is shown in SEQ ID NO. 6; the maternal sequence of the protein glutaminase mature region is shown in SEQ ID NO. 1, and the first leucine is mutated into lysine.

[0018] Further, the recombinant expression vector further comprises a 6His protein purification tag.

[0019] In an embodiment of the present application, the nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO. 2.

[0020] A fifth object of the present application is to provide a Bacillus subtilis comprising the above-mentioned recombinant expression vector.

[0021] Further, the Bacillus subtilis is Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168.

[0022] A sixth object of the present application is to provide a microbial inoculant comprising the above-mentioned Bacillus subtilis.

[0023] A seventh object of the present application is to provide the use of the above-mentioned recombinant expression vector, the above-mentioned Bacillus subtilis or the above-mentioned microbial inoculant in the preparation of protein glutaminase.

[0024] Further, the method comprises the following steps:

[0025] (1) inoculating the recombinant microorganism on a plate in a liquid LB medium for 10-12 hours to obtain a seed liquid.

[0026] (2) inoculating the seed liquid in a 25 mL TB medium at a 4% inoculation amount, and performing shake flask fermentation for 48 hours.

[0027] Further, the method further comprises the step of purifying the fermentation product by nickel column affinity chromatography.

[0028] The present application has the following beneficial effects:

[0029] The application provides a mutant of protein glutaminase and a one-step activation method thereof, a specific site of protein glutaminase derived from Chryseobacterium proteolyticum is subjected to saturation mutation to obtain a mutant with improved enzyme activity, a fusion protein composed of, in sequence, super-fold green fluorescent protein sfGFP, a protein glutaminase Pro region, an intein Mth RIR1 and a protein glutaminase mature region is expressed in Bacillus subtilis, sfGFP is used to promote extracellular secretion of the protein, and the intein is used to cut the specific site to realize efficient spontaneous activation of the protein glutaminase, thus solving the problem that, in the prior art, the activation of the protein glutaminase zymogen cannot be realized due to the limitation of the protease of Bacillus subtilis itself, and the prepared mature protein glutaminase has an enzyme activity of up to 40.2 U / mL. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to make the content of the application more easily understood, the application will be further described in detail below according to specific embodiments of the application and in conjunction with the drawings, in which:

[0031] Figure 1 is a schematic diagram of the principle of the application;

[0032] Figure 2 is the saturation mutation result of the protein glutaminase in Example 1 of the application;

[0033] Figure 3 is the result of expressing the protein glutaminase using different promoters in Example 2 of the application;

[0034] Figure 4 is the recombinant plasmid map in Example 3 of the application;

[0035] Figure 5 is the growth curve of the recombinant strain in Example 4 of the application;

[0036] Figure 6 is the enzyme activity of the protein glutaminase in the fermentation product of the recombinant strain in Example 4 of the application;

[0037] Figure 7 is the activation result of the protein glutaminase verified by electrophoresis in Example 4 of the application, wherein lane 1 is a protein Marker and lane 2 is a purified protein;

[0038] Figure 8 is the optimal reaction temperature detection result in Example 5 of the application;

[0039] Figure 9 is the optimal reaction pH value detection result in Example 5 of the application;

[0040] Figure 10Enzyme activity and cell growth of Example 6 tank fermentation. DETAILED DESCRIPTION

[0041] The application will be further described in conjunction with the drawings and specific examples so that those skilled in the art can better understand and implement the application, but the examples are not intended to limit the application.

[0042] The enzyme activity definition and enzyme activity determination method in the following examples are as follows:

[0043] Enzyme activity definition: 1 unit of enzyme activity (U / mL) is defined as the amount of enzyme that hydrolyzes 1 μmol of substrate Cbz-Gln-Gly to release 1 μmol of ammonia per minute at 37°C.

[0044] Enzyme activity determination method:

[0045] (1) Prepare different concentrations of ammonia standard solution using NH4Cl as the solute and deionized water as the solvent. Take 12 μL of different concentrations of ammonia solution, and add 48 μL of distilled water, 60 μL of color developing solution A, 30 μL of color developing solution B, and 60 μL of color developing solution C, mix well, and incubate in a 37°C water bath for 20 min, and measure the absorbance value at OD 630 to draw the ammonia standard curve. The x-axis of the ammonia standard curve is the concentration of NH4Cl solution, and the y-axis is the absorbance.

[0046] (2) Take 1 mL of Cbz-Gln-Gly solution and incubate in a 37°C water bath for 10 min, add 100 μL of fermentation supernatant diluted by an appropriate number of times, mix well, and incubate in a 37°C water bath for 30 min. After the reaction is completed, add 1 mL of trichloroacetic acid solution (0.4 M) to terminate the reaction. For the control, first add 1 mL of trichloroacetic acid solution and 100 μL of fermentation supernatant, and then add 1 mL of Cbz-Gln-Gly solution after 30 min of reaction. Then, add 48 μL of distilled water, 60 μL of color developing solution A, 30 μL of color developing solution B, and 60 μL of color developing solution C to 12 μL of the reaction solution in the experimental group or the control group. The reaction is carried out at 37°C for 20 min, and the absorbance value is measured. 630

[0047] (3) Substitute the absorbance value of step (2) into the ammonia standard curve of step (1) to calculate the mPG enzyme activity of the sample to be tested.

[0048] Substrate preparation method: weigh 0.337 g of dipeptide Cbz-Gln-Gly, dissolve in 0.2 M PBS buffer (pH = 6.5), and dilute to 100 mL for immediate use.

[0049] Color developing solution A: 40.46 g / L phenol, 0.15 g / L sodium nitroprusside, and store in the dark.​

[0050] Color developing solution B: 49.94 g / L potassium hydroxide.

[0051] Color developing solution C: 204 g / L potassium carbonate, 8.34 mL / L sodium hypochlorite solution.

[0052] Example 1: Protein glutaminase saturated mutation

[0053] 1. Designing the primer for saturated mutation

[0054] The primer pair Mutant-F and Mutant-R was used to mutate the first amino acid of mPG with the amino acid sequence as shown in SEQ ID NO. 1.

[0055] Mutant-F: NNKGCCAGCGTCATTCCGGAC

[0056] Mutant-R: GAATGACGCTGGCMNNgttatgaacgatgaagccgtttgc

[0057] 2. Construction of mutant plasmid

[0058] PCR was performed on pP43NMK-P srfA -sfGFP-PPG-RIR1, and the PCR product was directly transformed into E. coli JM109 and verified by sequencing. The remaining 19 mutant plasmids were then transformed into B. subtilis competent cells to verify the enzyme activity of the mPG mutant, and the results are shown in Figure 2 . The enzyme activity of the wild type was recorded as 100%, and the enzyme activity increased to 1.65 times that of the wild type after the first leucine was mutated to lysine, so the mPG mutant L115K was used for subsequent experiments.

[0059] Example 2: Promoter screening

[0060] Different promoters were screened for expressing the mPG mutant L115K, and the types and sources of the promoters are shown in Table 1.

[0061] Classification of selected promoters in Table 1

[0062]

[0063] The initial promoter P srfA in pP43NMK-P srfA -sfGFP-PPG-RIR1 was replaced with the selected promoters in Table 1, respectively, for expression, and the enzyme activity of the fermentation product was detected, and the results are shown in Figure 3 . The enzyme activity of the expression product of the initial promoter P srfA was recorded as 100%, and the enzyme activity of the expression product of the replaced P nprEThe enzyme activity after the promoter was increased to 1.38 times that of the wild type, so P nprE The promoter was used for subsequent experiments.

[0064] Example 3: Construction of recombinant plasmid and recombinant bacteria

[0065] 1. Construction of recombinant plasmid pP43NMK-P nprE -sfGFP-PPG-RIR1

[0066] Recombinant plasmid pP43NMK-P nprE -sfGFP-PPG-RIR1 is a circular plasmid, the nucleotide sequence is shown as SEQ ID NO. 2, and the plasmid map is shown as Figure 4 The nucleotides 4160-4659 constitute the promoter P nprE , the nucleotides 4660-6699 are a complete open reading frame (encoding a fusion protein), wherein the nucleotides 4660-4662 are a start codon, the nucleotides 4663-5376 encode sfGFP, the nucleotides 5377-5718 encode the Pro region, the nucleotides 5719-6123 encode the Mth RIR1 intein, the nucleotides 6124-6126 encode the amino acid residue K, the nucleotides 6124-6678 encode the mPG mutant L115K, the nucleotides 6679-6696 encode the His6 tag, and the nucleotides 6697-6699 are a stop codon. The nucleotide sequence of the mPG-pro region is shown as SEQ ID NO. 5.

[0067] The amino acid sequence of the fusion protein is shown as SEQ ID NO. 3. Among them, the amino acid residues 2-239 constitute sfGFP, the amino acid residues 240-353 constitute the propeptide region, the amino acid residues 354-488 constitute the Mth RIR1 intein, the amino acid residue 489 is K (corresponding to the screening result of Example 1), the amino acid residues 489-673 constitute mPG, and the amino acid residues 674-679 constitute the 6His tag.

[0068] The Mth RIR1 intein causes the fusion zymogen to be self-activated (the cleavage site is between the amino acid residue 488 and the amino acid residue 489 of SEQ ID NO. 3), obtaining the mPG-6His protein with the amino acid sequence shown as SEQ ID NO. 4.

[0069] 2. Construction of recombinant bacteria

[0070] The super competent cells of Bacillus subtilis WB600 were prepared by xylose induction method, and the recombinant plasmid pP43NMK-P nprE-sfGFP-PPG-RIR1 and pP43NMK empty vector into B. subtilis WB600, to obtain recombinant bacteria, named WB600-pP43NMK-P nprE -sfGFP-PPG-RIR1 and WB600-pP43NMK.

[0071] Example 4: Shake flask fermentation of recombinant bacteria

[0072] I. WB600-pP43NMK-P nprE Shake flask fermentation of -sfGFP-PPG-RIR1

[0073] 1. The WB600-pP43NMK-P nprE -sfGFP-PPG-RIR1 was inoculated in 3 mL of LB medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, pH = 7.0) containing 50 μg / mL kanamycin, and incubated at 37°C, 220 r / min for 10-12 hours to obtain a seed solution.

[0074] 2. 1 mL of the seed solution was inoculated into 25 mL of TB medium (12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 0.17 M KH2PO4, 0.72 M K2HPO4) containing 50 μg / mL kanamycin in a 250 mL conical flask, and fermented at 37°C, 220 r / min for 48 hours to obtain a fermentation product.

[0075] The fermentation product was centrifuged at 4°C, 12000 x g for 20 minutes, and the upper liquid was named fermentation supernatant, and the expression product in the fermentation supernatant was named sfGFP-PPG-RIR1.

[0076] II. Shake flask fermentation of WB600-pP43NMK

[0077] The recombinant bacteria WB600-pP43NMK were fermented according to the above steps to obtain a fermentation supernatant.

[0078] III. Growth curve determination

[0079] During the fermentation process of the above steps I and II, 1 mL was taken at regular time intervals, centrifuged at 12000 x g for 5 minutes to obtain the supernatant, and diluted by an appropriate number of times to 0 D600 In the range of 0.2-0.8, the spectrophotometer reading was recorded, and the growth curve was as shown in Figure 5

[0080] The results showed that the WB600-pP43NMK-P nprE ​The growth curve of sfGFP-PPG-RIR1 was similar to that of WB600-pP43NMK, indicating that the production of extracellular mPG did not cause obvious damage to the growth of the cells.

[0081] IV. Enzyme activity determination

[0082] During the fermentation process of step one, 1 mL was taken at regular time intervals, centrifuged at 12000 x g for 5 minutes to obtain the supernatant, which was used as the sample to be tested, and the enzyme activity of mPG was detected to calculate the enzyme activity of the fermentation product.

[0083] The results are shown in Table 1. Figure 6 The highest enzyme activity of mPG was 15.6 U / mL.

[0084] V. Protein purification and electrophoresis verification

[0085] 1. The fermentation supernatant obtained in step one was filtered through a 0.45 μm microporous filter, and then subjected to nickel column affinity chromatography to collect the eluate containing the target protein mPG-6His.

[0086] 2. The eluate was desalted using a 3 kD pore size column ultrafiltration membrane to replace it with PBS buffer at pH = 7.4. The protein solution with PBS buffer at pH = 7.4 as the solvent was named mPG-6His solution.

[0087] 3. 30 μL of the mPG-6His sample after nickel column purification and ultrafiltration desalting was mixed with 10 μL of sample buffer and heated at 70°C for 10 minutes. After instant centrifugation and cooling to room temperature, the sample was loaded and subjected to denaturing gel electrophoresis at a constant voltage of 120 V, with an electrophoresis time of 50 minutes.

[0088] 4. The obtained protein gel was stained with polyacrylamide protein gel fast staining solution.

[0089] The electrophoresis results are shown in Figure 2. Figure 7 , indicating that sfGFP-PPG-RIR1 can be completely activated to obtain mPG under the synergistic action of the intein and the extracellular protease.

[0090] Example 5: Determination of the enzymatic properties of mPG

[0091] The mPG-6His solution was prepared according to Example 4.

[0092] I. N-terminal amino acid sequencing of the protein

[0093] The stained protein gel prepared in Example 4 was cut with a knife to obtain the target band (20 kD). After washing with ddH2O for 3 times and drying with a paper towel, the sample was sent to Shanghai Sunred Bioengineering Co., Ltd. for determination of the N-terminal amino acid sequence by LC-MS technology. The first five N-terminal amino acids were KASVI.

[0094] The results show that the expression plasmid pP43NMK-P nprE Under the synergistic action of extracellular protease and intein Mth RIR1, the zymogen is self-activated, the activation site is between the 488th and 489th amino acid residues of SEQ ID NO. 3, and the mPG-6His protein shown in SEQ ID NO. 4 is obtained. This process can achieve 100% activation of the glutaminase zymogen without exogenous addition of protease activation, and a new method of one-step fermentation to obtain mPG in Bacillus subtilis WB600 is developed for the first time.

[0095] II. Determination of optimal reaction temperature

[0096] Take mPG-6His solution as the detection sample, and determine the enzyme activity of mPG at different temperatures. The reaction temperatures are: 30℃, 37℃, 40℃, 50℃, 60℃, 70℃, and 80℃.

[0097] The highest enzyme activity is taken as 100% activity, and the relative enzyme activity at other temperatures is calculated. The results are shown in Table 1. Figure 8 The highest enzyme activity is taken as 100% activity, and the relative enzyme activity at other temperatures is calculated. The results are shown in Table 1.

[0098] III. Determination of optimal reaction pH

[0099] Prepare the substrate buffer solution, respectively:

[0100] pH = 3.0, 0.2M acetic acid-sodium acetate buffer;

[0101] pH = 4.0, 0.2M acetic acid-sodium acetate buffer;

[0102] pH = 5.0, 0.2M acetic acid-sodium acetate buffer;

[0103] pH = 6.0, 0.2M Na2HPO4-NaH2PO4 buffer;

[0104] pH = 7.0, 0.2M Na2HPO4-NaH2PO4 buffer;

[0105] pH = 8.0, 0.2M Na2HPO4-NaH2PO4 buffer;

[0106] pH = 9.0, 0.2M Na2HPO4-NaH2PO4 buffer;

[0107] pH = 10, 0.2M glycine-NaOH buffer;

[0108] pH = 11, 0.2 M glycine-NaOH buffer.

[0109] The mPG-6His solution was diluted 10 times with different pH value substrate buffer solution, mixed uniformly as a test sample, and the enzyme activity of mPG was determined.

[0110] Preparation method of different pH substrate solution: 0.337 g dipeptide Cbz-Gln-Gly was weighed and dissolved in 0.2 M PBS buffer (pH = 6.5) and made up to 100 mL. 5 mL of the above substrate solution was taken and diluted 10 times with different pH value substrate buffer solution to obtain 50 mL of different pH value substrate solution. The highest enzyme activity was taken as 100% activity, and the relative enzyme activity at other pH values was calculated, and the results are shown in Table 1. Figure 9 The highest enzyme activity was taken as the optimum reaction pH. The optimum reaction pH was 5.0.

[0111] Example 6: 5L tank fermentation in Bacillus subtilis WB600

[0112] On the basis of Example 4 (I), fermentation scale-up verification was carried out in a 5L tank. The culture of recombinant Bacillus was as follows:

[0113] Fermentation medium (g / L): glucose 10, yeast powder 30, sodium chloride 8, potassium dihydrogen phosphate 2.5, dipotassium hydrogen phosphate 12.5, calcium chloride 0.3, manganese sulfate 0.2, ferrous sulfate heptahydrate 0.02, zinc sulfate heptahydrate 0.02, magnesium sulfate heptahydrate 1.

[0114] Feed medium (g / L): yeast powder 100, glucose 400, magnesium sulfate heptahydrate 7.8.

[0115] 1. Primary seed culture: the competent cells of recombinant plasmid transformed Bacillus subtilis were coated on a plate containing kanamycin resistance and cultured at 37°C for 12 hours to obtain single colonies. The single colonies were inoculated into 50 mL of LB medium and cultured at 37°C, 220 rpm for 10 hours to obtain seed liquid.

[0116] 2. Secondary seed culture: the above primary seed was inoculated into secondary LB medium at a inoculation amount of 4% (v / v) and cultured at 37°C, 220 rpm for 10 hours to obtain secondary seed liquid.

[0117] 3.5L tank fermentation: initial liquid volume 2L, after sterilization, temperature dropped to 37℃, initial pH 7.0, calibrated dissolved oxygen 100%, using flame inoculation method to add 10% (v / v) of secondary seed liquid, final concentration 10g / L of glucose solution, final concentration 50μg / mL of kanamycin solution. During fermentation, maintain fermentation temperature at 37℃, use 5M NaOH solution and 20% phosphoric acid solution to maintain pH at about 7.0, adjust stirring speed to keep DO between 20-30%.

[0118] 4. On the basis of the above medium, tank fermentation optimization was carried out again, the best nitrogen source of the feeding medium was yeast powder and soybean peptone compounded according to 1:2, the best carbon source of the feeding medium was glycerol, linear feeding strategy was used in the early stage of fermentation, and feeding was coupled with pH in the later stage of fermentation, other conditions were unchanged, tank cell enzyme production and growth were shown in Figure 10 .

[0119] The experimental results showed that the highest enzyme activity in tank reached 40.2U / mL in 48 hours, and the cell entered the stationary phase in about 24 hours.

[0120] Obviously, the above examples are only examples for clearly illustrating, not limiting the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, all the embodiments need not and cannot be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A mutant of protein glutaminase, characterized in that: The amino acid sequence is shown in SEQ ID NO. 1, wherein the first leucine of the parent sequence is mutated to lysine.

2. A gene encoding the mutant according to claim 1.

3. A one-step activation method for protein glutaminase, characterized in that: A fusion protein consisting of a superfolded green fluorescent protein, a protein glutaminase propeptide region, an intein, and a protein glutaminase mature region is sequentially expressed in Bacillus subtilis, wherein the intein spontaneously cleaves the fusion protein to obtain activated protein glutaminase; Among them, the nucleotide sequence of the propeptide region of the protein glutaminase is shown as SEQ ID NO.5; the intein is Mth RIR1, and the nucleotide sequence is shown as SEQ ID NO.6; the maternal sequence of the mature region of the protein glutaminase is shown as SEQ ID NO.1, and the leucine at position 1 is mutated to lysine.

4. The one-step activation method according to claim 3, wherein: The expression uses P nprE 、P amyQ or P lytR For the promoter.

5. The one-step activation method according to claim 3, wherein: The expression vector pP43NMK was used.

6. The one-step activation method according to claim 3, wherein: The Bacillus subtilis is Bacillus subtilis WB600, Bacillus subtilis WB800 or Bacillus subtilis 168.

7. A recombinant expression vector, characterized in that: The recombinant expression vector uses pP43NMK as the backbone and P nprE 、P amyQ or P lytR As the promoter, superfolded green fluorescent protein, protein glutaminase propeptide region, intein and protein glutaminase mature region are expressed in sequence; Among them, the nucleotide sequence of the propeptide region of the protein glutaminase is shown as SEQ ID NO.5; the intein is Mth RIR1, and the nucleotide sequence is shown as SEQ ID NO.6; the maternal sequence of the mature region of the protein glutaminase is shown as SEQ ID NO.1, and the leucine at position 1 is mutated to lysine.

8. Bacillus subtilis comprising the recombinant expression vector according to claim 7.

9. A microbial agent comprising the Bacillus subtilis according to claim 8.

10. Use of the recombinant expression vector according to claim 7, the Bacillus subtilis according to claim 8, or the microbial agent according to claim 9 in the preparation of protein glutaminase.

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