Bacillus subtilis with high yield of protein glutamine enzyme and application thereof

Abstract

The present application relates to a kind of high protein glutamine enzyme bacillus subtilis and its application, by detecting enzyme activity characterization extracellular protease AprE, Bpr and Epr can be activated to the zymogen of protein glutamine enzyme, to reach the effect of improving protein glutamine enzyme activity, and the knockout of extracellular protease WprA can improve the enzyme activity of protein glutamine enzyme, based on the above experimental findings constructs a kind of high protein glutamine enzyme bacillus subtilis BS1-PG, after 96 hours of shake flask fermentation, enzyme activity is 18.05U / mL, after 60 hours of fermentation in 5L fermenter, enzyme activity reaches 45.58U / mL, are significantly improved compared with original strain.For this, knockout protease related genes lytC, spoIVB, amyE, malA, srfAC and mdxL in bacillus subtilis genome, further improve enzyme activity to 65.68U / mL in 5L fermenter, it is favorable to reduce the production cost of protein glutamine enzyme, promote its large-scale production and application in food industry field.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a Bacillus subtilis strain that produces high levels of protein glutaminase and its applications. Background Technology

[0002] Protein-glutaminase (EC 3.5.1.44, PG) is a novel protein modification tool, belonging to the new enzyme preparations used in the food industry, playing an important role in both biological and industrial applications. It catalyzes the formation of glutamine groups in the side chains of proteins or long peptides, producing glutamate and releasing ammonia, without affecting free asparagine and glutamine residues. Protein-glutaminase exhibits excellent deamidation activity and acts only on the glutamine residues of proteins, without causing protein hydrolysis. In food processing, protein-glutaminase can improve the texture and mouthfeel of food, such as enhancing the gluten and extensibility of dough, and improving the elasticity and tenderness of food. Furthermore, it can promote the release of amino acids in food, increasing its nutritional value and flavor. Simultaneously, because this enzyme does not introduce unpleasant odors or alter the color of food, it has broad applicability in food processing. Besides the food industry, protein-glutaminase also shows potential value in other fields. For example, in the pharmaceutical field, it can be used to prepare peptide drugs with specific structures; in the feed industry, it can be used to improve feed utilization and animal growth performance. In summary, protein glutaminase, as an enzyme with unique catalytic function, plays an important role in multiple fields and has broad application prospects.

[0003] In 2000, Yamaguchi and Yokoe first discovered Chryseobacterium proteolyticum 9670 in *Chryseobacterium proteolyticum*. TProtein glutaminase, with industrial application potential, was discovered and purified from the culture supernatant. To this day, the main method for preparing protein glutaminase in industrial production is still microbial fermentation. The main drawbacks of using *Chlorella vulgaris* for fermentation to produce protein glutaminase include: (1) Due to the metabolic characteristics of the *Chlorella vulgaris* strain itself, the yield of protein glutaminase is low, limiting large-scale production and application; (2) The fermentation cycle of *Chlorella vulgaris* is long, usually requiring a long time to reach a high enzyme activity level, which increases production and time costs, and is not conducive to industrial production; (3) Due to the low yield and long cycle of *Chlorella vulgaris* fermentation to produce protein glutaminase, the overall production cost is high. This includes the culture of the strain, the maintenance of fermentation equipment, and the costs of reagents and labor in the downstream purification process; (4) *Chlorella vulgaris* is quite sensitive to fermentation conditions (such as temperature, pH, dissolved oxygen, etc.). Small changes in these conditions may affect the growth of the strain and the yield of the enzyme. Therefore, it is necessary to strictly control the fermentation conditions to ensure stable enzyme production. Given the above issues, achieving efficient heterologous expression of protein glutaminase is urgently needed. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention attempts to heterologously express protein glutaminase in Bacillus subtilis. By constructing extracellular protease knockout strains and overexpression strains, it was found that extracellular proteases AprE, Bpr, and Epr can activate the zymogen of protein glutaminase, thereby increasing the activity of protein glutaminase. Knockout of the extracellular protease WprA can enhance the production of protein glutaminase. Based on the above experiments, a Bacillus subtilis strain with high protein glutaminase production was constructed.

[0005] The first objective of this invention is to provide a Bacillus subtilis strain that produces a high level of protein glutaminase, wherein the Bacillus subtilis strain heterologously expresses protein glutaminase by overexpressing the extracellular protease encoding gene AprE, Bpr, or Epr, wherein the nucleotide sequence of AprE is shown in SEQ ID NO.1, the nucleotide sequence of Bpr is shown in SEQ ID NO.2, and the nucleotide sequence of Epr is shown in SEQ ID NO.3.

[0006] Extracellular proteases AprE, Bpr, and Epr can activate the protein glutaminase proenzyme, with AprE being the main activating protease.

[0007] In one embodiment of the present invention, the Bacillus subtilis overexpresses the extracellular protease encoding gene AprE.

[0008] Furthermore, the Bacillus subtilis genome contains knockout or silencing of the relevant genes lytC, spoIVB, amyE, malA, srfAC, and mdxL.

[0009] Furthermore, the protein glutaminase is derived from *Lactobacillus glutaminase*, and its nucleotide sequence is shown in SEQ ID NO. 10.

[0010] Furthermore, the Bacillus subtilis knockout extracellular protease encoding gene WprA, the nucleotide sequence of which is shown in SEQ ID NO.8.

[0011] The absence of WprA protease is beneficial for increasing the activity of protein glutaminase.

[0012] Furthermore, the Bacillus subtilis is Bacillus subtilis 168, Bacillus subtilis WB600, or Bacillus subtilis WB800.

[0013] In one embodiment of the present invention, the Bacillus subtilis is Bacillus subtilis WB800.

[0014] Furthermore, the overexpression uses P glvc The promoter, the P glvc The nucleotide sequence of the promoter is shown in SEQ ID NO. 9.

[0015] A second objective of the present invention is to provide a microbial preparation comprising the aforementioned Bacillus subtilis.

[0016] A third objective of this invention is to provide the application of the aforementioned Bacillus subtilis or the aforementioned microbial preparation in the production of protein glutaminase.

[0017] A fourth objective of this invention is to provide a method for producing protein glutaminase by adding the aforementioned Bacillus subtilis or the aforementioned microbial preparation to a fermentation system.

[0018] Furthermore, the fermentation time is 50-66 hours.

[0019] Furthermore, the fermentation system comprises 20 g / L glucose, 24 g / L yeast powder, 24 g / L peptone, 12.5 g / L K2HPO4, 2.5 g / L KH2PO4 and 8 g / L urea.

[0020] The beneficial effects of this invention are:

[0021] This invention provides a heterologous expression of protein glutaminase derived from *Chlorella vulgaris* in *Bacillus subtilis*, significantly increasing glutaminase production. After 96 hours of shake-flask fermentation, the enzyme activity reached 18.05 U / mL, and after 60 hours of fermentation in a 5L fermenter, the activity reached 45.58 U / mL, both significantly higher than the original strain. This is beneficial for reducing the production cost of protein glutaminase and promoting its large-scale production and application in the food industry. Furthermore, this invention, through the construction of extracellular protease knockout and overexpression strains for heterologous expression of *Chlorella vulgaris*-derived protein glutaminase, demonstrates that extracellular proteases AprE, Bpr, and Epr can activate the protein glutaminase prozymogen, while the deletion of WprA positively increases the enzyme activity of protein glutaminase. This discovery provides a novel approach and direction for the heterologous expression of protein glutaminase in *Bacillus subtilis*. To further increase the yield of protein glutamine, this invention knocked out six protease-related genes lytC, spoIVB, amyE, malA, srfAC, and mdxL in the Bacillus subtilis genome, resulting in an increase in protein glutaminase activity of the recombinant strain to 65.68 U / mL in a 5L fermenter, revealing the important role of protease-related genes in the expression and production of protein glutaminase. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0023] Figure 1 This is a map of the extracellular protease knockout plasmid in Example 1 of the present invention;

[0024] Figure 2 This is a map of the extracellular protease expression plasmid in Example 2 of the present invention;

[0025] Figure 3 This is a map of the protein glutaminase expression plasmid in Example 3 of the present invention;

[0026] Figure 4 This is the expression result of protein glutaminase in the extracellular protease knockout strain in Example 4 of the present invention;

[0027] Figure 5 The expression results of protein glutaminase in the extracellular protease overexpressing strain in Example 4 of the present invention;

[0028] Figure 6 The results of shake-flask fermentation of the recombinant strain BS1-PG in Example 5 of this invention;

[0029] Figure 7The results of fermentation in a 5L fermenter using the recombinant strain BS1-PG in Example 5 of this invention;

[0030] Figure 8 This is a schematic diagram of the gene knockout fragment fusion construction in Example 6 of the present invention;

[0031] Figure 9 The results of fermentation in a 5L fermenter for the recombinant strain W8ΔMT6-Pro-PPG in Example 7 of this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0033] The culture media involved in the following examples are shown below:

[0034] LB liquid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0035] LB solid medium: peptone 10g / L, yeast extract 5g / L, NaCl 10g / L, agar 10g / L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20min.

[0036] TB liquid culture medium: peptone 12g / L, yeast extract 24g / L, glycerol 5g / L, KH2PO4 2.31g / L, K2HPO4 12.54g / L, adjust pH to 7.2-7.4, autoclave at 115℃ for 20min.

[0037] The enzyme activity detection methods involved in the following examples are as follows:

[0038] The enzyme activity of protein glutaminase is defined as the amount of enzyme required to hydrolyze the substrate Z-Gln-Gly (benzyloxycarboxyl-glutamine-glycine) to produce 1 μmol of ammonium ions in 30 min at 37 °C and pH = 6.5. It is defined as one unit of protein glutaminase activity, denoted by U.

[0039] Weigh 0.314 g of NH4Cl and dry it in an oven for 1 hour. Add water to make up to 1 L and dilute according to Table 1 to obtain ammonia standard solutions of different concentrations.

[0040] Table 1. Ammonia standard solutions of different concentrations

[0041] <![CDATA[ddH2O(μL)]]> 2000 1920 1840 1760 1680 1600 Concentration μg / mL 0 4 8 12 16 20

[0042] Take 60 μL of ammonia solutions of different concentrations, add 240 μL of ddH2O, 300 μL of colorimetric reagent A, 150 μL of colorimetric reagent B, and 300 μL of colorimetric reagent C respectively, mix thoroughly, and develop in a 37℃ water bath for 20 min. Then, measure the absorbance at 630 nm using an ELISA reader. Plot a standard curve of ammonia with ammonium chloride concentration on the x-axis and absorbance on the y-axis based on the results.

[0043] Colorimetric solution A: Weigh 2.023g of phenol and 7.5mg of sodium nitrosoferricyanide, dissolve them in double-distilled water and bring the volume to 50mL.

[0044] Colorimetric solution B: Weigh 2.5g KOH, dissolve it in double-distilled water, and bring the volume to 50mL.

[0045] Colorimetric solution C: Weigh 10.2g of anhydrous K2CO3 and dissolve it in double-distilled water, add 417μL of NaClO solution, and dilute to 50mL with water.

[0046] Substrate Cbz-Gln-Gly solution: Weigh 0.337g of dipeptide Cbz-Gln-Gly, dissolve it in 0.176mol / L PBS buffer (pH=6.5), and bring the volume to 100mL.

[0047] Trichloroacetic acid solution (0.4 mol / L): Weigh 6.536 g of trichloroacetic acid and dissolve it in 90 mL of distilled water, then dilute to 100 mL.

[0048] Sample reaction: Incubate 1 mL of dipeptide Cbz-Gln-Gly solution in a 37°C water bath for 10 min. Add 100 μL of diluted fermentation supernatant, mix well, and incubate in a 37°C water bath for 30 min. After the reaction, add 1 mL of 0.4 mol / L trichloroacetic acid solution, mix well, and terminate the reaction. Take 60 μL of the reaction solution and mix thoroughly with 240 μL of distilled water, 300 μL of colorimetric reagent A, 150 μL of colorimetric reagent B, and 300 μL of colorimetric reagent C. Incubate in a 37°C water bath for 20 min, cool, and measure the absorbance of the system at 630 nm.

[0049] Blank reaction: Take 100 μL of diluted fermentation broth and boil it at 100℃ for 5 min as a blank group. The enzyme activity detection method is the same as above.

[0050] Enzyme activity (U / mL):

[0051] Where OD1 is the light absorbance of the experimental system, OD2 is the light absorbance of the control system, a is the slope of the ammonia solution standard curve, 30 is the reaction time in min, 2.1 / 0.1 is the volume ratio of the reaction solution to the enzyme solution, and 17.03 is the relative molecular mass of ammonia.

[0052] Example 1: Construction of a Bacillus subtilis protease-deficient strain

[0053] Using the primers listed in Table 2, amplification was performed on the upstream and downstream homologous arms of the extracellular protease-related genes (aprE, bpr, epr, mpr, vpr, nprB, nprE, and wprA, nucleotide sequences of which are shown in SEQ ID NO. 1-8, respectively), the chloramphenicol resistance gene fragment (containing the lox site), and the T-vector fragment derived from Bacillus subtilis 168. Seamless amplification was then performed. The CloningMaster Seamless Cloning Kit was used to ligate homologous arm fragments, chloramphenicol resistance gene (containing the lox site) fragment, and T vector fragment. The resulting fragments were transformed into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Successfully transformed transfectants were selected and inoculated into LB broth for 12 h. Plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis, yielding the protease expression plasmid T-AprE. Other protease knockout fragments were constructed in the same manner. Through ligation and screening, other protease knockout fragment plasmids T-Bpr, T-Epr, T-Mpr, T-Vpr, T-NprB, T-NprE, and T-WprA were obtained. The plasmid map is shown below. Figure 1 As shown.

[0054] The protease knockout fragment (including the upper and lower homologous arms of the protease and the chloramphenicol resistance gene) in the above recombinant plasmid was amplified by PCR and purified using primers. It was then transformed into Bacillus subtilis 168 using competent cell transformation. The specific procedure is as follows: Freshly activated Bacillus subtilis 168 was inoculated into an appropriate amount of fresh LB liquid medium and cultured overnight at 37°C and 220 rpm with shaking. The next day, the bacterial culture was diluted to OD. 600 The concentration was approximately 1.0. Xylose (50%) was added as an inducer to a final concentration of 3% for induction. After culturing at 37°C and 220 rpm for 2 hours with shaking, competent cells were obtained. 0.2 mL of competent cells were taken, and 10 μL of a protease knockout fragment was added. After culturing at 37°C and 220 rpm for 90 minutes with shaking, the cells were plated on LB agar plates containing 5 μg / mL chloramphenicol and incubated overnight at 37°C to obtain Bacillus subtilis strains with different protease knockouts. These strains were named BD1, BD2, BD3, BD4, BD5, BD6, BD7, and BD8, respectively.

[0055] Table 2 lists the primers involved.

[0056]

[0057]

[0058]

[0059] Example 2: Construction of Bacillus subtilis protease overexpression strain

[0060] The protease-related genes (aprE, bpr, epr, mpr, vpr, nprB, nprE, and wprA, with nucleotide sequences as shown in SEQ ID NO. 1-8, respectively) were amplified using the primers listed in Table 3. glvc The promoter gene fragment (nucleotide sequence shown in SEQ ID NO. 9) and the pHT01 vector fragment were first ligated using a seamless cloning master to the protease AprE gene fragment, promoter P glvc The gene fragment and the pHT01 gene vector fragment were transformed into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and cultured overnight at 37°C. Successfully transformed transfectants were selected and inoculated into LB broth for 12 h. Plasmids were extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis, yielding the protease expression plasmid pHT01-AprE. Then, using pHT01-AprE plasmid as a vector template, the aprE gene fragment was replaced with other protease genes. Through ligation and screening, other protease expression plasmids pHT01-Bpr, pHT01-Epr, pHT01-Mpr, pHT01-Vpr, pHT01-NprB, pHT01-NprE, and pHT01-WprA were obtained. The plasmid map is shown below. Figure 2 As shown.

[0061] The recombinant plasmid was co-transformed into the host Bacillus subtilis WB800 using a competent transformation method. The specific procedure is as follows: Freshly activated Bacillus subtilis WB800 was inoculated into an appropriate amount of fresh LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm. The next day, the bacterial culture was diluted to OD. 600 When the concentration reaches approximately 1.0, xylose (50%) is added as an inducer to a final concentration of 3% for induction. After incubation at 37°C and 220 rpm for 2 hours, competent cells are obtained. Then, 0.2 mL of competent cells are taken, and 10 μL of protease expression plasmid is added. After incubation at 37°C and 220 rpm for 90 min, the cells are plated on LB agar plates containing 10 μg / mL chloramphenicol and incubated overnight at 37°C to obtain Bacillus subtilis strains containing different protease expression plasmids, which are named BS1, BS2, BS3, BS4, BS5, BS6, BS7, and BS8, respectively.

[0062] Table 3 lists the primers involved.

[0063]

[0064]

[0065] Example 3: Construction of recombinant protein glutaminase strain

[0066] The protein glutaminase derived from *Chlorella vulgaris* was named PG, and its encoding nucleotide sequence is shown in SEQ ID NO. 10. The PG gene was synthesized by Sangon Biotech (Shanghai) Co., Ltd. Then, using the synthesized gene fragment as a template, the PG gene fragment was expanded using primers PG-F and PG-R, while the pP43NMK vector fragment was amplified using PV-F and PV-R.

[0067] The PCR primers are as follows:

[0068] PG-F:5'-cttgctggatgtacgGATTCAAATGGCAATCAAGAAATTAATGGC-3';

[0069] PG-R:5'-gctttcatcaTTAatgatgatgatgatgatgAAAGCCGCATGATGAAACAT CCG-3';

[0070] PV-F:5'-catcatcatTAAtgatgaaagcttggccaggac-3';

[0071] PV-R:5'-CATTTGAATCgtacatccagcaagcaaaatcataag-3'.

[0072] The protein glutaminase gene and the pP43NMK vector fragment were amplified using the primers described above. The vector fragment and the PG gene were ligated using the Seamless Cloning Master kit. The resulting fragment was transformed into *E. coli* JM109, plated on LB agar plates containing 100 μg / mL ampicillin, and incubated overnight at 37°C. Successfully transformed transfectants were selected and inoculated into LB broth for 12 hours. The plasmid was extracted and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis, yielding the protein glutaminase expression plasmid pP43NMK-PG. The plasmid map is shown below. Figure 3 As shown.

[0073] The recombinant plasmid pP43NMK-PG was transformed into Bacillus subtilis host cells deficient in different proteases using the same transformation method as above. After preparing competent cells from Bacillus subtilis strains containing different proteases, 0.2 mL of competent cells were taken and 10 μL of protein glutaminase expression plasmid was added. After incubation at 37°C and 220 rpm for 90 min with shaking, the cells were plated on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin and incubated overnight at 37°C to obtain Bacillus subtilis strains containing different protease expression plasmids and protein glutaminase expression plasmids, which were named BD1-PG, BD2-PG, BD3-PG, BD4-PG, BD5-PG, BD6-PG, BD7-PG, and BD8-PG, respectively.

[0074] The recombinant plasmid pP43NMK-PG was transformed into Bacillus subtilis host cells overexpressing different proteases using the same transformation method as above. After preparing competent cells from Bacillus subtilis strains containing different proteases, 0.2 mL of competent cells were taken and 10 μL of protein glutaminase expression plasmid was added. After incubation at 37°C and 220 rpm for 90 min with shaking, the cells were plated on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin and incubated overnight at 37°C to obtain Bacillus subtilis strains containing different protease expression plasmids and protein glutaminase expression plasmids, which were named BS1-PG, BS2-PG, BS3-PG, BS4-PG, BS5-PG, BS6-PG, BS7-PG, and BS8-PG, respectively.

[0075] Example 4: Screening of recombinant strains of protein glutaminase

[0076] Colonies obtained from the above plates were picked and inoculated into 2 mL of LB liquid containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin, respectively. The cultures were incubated at 37°C and 220 rpm for 12 h to obtain seed culture. Then, 1 mL of the seed culture was inoculated into 25 mL of TB liquid medium and incubated at 37°C and 220 rpm for 24 h. Maltose, an inducer, was added to a final concentration of 2%, and the cultures were continued for 36 h. The supernatant was obtained by centrifugation and the activity of protein glutaminase was detected.

[0077] The expression results of protein glutaminase in the protease-deficient strains constructed above are as follows: Figure 4 As shown, the PG enzyme activity of strain BD1-PG decreased significantly, while that of strain BD8-PG increased slightly. This indicates that the absence of the AprE protease is detrimental to the production of active protein glutaminase, while the absence of the WprA protease is beneficial to the production of active protein glutaminase.

[0078] The expression results of protein glutaminase in the protease overexpression strains constructed above are as follows: Figure 5 As shown, protein glutaminase activity was detected in the fermentation supernatant of BS1-PG, BS2-PG, and BS3-PG after 36 hours of fermentation, with values ​​of 6.7 U / mL, 4.6 U / mL, and 3.8 U / mL, respectively. This indicates that AprE, Bpr, and Epr can activate protein glutaminase prozymogen, and that AprE protease is the main activating protease, consistent with the results of the knockout strains mentioned above.

[0079] Example 5: Fermentation culture of recombinant protein glutaminase strain BS1-PG

[0080] Single colonies of the recombinant protein glutaminase strain were picked and inoculated into 2 mL of LB broth containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin, respectively. The cultures were incubated at 37°C and 220 rpm for 12 h to obtain seed culture. Then, 1 mL of the seed culture was inoculated into 25 mL of TB broth and incubated at 37°C and 220 rpm for 24 h. An inducer was then added. Starting from 36 h of culture, samples were taken every 12 h, and the supernatant was obtained by centrifugation for protein glutaminase activity detection.

[0081] The enzyme activity changes of the recombinant protein glutaminase strain BS1-PG constructed above during shake-flask fermentation are as follows: Figure 6 As shown, the activity of protein glutaminase reached its peak at 96 h of fermentation, with a maximum activity of 18.05 U / mL.

[0082] Single colonies of the recombinant glutaminase strain were picked and inoculated into 10 mL of LB broth containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin, respectively. The cultures were incubated at 37°C and 220 rpm for 12 h with shaking to obtain seed culture. Then, 2 mL of the seed culture was inoculated into 200 mL of LB broth and incubated at 37°C and 220 rpm for 12 h with shaking. Finally, the culture was transferred to a 5 L fermenter containing 2 L of fermentation medium. The fermentation medium composition was: glucose 20 g / L, yeast extract 24 g / L, peptone 24 g / L, K₂HPO₄ 12.5 g / L, KH₂PO₄ 2.5 g / L, urea 8 g / L, pH 7.4. An inducer was added after 30 h of fermentation. Fermentation process: temperature 37°C, stirring speed 500-600 rpm, aeration rate 1.5 m³ / h. 3 / h, inoculation amount is 5%, dissolved oxygen is controlled above 25%.

[0083] The enzyme activity and strain OD of the recombinant protein glutaminase strain BS1-PG constructed above were measured during fermentation in a 5L fermenter. 600 The results of the changes are as follows Figure 7 As shown, the enzyme activity reached its peak after 60 hours of fermentation, with a maximum activity of 45.58 U / mL.

[0084] Example 6: Construction of Bacillus subtilis engineered strain W8ΔMT6-Pro-PPG

[0085] To further increase the yield of protein glutaminase, the upstream and downstream homologous arms of the gene fragments lytC (Gene ID: 936777), spoIVB (Gene ID: 938654), amyE (Gene ID: 938356), malA (Gene ID: 936161), srfAC (Gene ID: 938308), and mdxL (Gene ID: 938623) on the Bacillus subtilis genome were amplified using the primers listed in Table 4. The chloramphenicol resistance gene fragment (containing the lox site) was also amplified. Homologous recombination fragments were obtained by multi-fragment PCR fusion. Figure 8 As shown in the figure, the homologous recombination fragment was purified and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis.

[0086] The verified homologous recombination fragment constructed above was transformed into Bacillus subtilis WB800. The specific procedure is as follows: Freshly activated Bacillus subtilis 168 was inoculated into an appropriate amount of fresh LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm. The next day, the bacterial culture was diluted to OD. 600 To induce gene knockout, xylose was added to a final concentration of 3% at approximately 1.0. The cells were then cultured at 37°C with shaking at 220 rpm for 2 hours to obtain competent cells. 0.2 mL of each competent cell was then added to 10 μL of a protease knockout fragment. After culturing at 37°C with shaking at 220 rpm for 90 minutes, the cells were plated on LB agar plates containing 5 μg / mL chloramphenicol and incubated overnight at 37°C to obtain Bacillus subtilis strains with different protease knockouts. After verifying successful gene knockout using primers, the chloramphenicol marker was eliminated using the pDG148-cre plasmid. Strains with the plasmid eliminated underwent the next round of gene editing. After six rounds of gene editing, the engineered strain W8ΔMT6 with six gene deletions was obtained.

[0087] Table 4 lists the primers involved.

[0088]

[0089]

[0090]

[0091] The recombinant plasmid pHT01-AprE constructed in Example 2 was co-transformed into the host Bacillus subtilis W8ΔMT6 using a competent transformation method. The specific process is as follows: Freshly activated strains were inoculated into an appropriate amount of fresh LB liquid medium and cultured overnight at 37°C with shaking at 220 rpm. The next day, the bacterial culture was diluted to OD. 600 To induce cell growth, xylose was added to a final concentration of 3% at approximately 1.0. The cells were then cultured at 37°C with shaking at 220 rpm for 2 hours to obtain competent cells. 0.2 mL of each competent cell was then added to 10 μL of a protease expression plasmid. After culturing at 37°C with shaking at 220 rpm for 90 minutes, the cells were plated onto LB agar plates containing 10 μg / mL chloramphenicol and cultured overnight at 37°C to obtain Bacillus subtilis strains containing different protease expression plasmids, named W8ΔMT6-Pro.

[0092] The recombinant plasmid pP43NMK-PG obtained in Example 3 was transformed into strain W8ΔMT6-Pro using the same transformation method as above. After preparing competent cells from Bacillus subtilis strains containing different proteases, 0.2 mL of competent cells were taken and 10 μL of protein glutaminase expression plasmid was added. After incubation at 37°C and 220 rpm for 90 min with shaking, the cells were plated on LB plates containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin and incubated overnight at 37°C to obtain an engineered strain containing both protease expression plasmid and protein glutaminase expression plasmid, named W8ΔMT6-Pro-PPG.

[0093] Example 7: Fermentation production of protein glutaminase by recombinant strain W8ΔMT6-Pro-PPG

[0094] Single colonies of the engineered strain W8ΔMT6-Pro-PPG constructed above were picked and inoculated into 2 mL of LB broth containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin, respectively. The cultures were incubated at 37°C and 220 rpm for 12 h to obtain seed culture. Then, 1 mL of the seed culture was inoculated into 25 mL of TB broth and incubated at 37°C and 220 rpm for 36 h. The bacterial cells were sampled and centrifuged to obtain the supernatant, which was then used to detect the enzyme activity of protein glutaminase.

[0095] The results showed that the protein glutaminase activity of the engineered strain constructed above was 15.05 U / mL after 36 h of fermentation in shake flasks.

[0096] Single colonies of the recombinant glutaminase strain were picked and inoculated into 10 mL of LB broth containing 10 μg / mL chloramphenicol and 50 μg / mL kanamycin, respectively. The cultures were incubated at 37°C and 220 rpm for 12 h with shaking to obtain seed culture. Then, 2 mL of the seed culture was inoculated into 200 mL of LB broth and incubated at 37°C and 220 rpm for 12 h with shaking. Finally, the culture was transferred to a 5 L fermenter containing 2 L of fermentation medium. The fermentation medium composition was: glucose 20 g / L, yeast extract 24 g / L, peptone 24 g / L, K₂HPO₄ 12.5 g / L, KH₂PO₄ 2.5 g / L, urea 8 g / L, pH 7.4. An inducer was added after 30 h of fermentation. Fermentation process: temperature 37°C, stirring speed 500-600 rpm, aeration rate 1.5 m³ / h. 3 / h, inoculation amount is 3%, dissolved oxygen is controlled above 15%.

[0097] The results are as follows Figure 9 The results showed that the constructed protein glutaminase engineered strain W8ΔMT6-Pro-PPG reached its highest enzyme activity after 64 hours of fermentation, with a maximum activity of 65.68 U / mL.

[0098] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A Bacillus subtilis strain that produces high levels of protein glutaminase, characterized in that: The Bacillus subtilis heterologously expresses the protein glutaminase and overexpresses the extracellular protease encoding gene. AprE, The AprE The nucleotide sequence is shown in SEQ ID NO.1, and the protein glutaminase is derived from *Aureobacterium glutamicum*, whose nucleotide sequence is shown in SEQ ID NO.

10.

2. The Bacillus subtilis according to claim 1, characterized in that: Knockout gene in the genome of Bacillus subtilis lytC , spoIVB , amyE , malA , sr fAC and mdxL .

3. The Bacillus subtilis according to claim 1, characterized in that: The Bacillus subtilis is Bacillus subtilis 168, Bacillus subtilis WB600, or Bacillus subtilis WB800.

4. The Bacillus subtilis according to claim 1, characterized in that: The overexpression uses P glvc The promoter, the P glvc The nucleotide sequence of the promoter is shown in SEQ ID NO.

9.

5. A microbial preparation comprising any one of the Bacillus subtilis according to claims 1-4.

6. The use of Bacillus subtilis according to any one of claims 1-4 or the microbial preparation according to claim 5 in the production of protein glutaminase.

7. A method for producing protein glutaminase, characterized in that: Add Bacillus subtilis according to any one of claims 1-4 or the microbial preparation according to claim 5 to the fermentation system.

8. The method according to claim 7, characterized in that: Fermentation time is 50-66 hours.

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