A method for increasing the secretory expression of protein glutaminase using NprB signal peptide

Abstract

This divisional application discloses a method for increasing the secretory expression of protein glutaminase using NprB signal peptide, and belongs to the field of genetic engineering technology. In the present invention, by modifying the signal peptide, efficient extracellular expression of protein glutaminase in Bacillus subtilis is achieved, solving the problem of insufficient extracellular secretion of protein glutaminase. The present invention increases the secretory enzyme activity of protein glutaminase from 440U / L to 2020U / L by modifying the signal peptide, and the enzyme activity is increased by 4.59 times. The application of the signal peptide in the present invention significantly increases the extracellular secretion of protein glutaminase, reduces the cost of industrial production of protein glutaminase, and also lays a foundation for increasing the expression of exogenous secretory proteins in Bacillus subtilis.

Description

[0001] This application is a divisional application of the invention patent with patent number "202110132935.5", application date "February 1, 2021", and name "A method for increasing the secretory expression of protein glutaminase". Technical Field

[0002] The invention relates to a method for improving the secretion expression of protein glutaminase, belonging to the technical field of genetic engineering. Background Art

[0003] Protein-glutaminase (EC3.5.1.44, PG for short) is a type of specific glutamine deamidating hydrolase. Protein-glutaminase is mainly used in the food industry. A large number of studies have verified that the enzyme can significantly improve the solubility, emulsification and foaming properties of plant proteins such as wheat gluten and rice gluten. The deamidation of protein-glutaminase does not require pretreatment of the protein in advance and has strong specificity. It only acts on the glutamine on the side chain of the protein, has no deamidation effect on asparagine, and does not cause adverse changes to the structure of the protein. However, the application of protein-glutaminase is currently restricted, and low protein secretion expression is one of the important factors.

[0004] Compared with the wild Chryseobacterium utilis that produces protein glutaminase, Bacillus subtilis, as a traditional industrial production bacterium, has a strong protein secretion ability and is a good system for the expression and secretion of protein glutaminase. Current studies have shown that the selection and optimization of expression elements such as promoters and signal peptides of Bacillus subtilis have an important influence on the expression of exogenous proteins. Among them, the most important function of the signal peptide is to guide secreted proteins or membrane proteins to correctly locate in different regions or organelles in the cell, which can improve the solubility of exogenous proteins and avoid difficulties such as product purification caused by inclusion body renaturation. However, signal peptides are often specific to protein expression and secretion. The expression and secretion efficiency of the same protein under the action of different signal peptides is often very different, and there is a lack of universal rules.

[0005] Therefore, it is very difficult to obtain an efficient and specific signal peptide sequence, improve the protein glutaminase secretion pathway, and increase the expression efficiency of protein glutaminase, but it has important application value. Summary of the invention

[0006] The present invention provides a method for improving the secretory expression of protein glutaminase, which comprises: using a signal peptide containing any one of the amino acid sequences shown in SEQ ID NO.1-8 to guide the secretory expression of protein glutaminase.

[0007] In one embodiment of the present invention, the amino acid sequence of the protein glutaminase is shown in GenBank Accession No.: AB046594.1, and the nucleotide sequence encoding the protein glutaminase is shown in SEQ ID NO.9.

[0008] In one embodiment of the present invention, the nucleotide sequence encoding the signal peptide shown in SEQ ID NO.1 is shown as SEQ ID NO.22. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.2 is shown as SEQ ID NO.23. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.3 is shown as SEQ ID NO.24. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.4 is shown as SEQ ID NO.25. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.5 is shown as SEQ ID NO.26. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.6 is shown as SEQ ID NO.27. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.7 is shown as SEQ ID NO.28. The nucleotide sequence encoding the signal peptide shown in SEQ ID NO.8 is shown as SEQ ID NO.29.

[0009] In one embodiment of the present invention, the secretory expression is achieved by constructing a recombinant bacterium.

[0010] In one embodiment of the present invention, the recombinant bacteria uses bacteria as hosts.

[0011] In one embodiment of the present invention, the recombinant bacterium is a recombinant Bacillus subtilis.

[0012] In one embodiment of the present invention, the starting strain of the recombinant Bacillus subtilis is Bacillus subtilis WB600.

[0013] In one embodiment of the present invention, the recombinant Bacillus subtilis uses Bacillus subtilis WB600 as the starting strain, and expresses a recombinant gene whose N-terminal fusion nucleotide sequence of the protein glutaminase gene encoded by the gene shown in SEQ ID NO.9 is a signal peptide shown in SEQ ID NOs. 22 to 29.

[0014] The second object of the present invention is to provide a recombinant bacterium, wherein the recombinant bacterium uses a signal peptide containing an amino acid sequence shown in any one of SEQ ID NOs. 1 to 8 to guide the secretory expression of protein glutaminase.

[0015] In one embodiment of the present invention, the recombinant bacterium is a recombinant Bacillus subtilis.

[0016] In one embodiment of the present invention, the method for constructing the recombinant Bacillus subtilis is to fuse the signal peptide shown in any one of SEQ ID NOs. 1 to 8 to the N-terminus of the gene encoding protein glutaminase to obtain a recombinant gene, and the recombinant plasmid formed by connecting the recombinant gene with the expression plasmid pP43NMK is transformed into Escherichia coli JM109, and the extracted plasmid is transformed into Bacillus subtilis WB600.

[0017] In one embodiment of the present invention, the amino acid sequence of the protein glutaminase is shown in GenBank Accession No. AB046594.1; the gene sequence encoding the protein glutaminase is shown in SEQ ID NO.9.

[0018] In one embodiment of the present invention, the recombinant gene is a recombinant gene containing a modified signal peptide and a protein glutaminase gene sequence obtained by gene synthesis and one-step cloning.

[0019] In one embodiment of the present invention, the construction method of the recombinant Bacillus subtilis is as follows: synthesize the signal peptide shown in any one of SEQ ID NO.22 to 29, insert the DNA fragment of each signal peptide into the RBS of the target vector by a one-step cloning method, the RBS sequence is as shown in SEQ ID NO.21, obtain a recombinant plasmid, transfer it into E.coli JM109, and after the plasmid is extracted, introduce it into the host bacterial cell to obtain the recombinant Bacillus subtilis.

[0020] The present invention further provides a method for producing protein glutaminase, which is to use the above-mentioned recombinant bacteria to ferment and produce protein glutaminase. Optionally, the recombinant bacteria is a recombinant Bacillus subtilis.

[0021] In one embodiment of the present invention, the method is to culture the recombinant Bacillus subtilis at 37° C. for 36 hours.

[0022] In one embodiment of the present invention, the method is to inoculate the recombinant Bacillus subtilis into a fermentation medium for fermentation.

[0023] In one embodiment of the present invention, the inoculation is to inoculate 4% seed liquid by volume.

[0024] In one embodiment of the present invention, the seed solution is obtained by inoculating the recombinant bacteria into LB culture medium and culturing at 37° C. and 220 rpm for 8 to 12 hours.

[0025] In one embodiment of the present invention, the method is to first perform seed culture and then ferment; the seed culture is to transform the constructed recombinant plasmid into Bacillus subtilis WB600, and transform it immediately after use. Pick a single colony of Bacillus subtilis from a plate, inoculate it into a 250mL triangular flask with a liquid volume of 25mL LB medium, and culture it at 37°C and 220r / min for 8 to 12h; the shake flask fermentation is to inoculate the seed liquid cultured for 10h into a fermentation medium containing 50mg / L kanamycin at an inoculum volume of 4% (V / V), with a liquid volume of 25mL / 250mL, and culture it at 37°C and 220r / min for 36h.

[0026] The present invention also claims to protect the application of the recombinant bacteria in the food and biological fields.

[0027] The present invention also claims to protect the use of the signal peptide (NprB) described in SEQ ID NO.7 in improving the secretory expression effect of protein glutaminase or in the production of protein glutaminase.

[0028] Beneficial effects:

[0029] In the present invention, by modifying the NprB signal peptide, efficient extracellular expression of protein glutaminase in Bacillus subtilis is achieved, and the problem of insufficient extracellular secretion of protein glutaminase is solved. The present invention improves the secretion activity of protein glutaminase from 440U / L to 2020U / L by modifying the signal peptide, and the enzyme activity is increased by 4.59 times. The application of the signal peptide in the present invention significantly improves the extracellular secretion of protein glutaminase, reduces the cost of industrial production of protein glutaminase, and also lays a foundation for increasing the expression amount of exogenous secretory protein of Bacillus subtilis. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Flow chart for constructing recombinant plasmids;

[0031] Figure 2 is an electrophoresis diagram; wherein GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB, and YdeJ are recombinant strains obtained by transforming a recombinant plasmid containing a SecA promoter and a signal peptide of GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB, and YdeJ into a competent Bacillus subtilis WB 600;

[0032] Figure 3 It is the fermentation enzyme activity of the recombinant strain. DETAILED DESCRIPTION

[0033] The amino acid sequences of the signal peptides GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB, and YdeJ are shown in SEQ ID NOs. 1 to 8, and the nucleotide sequences of the genes encoding the above signal peptides are shown in SEQ ID NOs. 22 to 29, respectively.

[0034] The nucleotide sequence of the gene PG encoding protein glutaminase is shown in SEQ ID NO.9.

[0035] LB / seed medium (g / L): tryptone 10, yeast powder 5, NaCl 10. Fermentation medium (g / L): yeast extract 24, tryptone 12, glycerol 10, K 2HPO 4 16.43, KH 2PO 4 2.32.

[0036] Method for determining the activity of protein glutaminase: Protein glutaminase can specifically hydrolyze the aminoacyl group on the glutamine residue in protein to release free ammonia. Under the catalysis of sodium nitroprusside and with sodium hypochlorite as an oxidant, ammonia reacts with phenol 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 OD 630.

[0037] Enzyme activity definition: 100 μL enzyme solution hydrolyzes dipeptide Cbz-Gln-Gly to release 1 μmol of ammonia per minute as 1 enzyme activity unit (U / ml). Method: Take 1 mL dipeptide Cbz-Cln-Gly solution (weigh 0.337 g dipeptide Cbz-Gln-Gly, dissolve in 0.176 mol / L PBS buffer (pH 6.5), and dilute to 100 mL), add 100 μL of the sample to be tested (100 μL of inactive enzyme solution as blank control), mix well, place in a 37°C incubator for 30 min, add 1 mL 0.4 mol / L trichloroacetic acid solution, mix well and terminate the reaction. Take 60 μL of the reaction solution and 240 μL of distilled water, 300 μL of color reagent A (weigh 2.023 g of phenol and 7.5 mg of sodium nitrosoferricyanide, dissolve in double distilled water and make up to 50 mL), 150 μL of color reagent B (weigh 2.5 g of KOH, dissolve in double distilled water and make up to 50 mL), and 300 μL of color reagent C (weigh 10.2 g of anhydrous K 2CO 3, dissolve in double distilled water, add 417 μL of NaClO solution, and add water to make up to 50 mL), mix thoroughly, place in a 37°C incubator for 20 min, cool, and measure the light absorbance of the system at 630 nm.

[0038] Example 1: Construction of recombinant plasmid of starting strain

[0039] A 6×His histidine tag with a sequence as shown in SEQ ID NO.31 was added to the C-terminus of protein glutaminase (GenBank: AB046594.1), and a nucleotide sequence of 981 bp in total was synthesized (completed by Shanghai Biotechnology Co., Ltd.). Two pairs of primers SEQ ID NO.11 and SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.14 were designed, and the target fragment with a sequence of SEQ ID NO.9 and the pP43NMK vector fragment with sticky ends of SEQ ID NO.10 were obtained by one-step PCR, respectively. The fragments were connected at 37°C for 30 minutes using a one-step cloning method, and the connected products were chemically transformed into Escherichia coli JM109 competent cells. The transformation solution was spread on an LB plate containing ampicillin (100 mg / L). The sequencing of the transformants was completed by Shanghai Biotechnology Co., Ltd., and the recombinant plasmid of the starting strain was obtained after the sequencing was correct.

[0040] Example 2: Preparation of competent Bacillus subtilis

[0041] The recombinant plasmid of the starting strain is recovered by the column and transformed into Bacillus subtilis WB600. The specific operation is as follows:

[0042] (1) Pick a loop of Bacillus subtilis WB600 culture medium from the glycerol tube, streak it on an LB plate, and culture it at 37°C overnight;

[0043] (2) Pick a single colony of Bacillus subtilis WB600 from the plate, inoculate it into a 14 mL shaking tube containing 2 mL SPI, and culture it at 37°C, 220 rpm overnight;

[0044] (3) Take 200 μL of the overnight culture in step 2 and inoculate it into a 14 mL shake tube containing 2 mL of SPII. Cultivate at 37°C and 220 rpm for 1.5 to 2 h.

[0045] (4) Add 20 μL EGTA 10 min before aliquoting in step 3.

[0046] (5) Aliquot 500 μL of the culture obtained in step 3 into a 1.5 mL EP tube to obtain competent Bacillus subtilis WB600.

[0047] Example 3: Construction of a recombinant plasmid containing the promoter SecA

[0048] Primers were designed, and the sequences were shown in SEQ ID NO.15 and SEQ ID NO.16. The recombinant plasmid constructed in Example 1 was used as a template for one-step PCR to obtain the pP43NMK fragment with sticky ends. Primers SEQ ID NO.17 and SEQ ID NO.18 were designed at the same time. The promoter SecA fragment was obtained from the Bacillus subtilis genome by one-step PCR. The two fragments obtained by the above PCR were cloned in one step, and the ligation product was transformed into Escherichia coli JM109 competent cells by a chemical method. The transformation liquid was spread on an LB plate containing ampicillin (100 mg / L). The sequencing of the transformant was completed by Shanghai Biotech, and the promoter P43 was finally replaced with the promoter SecA after the sequencing was correct.

[0049] Example 4: Construction of recombinant Bacillus subtilis containing different signal peptides

[0050] The upstream and downstream primers were designed, the upstream primer sequence was shown in SEQ ID NO.19, and the downstream sequence was shown in SEQ ID NO.20. The recombinant plasmid of Example 3 was used as a template for PCR amplification, and the obtained fragment sequence was shown in SEQ ID NO.30, which was used as the vector fragment for the subsequent one-step cloning; the genes encoding the signal peptides shown in SEQ ID NOs.1 to 8 were synthesized, and the homologous fragment gtaagagaggaatgtacac shown in SEQ ID NO.32 was added upstream of each signal peptide, and the homologous fragment GATTCAAATGGCAATCAAGAAATTAATG was added downstream, and they were named GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB, and YdeJ (the nucleotide sequences were shown in SEQ ID NOs.22 to 29, respectively) as the insert fragments for one-step cloning and inserted into the target vector RBS. The RBS sequence was shown in SEQ ID NO.21. After connection by the one-step cloning method, transformation and sequencing were performed to obtain the recombinant plasmid after signal peptide modification. 5-10 μL of the recombinant plasmid was transferred into the competent medium of Bacillus subtilis WB600 prepared in Example 2, cultured at 37°C and 220 r / min for 2 h, centrifuged at 8000 rpm for 2 min, 400 μL of the supernatant was discarded and mixed, and then spread on an LB plate containing kanamycin (50 mg / L), and cultured at 37°C overnight to obtain transformants; the transformants on the LB plate were picked for colony PCR, and the bands of the correct size were obtained by nucleic acid electrophoresis ( Figure 2 ), indicating that recombinant Bacillus subtilis was obtained.

[0051] Example 5: Construction of shake flask fermentation culture of recombinant Bacillus subtilis

[0052] The signal peptides GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB, and YdeJ of Example 4 were used in recombinant Bacillus subtilis as production strains, and the GlmU recombinant Bacillus subtilis constructed in Example 4 was used as a control. The 8 recombinant Bacillus subtilis single colonies in Example 4 were picked and inoculated into a 250 mL Erlenmeyer flask containing 25 mL of LB medium, and cultured at 37° C. and 220 r / min for 8 to 12 h. Then, they were transferred to a 250 mL Erlenmeyer flask containing 25 mL of fermentation medium according to an inoculum size of 4%, and cultured at 37° C. and 220 r / min for 36 h, and then the enzyme activity of the fermentation broth supernatant was measured.

[0053] The results are as follows Figure 3 As shown, the fermentation enzyme activities of the recombinant Bacillus subtilis GlmU, LipA, YmzC, NprE, YfkN, SpoIIIJ, NprB and YdeJ were 440U / L, 480U / L, 20U / L, 570U / L, 910U / L, 540U / L, 2020U / L and 3260U / L, respectively. Among them, the enzyme activity of strain NprB reached 2020U / L, which was 4.59 times higher than that of strain GlmU (control).

[0054] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

[0055] SEQ ID NO.1

[0056] MDRRDNGGQYMDKRFAVVLAAGQGTRMKSKLYKVLHP

[0057] SEQ ID NO.2

[0058] MKFVKRRIIALVTILMLSVTSLFALQPSAKA

[0059] SEQ ID NO.3

[0060] MFESEAELRRIRIALVWIAVFLLFGACG

[0061] SEQ ID NO.4

[0062] MGLGKKLSVAVAASFMSLSISLPGVQA

[0063] SEQ ID NO.5

[0064] MRIQKRRTHVENILRILLPPIMILSLILPTPPIHA

[0065] SEQ ID NO.6

[0066] MLLKRRIGLLLSMVGVFMLLAGCS

[0067] SEQ ID NO.7

[0068] MRNLTKTSLLLAGLCTAAQMVFVTHASA

[0069] SEQ ID NO.8

[0070] MKKRRKICYCNTALLLMILLAGCT

[0071] SEQ ID NO.9

[0072] ATGAAAAACCTTTTTCTGAGCATGATGGCGTTTGTTACAGTTCTGACCTTTAATAGCTGCGCTGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTTTCCGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACGGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTCTCTTTTATGCTGACAGCACAATTTTATGAGATTAAACCTACAAAAGAAAATGAACAATATATTGGTATGCTTAGACAAGCAGTTAAAAATGAATCACCGGTCCATATTTTTCTTAAACCGAATAGCAATGAAATTGGCAAAGTTGAATCAGCGAGCCCTGAAGATGTTAGATACTTTAAAACAATTCTCACGAAGGAAGTGAAAGGCCAGACGAATAAACTGGCCAGCGTCATTCCGGACGTGGCGACGCTGAATAGCCTTTTTAACCAAATCAAGAATCAAAGTTGTGGAACATCAACAGCATCAAGCCCGTGCATTACATTTAGATATCCGGTTGATGGCTGCTATGCGAGAGCACATAAAATGAGACAGATTCTTATGAATAATGGCTATGATTGCGAAAAACAATTTGTCTATGGCAATCTGAAAGCAAGCACAGGCACATGCTGCGTCGCATGGAGCTATCATGTGGCAATTCTGGTTTCATATAAAAATGCGTCAGGCGTAACAGAAAAACGCATTATTGATCCGTCATTATTTTCATCAGGCCCGGTGACAGATACGGCATGGAGAAATGCGTGCGTGAATACAAGCTGCGGCTCAGCAAGCGTTAGCAGCTATGCAAATACAGCGGGAAATGTTTATTATCGTTCACCGTCAAATAGCTATCTTTATGATAATAATCTGATTAATACAAATTGCGTTCTGACAAAATTTAGCCTTCTGAGCGGCTGTAGCCCGAGCCCGGCACCGGATGTTTCATCATGCGGCTTTcatcatcatcatcatcatTAA

[0073] SEQ ID NO.10

[0074]

[0075] SEQ ID NO.11

[0076] ATGAAAAACCTTTTTCTGAGCATGATGGCGTTTG

[0077] SEQ ID NO.12

[0078] TTAatgatgatgatgatgatgAAAGCCGCATGATG

[0079] SEQ ID NO.13

[0080] GAAAAAGGTTTTTCATgtgtacattcctctcttacctataatg

[0081] SEQ ID NO.14

[0082] catcatcatTAAtgatgaaagcttggcgtaatc

[0083] SEQ ID NO.15

[0084] GAGGTATACATGGACTAGgtaagagaggaatg

[0085] SEQ ID NO.16

[0086] CAAGCTCTCCTCggatccagttgctcaaaaaaatctcg

[0087] SEQ ID NO.17

[0088] GAGGAGAGCTTGGACATCGTC

[0089] SEQ ID NO.18

[0090] CTAGTCCATGTATACCTCTCATTATC

[0091] SEQ ID NO.19

[0092] GATTCAAATGGCAATCAAGAAATTAATG

[0093] SEQ ID NO.20

[0094] Gtgtacattcctctcttac

[0095] SEQ ID NO.21

[0096] Gtaagagaggaatgtacac

[0097] SEQ ID NO.22

[0098] ATGGATAggAGGGATAATGGAGGCCAATACATGGATAAGCGGTTTGCAGTTGTGTTAGCAGCTGGTCAAGGAACAAGAATGAAATCAAAGCTATATAAAGTTCTTCATCCT

[0099] SEQ ID NO.23

[0100] atgaaatttgtaaaaagaaggatcattgcacttgtaacaattttgatgctgtctgttacatcgctgtttgcgttgcagccgtcagcaaaagcc

[0101] SEQ ID NO.24

[0102] Atgtttgaaagtgaagcagaactgagacgaatcaggattgcacttgtatggatagctgtctttttactgttcggggcgtgcggg

[0103] SEQ ID NO.25

[0104] Atgggtttaggtaagaaattgtctgttgctgtcgctgcttcgtttatgagtttatcaatcagcctgccaggtgttcaggct

[0105] SEQ ID NO.26

[0106] Atgagaatacagaaaagacgaacacacgtcgaaaacattctccgtattcttttgcccccaattatgatacttagcctaatcctcccaacaccacccattcatgca

[0107] SEQ ID NO.27

[0108] atgttgttgaaaaggagaatagggttgctattaagtatggttggcgtattcatgcttttggctggatgctcg

[0109] SEQ ID NO.28

[0110] Atgcgcaacttgaccaagacatctctattactggccggcttatgcacagcggcccaaatggtttttgtaacacatgcctcagct

[0111] SEQ ID NO.29

[0112] Atgaagaaacgcagaaagatatgttattgcaatactgccctgctgcttatgattttgcttgctggatgtacg

[0113] SEQ ID NO.30

[0114]

[0115] SEQ ID NO.31

[0116] catcatcatcatcatcat

[0117] SEQ ID NO.32

[0118] Gtaagagaggatgtacac

[0119] SEQ ID NO.33

[0120] GATTCAAATGGCAATCAAGAAATTAATG

Claims

1. A method for increasing the secretory expression of protein glutaminase using NprB signal peptide, characterized in that: The method comprises: using a signal peptide containing the amino acid sequence shown in SEQ ID NO.7 to guide the secretory expression of protein glutaminase.

2. The method according to claim 1, characterized in that The amino acid sequence of the protein glutaminase is shown in GenBank accession number: AB046594.

1.

3. The method according to claim 1, characterized in that The secretory expression is achieved by constructing a recombinant bacterium.

4. The method according to claim 3, characterized in that The recombinant bacteria uses bacteria as hosts.

5. A recombinant bacterium, characterized in that: The recombinant bacteria guides the secretory expression of protein glutaminase with a signal peptide containing the amino acid sequence shown in SEQ ID NO.

7.

6. The recombinant bacterium according to claim 5, characterized in that The construction method of the recombinant Bacillus subtilis is as follows: the signal peptide shown in SEQ ID NO.7 is fused to the N-terminus of the gene encoding protein glutaminase to obtain a recombinant gene, and the recombinant plasmid formed by connecting the recombinant gene with the expression plasmid pP43NMK is transformed into Escherichia coli JM109, and the extracted plasmid is transformed into Bacillus subtilis WB600.

7. The recombinant bacterium according to claim 5, characterized in that The recombinant bacteria uses Bacillus subtilis WB600 as a starting strain, and expresses a recombinant gene formed by the N-terminal fusion nucleotide sequence of the protein glutaminase gene encoded by the gene shown in SEQ ID NO.9 and the signal peptide shown in SEQ ID NO.

28.

8. A method for producing protein glutaminase, characterized in that: The invention relates to a method for producing protein glutaminase by fermentation using the recombinant bacteria described in any one of claims 5 to 7.

9. Use of the recombinant bacteria according to any one of claims 5 to 7 in the food and biological fields.

10. Application of the signal peptide shown in SEQ ID NO.7 in improving the secretory expression effect of protein glutaminase or in the production of protein glutaminase.

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