Bacillus licheniformis of high-yield protein glutaminase as well as construction method and application of bacillus licheniformis

Abstract

The invention provides bacillus licheniformis for high-yield protein glutaminase as well as a construction method and application of the bacillus licheniformis, a target strain is obtained by knocking out an alkaline protease gene aprE and extracellular protease genes bpr, epr, vpr or wpr and heterologously expressing the protein glutaminase gene, an EPE strain is optimal in expression, PG enzyme activity reaches 0.358 U / mL and is improved by 3.55 times compared with that of a wild type, and the bacillus licheniformis has the advantages that the bacillus licheniformis is high in yield and high in protein glutaminase yield. In addition, the growth condition of each mutant strain is not obviously abnormal, and the growth speed is obviously increased. It is fully proved that the expression level of the heterologous protein PG can be remarkably improved by targeted knockout of the extracellular protease gene of the bacillus licheniformis, and a theoretical basis is laid for construction of an efficient protein expression host. The invention not only provides a new scheme for high-efficiency expression of PG, but also provides important reference for expression optimization of heterologous proteins of other gram-positive bacteria, and shows a wide industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a Bacillus licheniformis strain that produces high levels of protein glutaminase, its construction method, and its applications. Background Technology

[0002] Protein-glutaminase (EC 3.5.1.44, abbreviated as PG) is a deamidase that has received widespread attention in recent years, belonging to the class of amide hydrolases. PG can hydrolyze glutamine on the side chains of large protein molecules and some small peptides to produce L-glutamate and ammonia, thereby improving various functional properties of proteins and reducing the allergenicity of plant proteases. In 2020, PG was officially included in my country's "Standard for Use of Food Additives GB2760," becoming one of the most watched plant protein modification enzyme preparations. Therefore, PG is widely used in the food, cosmetics, pharmaceutical, and feed industries. PG is expressed in the form of inactive pro-PG, which contains a 114-amino acid propeptide and a 185-amino acid mature protein. The side chain of the propeptide is located at the entrance of the catalytic pocket, partially hindering substrate binding and forming an inactive pro-PG complex. Therefore, pro-PG containing the propeptide has no enzymatic activity and must be cleaved by an exogenous protease to be converted into active mPG. Currently, although some wild strains that produce PG have been screened, their PG enzyme activity is not high, and further efforts are needed to improve yield and enzyme activity through genetic engineering or fermentation optimization.

[0003] Heterologous expression by microorganisms is an effective strategy for improving the expression level of target enzyme proteins. Current research on prostaglandins (PGs) mainly focuses on applications of plant protein modification, with relatively little research on molecular engineering or efficient expression, failing to achieve significant increases in PG enzyme activity and expression levels. Currently, *Escherichia coli*, *Corynebacterium glutamicum*, *Pichia pastoris*, and *Bacillus subtilis* are primarily used as hosts for PG production. However, the current production level of PG enzyme activity remains low, limiting the production and widespread application of PGs.

[0004] Bacillus licheniformis is a generally regarded as safe (GRAS) Gram-positive bacterium. Its highly efficient protein secretion mechanism, short-cycle, high-density fermentation characteristics, and mature molecular genetic manipulation system provide a solid foundation for industrial fermentation production. However, the highly efficient extracellular protease secretion system of wild-type Bacillus licheniformis secretes high levels of extracellular protease activity, which degrades heterologously expressed target proteins, severely affecting the yield and activity of the target product.

[0005] Therefore, there is an urgent need to construct a new genetically engineered strain that can significantly increase PG enzyme activity and expression levels. Summary of the Invention

[0006] To address the aforementioned issues, this invention first employs homologous recombination technology to progressively knock out multiple extracellular protease genes (aprE, bpr, epr, vpr, and wpr) in the original *Bacillus licheniformis* strain, ultimately obtaining four mutant strains: *B. licheniformis* ΔaprE-Δbpr (BLAB), *B. licheniformis* ΔaprE-Δepr (BLAE), *B. licheniformis* ΔaprE-Δvpr (BLAV), and *B. licheniformis* ΔaprE-Δwpr (BLAW). Then, the PG expression plasmid pLY-3-PG was electroporated into each of these mutant strains. By plotting the growth curves of the four mutant strains and measuring PG enzyme activity, the *Bacillus licheniformis* mutant strain most suitable for PG enzyme expression was screened.

[0007] The specific technical solution of the present invention is as follows: A method for constructing a Bacillus licheniformis strain that produces high levels of protein glutaminase is provided. The method includes knocking out the alkaline protease gene aprE and the extracellular protease genes bpr, epr, vpr, or wpr, and heterologously expressing the protein glutaminase gene.

[0008] Furthermore, the aprE gene has the Gene ID CP083238.1 in NCBI, with Range 1: 3162388 to 3165527; the bpr gene has the Gene ID CP083238.1 in NCBI, with Range 1: 3933150 to 3939149; the vpr gene has the Gene ID CP083238.1 in NCBI, with Range 1: 1668509 to 1670929; the epr gene has the Gene ID CP083238.1 in NCBI, with Range 1: 3177842 to 3179617; and the wpr gene has the Gene ID CP083238.1 in NCBI, with Range 1: 610794 to 613936.

[0009] Furthermore, the method specifically includes: 1) Using Bacillus licheniformis as the initial strain, the alkaline protease gene aprE was knocked out to obtain a strain with the aprE gene knocked out, which was named B. licheniformis ΔaprE (BLA). 2) Using the Bacillus licheniformis ΔaprE (BLA) obtained in step 1) as the starting strain, the extracellular protease genes bpr, epr, vpr and wpr were knocked out respectively to obtain mutant strains B. licheniformis ΔaprE-Δbpr (BLAB), B. licheniformis ΔaprE-Δepr (BLAE), B. licheniformis ΔaprE-Δvpr (BLAV) and B. licheniformis ΔaprE-Δwpr (BLAW); 3) Using the Bacillus licheniformis ΔaprE-Δbpr (BLAB), B. licheniformis ΔaprE-Δepr (BLAE), B. licheniformis ΔaprE-Δvpr (BLAV), and B. licheniformis ΔaprE-Δwpr (BLAW) obtained in step 2) as starting strains, the protein glutaminase gene was introduced to obtain Bacillus licheniformis EPB, EPE, EPV, and EPW strains. The target strains were then screened by growth curve determination and PG enzyme activity detection.

[0010] A Bacillus licheniformis strain that produces a high level of protein glutaminase is provided, which is obtained by the above-described construction method.

[0011] Furthermore, the Bacillus licheniformis mutant strain is less prone to lysis and has an increased growth rate; the increased growth rate means that the time required for the Bacillus licheniformis mutant strain to enter the stable growth phase is reduced by half compared to the initial strain.

[0012] Furthermore, the Bacillus licheniformis mutant strain requires 9 hours to reach the stable growth phase, while the initial strain requires 18 hours.

[0013] A microbial preparation containing the aforementioned Bacillus licheniformis is provided.

[0014] This invention provides the application of Bacillus licheniformis or its microbial preparation in the production of protein glutaminase.

[0015] A method for producing protein glutaminase is provided, wherein the above-mentioned Bacillus licheniformis or its microbial preparation is added to a fermentation system.

[0016] Furthermore, the fermentation system is a 5L fermenter, the fermentation time is 50-60 hours, and the fermentation medium contains 24g / L yeast powder, 12g / L tryptone, 8.2g / L K2HPO4, 1.2g / L KH2PO4, 0.15g / L CaCl2, and 2.5g / L MgSO4·7H2O.

[0017] The present invention has the following advantages and effects compared with the prior art: This invention constructs four mutant strains of Bacillus licheniformis suitable for high-efficiency expression of the PG enzyme by knocking out some extracellular protease genes: B. licheniformis ΔaprE-Δbpr (BLAB), B. licheniformis ΔaprE-Δepr (BLAE), B. licheniformis ΔaprE-Δvpr (BLAV), and B. licheniformis ΔaprE-Δwpr (BLAW). These strains were then introduced into PG expression plasmids, and PG enzyme activity was measured. The most suitable B. licheniformis mutant strain, EPE, was selected for PG enzyme activity expression, achieving a PG enzyme activity of 0.358 U / mL, 3.55 times higher than the original strain. Furthermore, no significant abnormalities were observed in the growth of any of the mutant strains, and the growth rate was significantly improved. This fully demonstrates that targeted knockout of extracellular protease genes in Bacillus licheniformis can significantly enhance the expression level of the heterologous protein PG, laying a theoretical foundation for constructing a high-efficiency protein expression host. This invention not only provides a new approach for the efficient expression of PG, but also offers an important reference for optimizing the expression of heterologous proteins in other Gram-positive bacteria, demonstrating broad prospects for industrial applications. Attached Figure Description

[0018] Figure 1 In Figure A, the ammonia standard curve is shown; in Figure B, the activity comparison of two enzymes in B. licheniformis is shown. Figure 2 This is the construction of the aprE knockout vector, where M is the nucleic acid marker, lanes 1-2 show the upstream and downstream amplification results, and lanes 3-4 show the vector construction verification results, 1100 bp; Figure 3 This is the aprE gene knockout and validation, where M is the nucleic acid marker, lanes 1-6 are the single crossover validation results, 1100 bp, lane 1 is the negative control, no band; lanes 7-11 are the double crossover validation results, 1100 bp; Figure 4This is a validation of protease gene knockout. M is the nucleic acid marker. Lanes 1-3 in A are the validation results of BLAB double crossover; lanes 1-4 in B are the validation results of BLAE double crossover; lanes 1-4 in C are the validation results of BLAV double crossover; and lanes 1-3 in D are the validation results of BLAW double crossover. Figure 5 This section describes the validation of the expression host construction. M represents the nucleic acid marker. Lanes 1-3 in A show the BLAE electroporation validation results; lanes 1-3 in B show the BLAV electroporation validation results; lanes 1-3 in C show the BLAB electroporation validation results; and lanes 1-3 in D show the BLAW electroporation validation results. Figure 6 This is a growth curve diagram of four strains, EPB, EPE, EPV and EPW, in LB liquid medium; Figure 7 These are PG enzyme activity curves for four strains: EPB, EPE, EPV, and EPW. Figure 8 This is a growth curve diagram of four strains, EPB, EPE, EPV and EPW, in fermentation medium; Figure 9 This is a graph showing the enzyme activity of the EPE strain in a 5L fermenter. Detailed Implementation

[0019] The embodiments of the present invention will be described in detail below with reference to examples. However, the following examples are only for illustrating the present invention and should not be regarded as limiting the scope of the present invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products.

[0020] The culture media involved in the following examples are shown below: LB liquid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0021] LB solid medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar powder 2 g / L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0022] LBS liquid culture medium: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 5 g / L, D-sorbitol 0.5 mol / L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0023] Fermentation medium: yeast extract 24 g / L, tryptone 12 g / L, K2HPO4 8.2 g / L, KH2PO4 1.2 g / L, CaCl2 0.15 g / L, MgSO4·7H2O 2.5 g / L, adjust pH to 7.2-7.4, autoclave at 121℃ for 20 min.

[0024] The bacterial strains and plasmids involved in the following examples are shown below: The bacterial strains E. coli EC135 and E. coli EC135 pM.Bam were obtained from the Institute of Microbiology, Chinese Academy of Sciences. The starting strain B. licheniformis was obtained from our laboratory. The thermosensitive shuttle vector pTU and the expression vector ply-3-PG were obtained from our laboratory.

[0025] The growth curve determination methods involved in the following embodiments are as follows: The strain was streaked in three zones on an LB plate and incubated statically at 37°C for about 12 h. Newly activated single colonies were picked and placed in a shake flask containing 50 mL of LB medium and incubated at 37°C and 220 r / min for 12 h with shaking. The inoculum was then transferred at a rate of 2% to 12 test tubes containing 5 mL of LB medium and incubated at 37°C and 220 r / min with shaking for 24 h. Every 2 h, one tube was taken out, appropriately diluted, and the OD600 was measured to plot the growth curve.

[0026] The enzyme activity detection methods involved in the following examples are as follows: Weigh 0.314 g of NH4Cl (previously dried in an oven for 1 hour), dissolve and dilute to 1 L with ddH2O to prepare ammonia standard stock solution for later use. Dilute according to Table 1 to obtain ammonia standard solutions of different concentration gradients.

[0027] Table 1 NH4Cl Concentration Gradient <![CDATA[NH4Cl]]> 0 80 160 240 320 400 <![CDATA[ddH2O]]> 2000 1920 1840 1760 1680 1600 Concentration (μg / ml) 0 4 8 12 16 20 Add 12 μL of ammonia standard solutions of different concentrations to each well of a 96-well plate, then add 48 μL of ddH2O, 60 μL of chromogenic reagent A, 30 μL of chromogenic reagent B, and 60 μL of chromogenic reagent C sequentially. Mix thoroughly and incubate at 37°C for 20 min. Measure the absorbance at 630 nm using a microplate reader. Plot the ammonia standard curve.

[0028] PG enzyme activity assay: (1) Take 1 mL of fermentation broth, centrifuge and take the supernatant to obtain the enzyme solution.

[0029] (2) Experimental group: Take 10 μL of enzyme solution, add 100 μL of substrate solution to it, incubate the mixture at 37℃ for 30 min, aspirate 12 μL of the mixture into a 96-well plate, then add 48 μL of ddH2O, 60 μL of chromogenic agent A, 30 μL of chromogenic agent B and 60 μL of chromogenic agent C in sequence, mix them evenly, and incubate in a constant temperature incubator at 37℃ for 20 min. Measure the absorbance at 630 nm using an enzyme-linked immunosorbent assay reader.

[0030] (3) Control group: Take 10 μL of enzyme solution, add 100 μL of ddH2O to it, and incubate the mixture in a 37℃ water bath for 30 min until well mixed. Pipette 12 μL of the mixture into a 96-well plate, and then add 48 μL of ddH2O, 60 μL of chromogenic agent A, 30 μL of chromogenic agent B and 60 μL of chromogenic agent C in sequence, and mix them well. Incubate the plate in a 37℃ constant temperature incubator for 20 min, and measure the absorbance at 630 nm using an ELISA reader.

[0031] One unit of enzyme activity is defined as the amount of enzyme required to catalyze the production of 1 μmol of ammonia from the substrate per minute. Therefore, the protein glutaminase activity (U / mL) = (AB) x (2.1 / 0.1) / 17.03 / 30 / a; where A: absorbance of the experimental group at a wavelength of 630 nm; B: absorbance of the control group at a wavelength of 630 nm; a: slope of the ammonia standard curve.

[0032] Color developer A: Weigh 40.06 g of phenol and 0.15 g of sodium nitroprusside, dissolve in pure water, bring to a final volume of 1 L, mix well before use, and store at 4°C in the dark.

[0033] Color developer B: Weigh 49.94g KOH, dissolve in pure water, bring the volume to 1L, mix well and use.

[0034] Color developer C: 200.4 g anhydrous K2CO3, 8.33 mL NaClO (food grade), dissolved in pure water, and brought to a final volume of 1 L (prepare fresh before use), mix well before use.

[0035] The main primers used in the following examples are shown in Table 2: Table 2. Main primers The nucleic acid fragment amplification and ligation involved in the following embodiments are shown below: DNA amplification was performed using a three-step method, and the amplification system is shown in Table 3.

[0036] (1) Gene amplification system (50 μL) Table 3 Gene amplification reaction system System reagents Added amount <![CDATA[ddH2O]]> 35 μL 5 ng / μL amplification template 1 μL 10 μmol / LF end primer 2 μL 10 μmol / L-terminal primer 2 μL PrimeSTAR Max DNAPolymerase 0.2 μL Buffer 5 μL dNTP 5 μL

[0037] (2) Gene amplification reaction conditions

[0038] After PCR amplification, the correct PCR products were detected by agarose gel electrophoresis and then recovered by gel extraction.

[0039] The plasmid vector was subjected to double enzyme digestion, and the enzyme digestion system is shown in Table 4.

[0040] Table 4 Enzyme digestion reaction system (50 µL) reagents volume ddH2O 30 μL PCR amplification products / vector plasmids 10 μL 10×Buffer 5 μL Restriction enzyme I 2.5 μL Restriction enzyme II 2.5 μL

[0041] After purification and recovery, the linearized vector digested by enzymes can be ligated with the amplified gene fragment via recombinase. The ligation reaction system (20 μL) is shown in Table 5.

[0042] Table 5 Recombinant Linkage Reaction System reagents volume Linearized carrier 4 μL Gene fragment 1 2 μL Gene fragment 2 2 μL 5 × CE MultiS Buffer 4 μL Exnase II 2 μL ddH2O 6 μL

[0043] According to the reaction system in Table 5, add each component into the EP tube, mix gently, react at 37°C for 30 min, and store at 4°C for later use.

[0044] The colony PCR amplification system and reaction conditions involved in the following examples are shown below: Take an appropriate amount of bacterial cells into a small centrifuge tube containing 20 μL of ddH2O, and incubate in a boiling water bath for 15 min. Then, centrifuge at 12000 r / min for 1 min, and take the supernatant for colony PCR.

[0045] (1) Colony PCR amplification system (20 μL) System reagents Added amount ddH2O 7 μL Amplification template 1 μL 10 μmol / LF end primer 1 μL 10 μmol / L-terminal primer 1 μL 2×Es Taq MasterMix (CWBIO) 10 μL

[0046] (2) Colony PCR amplification reaction conditions

[0047] Example 1: Expression of PG in Bacillus licheniformis starting strain In this invention, the PG expression plasmid pLY-3-PG was electroporated into the starting strain *B. licheniformis*. Successfully electroporated single clones were picked and cultured in 5 mL of 50 μg / mL Kan LB medium at 37°C with shaking for approximately 12 h. 1 mL of the bacterial culture was then transferred to 100 mL (500 mL baffle flask) of LB medium under the same conditions and cultured at 37°C. The culture was then diluted according to Table 1, and an ammonia standard curve was obtained. Figure 1-A). Subsequently, its PG enzyme activity was measured and compared with the AprE enzyme activity of the starting strain B. licheniformis (previously measured in our laboratory). The results are shown in Figure 1-B; the PG enzyme activity was lower, while the AprE enzyme activity was significantly higher than the PG enzyme activity.

[0048] Although the expression of PG enzyme activity requires the cleavage of the propeptide by extracellular proteases secreted by Bacillus licheniformis to convert it into active mPG, high levels of AprE activity have more negative effects on PG expression. For example, high AprE expression may consume a large amount of transcription-translation resources (such as ribosomes and ATP), thus limiting the efficiency of PG synthesis; AprE, as an extracellular protease, may directly degrade PG. These results indicate that PG is successfully secreted and expressed in Bacillus licheniformis, but its enzyme activity is low, which is presumably related to the high expression of AprE enzyme activity. Therefore, a plan is to knock out the aprE gene to optimize and enhance the expression of PG enzyme activity.

[0049] Example 2: Knockout of the alkaline protease aprE gene in the starting strain of Bacillus licheniformis After amplifying the upstream and downstream homologous sequences of the target gene using the primer pairs aprE-UF / aprE-UR and aprE-DF / aprE-DR shown in Table 2, the purified and recovered upstream and downstream fragments were both approximately 500 bp in size. Figure 2 The pTU plasmid fragment, recovered from BamHI / SacII digestion, was recombined and ligated to form the knockout vector pTU-ΔaprE. PCR verification was performed using primers pTU-PF and pTU-PR, and the correct band size was approximately 1100 bp. Figure 2 After sequencing confirmation, the cells were transformed into E. coli EC135 pM.Bam for methylation modification, then electroporated into host competent cells, and cultured on 25 μg / mL Kan resistance plates at 37°C for about 12 h.

[0050] Transformants from the plate were transferred to 5 mL of LB medium with the same antibiotic concentration and incubated at 45°C for 10 h. Then, 10 μL of the bacterial culture was transferred to LB medium under the same conditions and incubated at 45°C for another 10 h. This process was repeated for 2-3 generations. The culture was then diluted and spread onto Kans plates and incubated statically at 45°C. The single colonies on the plates were the theoretically single-exchange strains. However, to avoid non-specific integration, colony PCR was performed using the primer pairs pTU-VF / aprE-GR or pTU-VR / aprE-GF shown in Table 2 to verify single exchange. The results are as follows: Figure 3 The DNA band size of the successfully single-exchange recombinant bacteria was approximately 1100 bp or 2100 bp, while the negative control using the competent genome as a template showed no band, indicating that a specific single exchange occurred.

[0051] Select correctly exchanged colonies and incubate them in 5 mL of antibiotic-free LB medium at 37°C with shaking for approximately 12 h. Transfer 10 μL of the culture to LB medium under the same conditions and continue incubating at 37°C for approximately 12 h. Repeat this process for 2-3 generations. Dilute the culture and spread it onto antibiotic-free LB plates, then incubate at 37°C for approximately 12 h. Use the primers aprE-MF / aprE-MR shown in Table 2 to perform colony PCR verification on the plates. Figure 3 As shown, due to the deletion of the aprE gene, the amplified fragment size of the mutant strain is approximately 1100 bp, indicating that a double crossover has been successfully achieved. Further sequencing confirmed that the strain with the aprE gene deleted was named B. licheniformis ΔaprE (BLA) and used as the starting strain for subsequent molecular modification.

[0052] Example 3: Host bacteria for high-efficiency PG expression Using the *Bacillus licheniformis* BL ΔaprE strain with the aprE gene knocked out as the starting strain, and following the steps shown in Example 2 of this invention, the protease genes bpr, epr, vpr, and wpr were knocked out respectively on the basis of BL ΔaprE using the corresponding primers shown in Table 2, thereby obtaining mutant strains *B. licheniformis* ΔaprE-Δbpr (BLAB), *B. licheniformis* ΔaprE-Δepr (BLAE), *B. licheniformis* ΔaprE-Δvpr (BLAV), and *B. licheniformis* ΔaprE-Δwpr (BLAW). The results are as follows: Figure 4 As shown.

[0053] Example 4: Construction and screening of recombinant bacteria for high-efficiency expression of PG enzyme To further investigate which extracellular protease knockouts are more conducive to PG expression, the expression of PG under different protease knockout bacteria was studied. The constructed pLY-3-PG plasmid was electroporated into BLAB, BLAE, BLAV, and BLAW strains, obtaining four strains: EPB, EPE, EPV, and EPW, respectively. Single clones were selected for colony PCR verification, and the results are as follows: Figure 5 As shown, a band of approximately 1300 bp was successfully amplified on the plasmid, indicating that the electroporation was successful.

[0054] Following the growth curve determination method described above, growth curves for four strains, EPB, EPE, EPV, and EPW, were obtained, and the results are as follows: Figure 6As shown in the figure, the four mutant strains exhibited similar growth patterns, with their growth curves almost completely overlapping. They reached the stationary phase in just 9 hours, while the initial strain required 18 hours. The four mutant strains achieved this in half the time compared to the initial strain, demonstrating a significant increase in growth rate. This suggests that appropriate protease knockout in the host strain is beneficial to its growth, while the loading of expression plasmids has no significant impact on the strain's growth.

[0055] To screen for the most suitable strains for PG expression, the PG enzyme activities of four strains (EPB, EPE, EPV, and EPW) were obtained according to the enzyme activity assay method described above. The results are as follows: Figure 7 As shown, the enzyme activities of all four strains began to increase slowly from 36 hours, and started to decrease around 53 hours. The highest enzyme activities of the four strains were EPB: 0.255 U / mL, EPE: 0.358 U / mL, EPV: 0.298 U / mL, and EPW: 0.255 U / mL, respectively. Compared with the initial strain (0.1007 U / mL) shown in Example 1, the enzyme activities of EPB, EPE, EPV, and EPW of the strains were significantly improved. Among them, strain EPE had the highest PG activity, reaching 0.358 U / mL, which was 355% higher than that of the initial strain.

[0056] Example 5: Fermentation production of recombinant bacteria with high-efficiency PG enzyme expression After replacing the LB liquid medium with fermentation medium, the PG enzyme activity of the four strains was measured again using the method described above. The results are as follows: Figure 8 As shown, the enzyme activities of all four strains began to increase slowly from 40 hours, and started to decrease around 52 hours. The highest enzyme activities of the four strains were EPB: 0.338 U / mL, EPE: 0.537 U / mL, EPV: 0.395 U / mL, and EPW: 0.350 U / mL, respectively. Compared with the initial strain (0.1007 U / mL), the enzyme activities of all four strains were significantly improved. Among them, strain EPE had the highest PG activity, reaching 0.537 U / mL, which was 533% higher than that of the initial strain.

[0057] 5 L fermenter culture: The seed culture of the PG enzyme-expressing recombinant strain EPE was transferred to a 5 L fermenter containing 2.5 L of fermentation medium by flame inoculation. Fermentation parameters were adjusted for culture. During the fermentation process, defoaming was performed online using a 4-fold diluted defoamer. An appropriate amount of fermentation sample was taken for testing.

[0058] The initial culture conditions in the fermenter were: temperature 37℃, stirring speed 500 r / min, aeration rate 4 L / min, seed age 12h, inoculum size 10%, and fermentation cycle 52h. Simultaneously, glucose (100 g / 300 mL) was added at a constant flow rate of 20 mL·h⁻¹ during fermentation from 4 to 16 h.

[0059] The results are as follows Figure 9 As shown, the EPE strain reached its peak enzyme activity at 48h, with a maximum enzyme activity of 1.56 U / mL, which is 2.9 times the maximum enzyme activity under normal fermentation conditions.

[0060] This study constructed a series of Bacillus licheniformis gene-deleted host bacteria by superimposed knockout of extracellular proteases in the host bacteria. These bacteria significantly promoted PG activity expression, providing a theoretical basis for the promotion and application of Bacillus licheniformis host chassis cells in PG production. This study has important research significance and potential application value.

[0061] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing a Bacillus licheniformis strain that produces high levels of protein glutaminase, characterized in that, The method includes knocking out the alkaline protease gene aprE and the extracellular protease genes bpr, epr, vpr or wpr, and heterologously expressing the protein glutaminase gene.

2. The construction method according to claim 1, characterized in that, The method specifically includes: 1) Using Bacillus licheniformis as the initial strain, the alkaline protease gene aprE was knocked out to obtain a strain with the aprE gene knocked out, which was named B. licheniformis ΔaprE (BLA). 2) Using the Bacillus licheniformis ΔaprE (BLA) obtained in step 1) as the starting strain, the extracellular protease genes bpr, epr, vpr and wpr were knocked out respectively to obtain mutant strains B. licheniformis ΔaprE-Δbpr (BLAB), B. licheniformis ΔaprE-Δepr (BLAE), B. licheniformis ΔaprE-Δvpr (BLAV) and B. licheniformis ΔaprE-Δwpr (BLAW); 3) Using the Bacillus licheniformis ΔaprE-Δbpr (BLAB), B. licheniformis ΔaprE-Δepr (BLAE), B. licheniformis ΔaprE-Δvpr (BLAV), and B. licheniformis ΔaprE-Δwpr (BLAW) obtained in step 2) as starting strains, the protein glutaminase gene was introduced to obtain Bacillus licheniformis EPB, EPE, EPV, and EPW strains. The target strains were then screened by growth curve determination and PG enzyme activity detection.

3. A Bacillus licheniformis species that produces a high level of protein glutaminase, characterized in that, The Bacillus licheniformis was obtained by the construction method described in claim 1 or 2.

4. The Bacillus licheniformis according to claim 3, characterized in that, The growth rate of Bacillus licheniformis is increased, and the increased growth rate reduces the time required for Bacillus licheniformis to enter the stable growth phase by half compared to the initial strain.

5. The Bacillus licheniformis according to claim 4, characterized in that, The Bacillus licheniformis requires 9 hours to reach a stable growth phase, while the initial strain requires 18 hours.

6. A microbial preparation comprising Bacillus licheniformis as described in claim 3, 4, or 5.

7. The use of the construction method according to any one of claims 1-2, or the Bacillus licheniformis according to claim 3, 4, or 5, or the microbial preparation according to claim 6, in the production of protein glutaminase.

8. A method for producing protein glutaminase, characterized in that: Add the Bacillus licheniformis of claim 3, 4, or 5 or the microbial preparation of claim 6 to the fermentation system.

9. The method according to claim 8, characterized in that: The fermentation system was a 5L fermenter, with a fermentation time of 50-60 hours. The fermentation medium contained 24g / L yeast extract, 12g / L tryptone, 8.2g / L K2HPO4, 1.2g / L KH2PO4, 0.15g / L CaCl2, and 2.5g / L MgSO4·7H2O.

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