A method for improving the expression level of exogenous proteins in bacillus

Abstract

This invention provides a method for increasing the expression level of exogenous proteins in Bacillus subtilis, relating to the field of bioengineering technology. The method involves using the glutaminase gene (a protein derived from *Chlorella vulgaris*)... pg Expression was performed in *Bacillus amyloliquefaciens*; overlap PCR was used to analyze the expression of the expression. pg The propeptide gene was mutated, and the mutants were screened to obtain protein glutaminase mutants with increased expression activity.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for increasing the expression level of exogenous proteins in Bacillus. Background Technology

[0002] Bacillus is a type of Gram-positive bacteria. Many strains, such as Bacillus subtilis, Bacillus amyloliquefaciens, and Bacillus licheniformis, have become ideal hosts for the expression of exogenous proteins due to their unique biological advantages. They have wide and important applications in many fields, such as industrial enzyme production, pharmaceutical and biological product research and development, and agricultural biocontrol.

[0003] Enzymes are highly efficient and specific biological catalysts, and some enzyme proteins are expressed in an inactive zymogen form. The zymogen can inhibit enzyme activity, preventing damage to the cell itself. For example, the alkaline protease in *B. subtilis* exists as a zymogen within the cell, where it is inactive. Only after being secreted extracellularly can it be activated into an active enzyme through cleavage of its propeptide by itself or other proteases, ensuring that the cell is not destroyed by its own products. The maturation process of an enzyme is crucial for its normal function. In the process of zymogenization into mature enzyme, the molecular chaperone—the propeptide—plays an important role. The propeptide can participate in guiding enzyme assembly through specific structural domains, such as a hydrophobic core.

[0004] Protein glutaminase (EC 3.5.1.44, PG) is a novel deamidase used for modifying food proteins. It catalyzes the deamidation of glutamine residues in substrate proteins or peptides to glutamate, releasing ammonia in the process. It has already met national food safety standards. However, the main problem with current protein glutaminase is insufficient expression levels, making its application in industrial production difficult.

[0005] Therefore, this invention is proposed. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for enhancing the expression level of exogenous proteins in Bacillus. The glutaminase gene (pg) derived from *Chlorella utilis* is expressed in *Bacillus amyloliquefaciens*. The pg precursor gene is mutated using overlap PCR, and the mutants are screened to obtain glutaminase mutants with enhanced expression activity.

[0007] In order to achieve the objective of this invention, the following technical solution is adopted:

[0008] This invention provides a mutant of the protein glutaminase propeptide, obtained by double mutation of E35I and E58A in the wild-type protein glutaminase propeptide; wherein the amino acid sequence of the wild-type protein glutaminase propeptide is shown in SEQ ID NO.1; and the amino acid sequence of the E35I mutant is shown in SEQ ID NO.2.

[0009] The amino acid sequence of the E58A mutant is shown in SEQ ID NO.3;

[0010] The amino acid sequence of the E35I / E58A mutant is shown in SEQ ID NO.4.

[0011] SEQ ID NO.1

[0012] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0013] SEQ ID NO.2

[0014] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDIENGMIKVSFMLTAQFYEIKPTKENEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0015] SEQ ID NO.3

[0016] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKANEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0017] SEQ ID NO.4

[0018] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDIENGMIKVSFMLTAQFYEIKPTKANEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0019] The present invention also provides a polynucleotide encoding the above-mentioned mutants, wherein the polynucleotide sequence encoding the E35I / E58A mutant is shown in SEQ ID NO.6; the polynucleotide sequence encoding the E35I mutant is shown in SEQ ID NO.7; and the polynucleotide sequence encoding the E58A mutant is shown in SEQ ID NO.8.

[0020] The nucleotide sequence encoding the wild type is shown in SEQ ID NO.5.

[0021] SEQ ID NO.5

[0022] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAAAATGAATCACCG GCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0023] SEQ ID NO.6

[0024] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCACAACAGTTCCGGTTGGCATTGATATTGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGCAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAATTGAATCACCG GCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0025] SEQ ID NO.7

[0026] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATATTGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAATTGAATCACCG GCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0027] SEQ ID NO.8

[0028] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGCAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAAAATGAATCACCGGCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTAGATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCCGGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0029] The present invention also provides a recombinant plasmid comprising the above polynucleotide.

[0030] Furthermore, the expression vector for the recombinant plasmid is pBSA43.

[0031] The present invention also provides a host cell comprising the above-mentioned polynucleotides or recombinant plasmids.

[0032] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell.

[0033] The present invention also provides a genetically engineered bacterium containing the above-mentioned polynucleotides or the above-mentioned recombinant plasmids.

[0034] The present invention also provides a method for improving the expression level of exogenous proteins in Bacillus, the method comprising: introducing the above-mentioned recombinant plasmid into Bacillus for expression.

[0035] Furthermore, the Bacillus is Bacillus amyloliquefaciens.

[0036] The present invention has the following technical effects:

[0037] This invention employs a method for hydrophobic modification of the propeptide, which enhances the expression activity of PG by hydrophobically designing the propeptide sequence. Based on this, the expression activity of the combined mutant propeptide E35I / E58A for PG is increased by 80% compared to the wild-type propeptide. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 Electrophoresis diagram of upstream fragment P1 and downstream fragment P2 of the PG target gene. Lane 1 is the E35I target gene, lane 2 is the upstream fragment P1, and lane 3 is the downstream fragment P2.

[0040] Figure 2 Electrophoresis images of pBSA43 plasmid and PG target gene after double enzyme digestion. Lane 1 shows the enzyme digestion results of expression plasmid pBSA43, and lane 2 shows the enzyme digestion results of E35I target gene.

[0041] Figure 3 : Electrophoresis diagram of double enzyme digestion of pBSA43-pg recombinant plasmid, where lane 1 is the electrophoresis diagram of double enzyme digestion of pBSA43-E35I recombinant plasmid;

[0042] Figure 4 : Expression activity of PG mutant propeptide. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0044] The following is a detailed explanation using specific embodiments:

[0045] The solutions and culture media used in the embodiments of this invention are as follows:

[0046] (1) LB medium: Weigh 10 g tryptone, 5 g yeast extract and 10 g NaCl, add deionized water to dissolve and bring the volume to 1 L; if preparing a solid medium, add an additional 15 g / L agar powder. After preparation, autoclave at 121 ℃ for 20 min.

[0047] (2) LBS medium: Weigh 10 g tryptone, 5 g yeast extract, 10 g NaCl and 91.0 g D-sorbitol, add deionized water to dissolve and bring the volume to 1 L. After preparation, autoclave at 121℃ for 20 min.

[0048] (3) Preparation of washing solution for preparing competent B. amyloliquefaciens: Weigh 91.0 g D-sorbitol and 91.0 g mannitol, add 600 mL deionized water to dissolve, then add 100 mL glycerol, and continue to add deionized water to make up to 1 L. After preparation, autoclave at 121 ℃ for 20 min.

[0049] (4) Preparation of electrotransfer competent cell preservation solution for B. amyloliquefaciens: Weigh 9.1 g D-sorbitol and 9.1 g mannitol, add 80 mL deionized water to dissolve, then add 1.0 mL glycerol and 1.4 g PEG-6000, and bring the volume to 100 mL. After preparation, autoclave at 121 °C for 20 min.

[0050] (5) Preparation of electroporation resuscitation solution for B. amyloliquefaciens: Add 0.5 M D-sorbitol and 0.38 M D-mannitol to LB medium, dissolve in deionized water and bring to a final volume of 1 L, and autoclave at 121 °C for 20 min.

[0051] (6) Preparation of phosphate-buffered saline (PBS): Weigh 0.176 mol of Na2HPO4·12H2O, dissolve it in pure water and bring the volume to 1L to obtain solution 1; weigh 0.176 mol of NaH2PO4·2H2O, dissolve it in water and bring the volume to 1L to obtain solution 2. Slowly add solution 1 to solution 2 and adjust the pH to 6.5.

[0052] (7) Preparation of CBZ-Gln-Gly solution as substrate for enzyme reaction: Weigh 0.337 g of CBZ-Gln-Gly, dissolve it in 0.176 M PBS buffer (pH 6.8) and bring the volume to 100 mL. Mix well before use.

[0053] (8) Preparation of trichloroacetic acid (TCA) solution: Weigh 65.36 g of TCA, add pure water to dissolve and make up to 1 L, mix well and it is ready to use.

[0054] (9) Preparation of colorimetric reagent A: Accurately weigh 40.06 g of phenol and 0.15 g of sodium nitroprusside, add pure water to dissolve and make up to 1 L, mix well and store at 4 ℃ in the dark.

[0055] (10) Preparation of colorimetric reagent B: Weigh 49.94 g KOH accurately, add pure water to dissolve and make up to 1 L, mix well and it is ready for use.

[0056] (11) Preparation of color reagent C: Take 200.4 g of anhydrous K2CO3 and 8.33 mL of food grade NaClO, add deionized water to dissolve and make up to 1 L. After mixing, it can be used.

[0057] Example 1: Obtaining the wild-type protein glutaminase gene

[0058] Using the extracted *Chlorella vulgaris* genome as a template, upstream and downstream primers F and R were designed to introduce two restriction enzyme sites, BamHI and SmaI. The amplification primers for the protein glutaminase gene pg of this invention are as follows:

[0059] F: 5'-CGCGGATCCATGGATTCAAATGGCAATCAAGAA -3'

[0060] R: 5'-TCCCCCGGGTTA GAAGCCGCATGAGCTAAC-3'

[0061] Using F and R as upstream and downstream primers, and the genome of *Aureobacterium utilis* as a template, amplification was performed. The amplification system is shown in Table 1.

[0062] Table 1: Amplification System

[0063] F 2 μL R 2 μL Genome template of *Cyperus pruritus* 2 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0064] The amplification program was as follows: 98℃ pre-denaturation for 30 min; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 5 s, for 30 cycles; 72℃ extension for 10 min. PCR products were subjected to 1.2% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit. The recovered products were double-digested with BamHI and SmaI and ligated into the vector pBSA43. The ligation product was first transformed into the cloning host *E. coli* JM109, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double-enzyme digestion verification. Successful transformations were then sequenced. The correctly verified plasmid was transformed into *B. amyloliquefaciens* TCCC111108, plated on LB agar, and the transformants were extracted for colony PCR verification. The obtained positive transformants were the pBSA43-pg recombinant strain.

[0065] Bioinformatics predictions were used to screen for two amino acids, Glu35 and Glu58, which were then mutated to E35I and E58A, respectively, as well as the combined mutation E35I / E58A. The mutants E35I, E58A, and E35I / E58A were obtained.

[0066] Example 2: Obtaining the mutant gene of protein glutaminase propeptide

[0067] The mutant was constructed using overlap PCR, and the primers required for the mutation are as follows:

[0068] f-E35I:GCCATTTTCAATATCAATGCCAAC

[0069] r-E35I:GTTGGCATTGATATTGAAATGGC

[0070] f-E58A: CCGACAAAAGAAATGAACAATATATT

[0071] r-E58A:AATATATTGTTCATTGGCTTTTGTCGG

[0072] The mutant E35I / E58A can be obtained by mutating the mutant E35I into the E58A mutant.

[0073] Taking the mutant E35I as an example, in the first step of the overlap PCR reaction system, F and r-E35I were used as upstream and downstream primers, respectively, and f-E35I and R were used as upstream and downstream primers, respectively. Using pBSA43-pg as a template, PCR1 reaction was performed to obtain the upstream fragment P1 and the downstream fragment P2, respectively.The upstream fragment P1 nucleotide sequence is: ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATATTGAAAATGGCATGATTAAAGTT; the downstream fragment P2 nucleotide sequence is: , and the experimental results are as follows. Figure 1 As shown.

[0074] The reaction systems for upstream fragment amplification are shown in Table 2, and the reaction systems for downstream fragment amplification are shown in Table 3.

[0075] Table 2: Reaction system for upstream fragment amplification

[0076] F 2 μL r-E35I 2 μL <![CDATA[pBSA43 -pg ]]> 2 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0077] Table 3: Reaction system for downstream fragment amplification

[0078] f-E35I 2 μL R 2 μL <![CDATA[pBSA43 -pg ]]> 2 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0079] The amplification program was as follows: pre-denaturation at 98℃ for 30 min; denaturation at 98℃ for 10 s, annealing at 54℃ for 20 s, extension at 72℃ for 5 s, for 30 cycles; and extension at 72℃ for 10 min.

[0080] After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system is shown in Table 4.

[0081] Table 4: Reaction systems of upstream and downstream segments

[0082] Upstream segment 2.0 μL Downstream segments 2.0 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 21 μL

[0083] The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 8 s, for 5 cycles; 72℃ extension for 10 min.

[0084] After PCR 2, 2 μL each of primers F and R were added to the system for PCR 3. The amplification program was as follows: 98℃ pre-denaturation for 30 s; 98℃ denaturation for 10 s, 54℃ annealing for 20 s, 72℃ extension for 10 s, for 30 cycles; 72℃ extension for 10 min. The PCR products were subjected to 1.2% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit.

[0085] The wild-type pg precursor gene was ligated to the pBSA43 vector using BamHI and SmaI. The ligation product was first transformed into the cloning host E. coli JM109, plated on LB solid medium, and plasmids were extracted from transformants for PCR and double enzyme digestion verification. Electrophoresis results are shown below. Figure 2 As shown, successful sequencing verification was performed. The plasmid that was verified to be correct was electroporated into B. amyloliquefaciensTCCC111108, plated on LB solid plates, and transformed was extracted for colony PCR verification. The obtained positive transformant was pBSA43-pg propeptide wild-type recombinant strain.

[0086] The pg precursor mutant gene was ligated to the pBSA43 vector using BamHI and SmaI. The ligation product was first transformed into the cloning host E. coli JM109, plated on LB solid medium, and plasmids were extracted from transformants for PCR and double enzyme digestion verification. Electrophoresis results are shown below. Figure 3 As shown, successful verification was performed by sequencing. The plasmid that was verified to be correct was electroporated into B. amyloliquefaciensTCCC111108, plated on LB solid plates, and transformed were extracted for colony PCR verification. The positive transformants obtained were pBSA43-E35I, pBSA43-E58A, and pBSA43-E35I / E58A precursor mutant recombinant strains.

[0087] Example 3: Expression and Enzyme Activity Assay of Protein Glutaminase

[0088] From overnight cultured antibiotic-resistant plates, select morphologically uniform single colonies free from contamination. Inoculate each colony into 3 mL of LB medium (with appropriate antibiotics added) and incubate at 37 °C and 220 r / min for 8–10 h. Transfer 1 mL of the inoculum to a 250 mL shake flask containing 50 mL of LB medium (with appropriate antibiotics added) and ferment at 37 °C and 220 r / min for 48 h. Collect the crude enzyme (supernatant) by centrifugation at 8,000 ×g for 30 min.

[0089] PG can specifically hydrolyze the amide group of glutamine residues in proteins, releasing free NH4. + Under the catalysis of sodium nitroprusside and with sodium hypochlorite as the oxidant, NH4+ + It reacts with phenol to produce a blue-green indophenol blue color reaction, which is effective at a wavelength of 630 nm (OD). 630 The spectrum of ) has a specific absorption effect, which can be obtained by measuring OD. 630 This can reflect the vitality of the PG (Positive Gainer). The specific method is as follows:

[0090] Experimental group: Take 10 µL of enzyme solution, preheat at 37 ℃ for 1 min, then add 100 µL of substrate solution (preheated for 10 min beforehand), react the mixture at 37 ℃ for 30 min, add 100 µL of CCl3COOH solution, mix well, and transfer 12 µL of the mixture to 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 sequentially, mix well, and incubate at 37 ℃ for 20 min. Finally, measure the wavelength at 630 nm (OD) using a microplate reader. 630 The absorbance value of ).

[0091] Control group: Take 10 µL of enzyme solution, preheat at 37 ℃ for 1 min, then add 100 µL of 400 mM CCl3COOH solution preheated at 37 ℃ for 10 min. Incubate the mixture in a 37 ℃ water bath for 30 min, then add 100 µL of substrate solution and mix thoroughly by pipetting. Transfer 12 µL of the mixture to 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 sequentially, mixing thoroughly by pipetting. Incubate at 37 ℃ for 20 min. Finally, measure the wavelength at 630 nm (OD) using a microplate reader. 630 The absorbance value of ).

[0092] The PG enzyme activity is defined as: under conditions of 37 ℃ and pH 6.8, the amount of NH4+ released per minute per unit volume of enzyme solution hydrolyzing the substrate CBZ-GIn-Gly. + As one unit of enzyme activity (U·mL) -1 ).

[0093] Enzyme activity formula: Enzyme activity (U) = (AB) × (2.1 / 0.1) / 17.03 / 30 / a,

[0094] A: Absorbance of the experimental group at a wavelength of 630 nm;

[0095] B: Absorbance of the control group at a wavelength of 630 nm;

[0096] a: NH4 + The slope of the standard curve.

[0097] like Figure 4 As shown, the relative activities of mutants E35I and E58A were both higher than those of WT propeptide. E35I showed an expression activity of 4.24 U / mL, a 57% increase compared to the WT propeptide (2.7 U / mL), while E58A showed an expression activity of 3.64 U / mL, a 35% increase compared to the WT propeptide. The combined expression activity of E35I / E58A was 4.86 U / mL, an 80% increase compared to the WT propeptide.

[0098] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A protein glutaminase precursor mutant, characterized in that, The mutant is obtained by causing E35I, E58A, or E35I / E58A mutations or any one of them on the basis of the wild-type protein glutaminase propeptide; wherein, the amino acid sequence of the wild-type protein glutaminase propeptide is shown in SEQ ID NO.1; and the amino acid sequence of the E35I mutant is shown in SEQ ID NO.2; The amino acid sequence of the E58A mutant is shown in SEQ ID NO.3; The amino acid sequence of the E35I / E58A mutant is shown in SEQ ID NO.

4.

2. A polynucleotide, characterized in that, The polynucleotide sequence encoding the mutant according to claim 1 is shown in SEQ ID NO. 6; the polynucleotide sequence encoding the E35I mutant is shown in SEQ ID NO. 7; and the polynucleotide sequence encoding the E58A mutant is shown in SEQ ID NO.

8.

3. A recombinant plasmid, characterized in that, It comprises the polynucleotide as described in claim 2.

4. The recombinant plasmid according to claim 3, characterized in that, The expression vector for the recombinant plasmid is pBSA43.

5. A host cell, characterized in that, It comprises the polynucleotide of claim 2 or the recombinant plasmid of any one of claims 3-4.

6. The host cell according to claim 5, characterized in that, The host cell is a prokaryotic cell or a eukaryotic cell.

7. A genetically engineered bacterium, characterized in that, It comprises the polynucleotide of claim 2, or the recombinant plasmid of any one of claims 3-4.

8. A method for increasing the expression level of exogenous proteins in Bacillus, characterized in that, The method includes: introducing the recombinant plasmid of claim 3 into Bacillus for expression.

9. The method for expressing exogenous proteins in Bacillus according to claim 8, characterized in that, The Bacillus mentioned is Bacillus amyloliquefaciens.

10. The application of a protein glutaminase precursor mutant as described in claim 1 in enhancing the expression level of exogenous proteins in Bacillus.

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