A protein glutaminase mutant, a gene thereof, and a preparation method of an engineered bacterium

Abstract

The application provides a protein glutaminase mutant and a preparation method of a gene and an engineering bacterium, and relates to the technical field of bioengineering. Chryseobacterium proteolyticum Through rational design on protein glutaminase from Chryseobacterium proteolyticum, protein glutaminase with improved activity and thermal stability is obtained, and then, the protein glutaminase mutant with higher activity and stability is prepared by using expression systems of Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and in particular to a method for preparing a protein glutaminase mutant and its gene, as well as engineered bacteria. Background Technology

[0002] Protein glutaminase (EC 3.5.1.44, PG) is a novel deamidase for modifying food proteins. It catalyzes the deamidation of glutamine residues in substrate proteins or peptides to glutamate, releasing ammonia in the process. PG was initially screened from the soil bacterium *Chryseobacterium proteolyticum* 9670T. PG's deamidation converts the amide group of the substrate protein's side chain to a carboxyl group, enhancing the electrostatic repulsion between protein molecules and causing protein depolymerization. This improves solubility and other functional properties, such as emulsifying and foaming properties. Traditional enzymatic deamidation, such as peptidase (EC 3.5.1.2), can also depolymerize proteins, but its substrates are limited to short peptides and are not suitable for both peptides and proteins. Chemical deamidation, on the other hand, involves protein hydrolysis, resulting in the loss of protein functional properties. PG acts on a single substrate, catalyzing only the deamidation of glutamine in the side chains of the substrate protein or polypeptide. It does not react with free glutamine or asparagine residues, exhibiting high specificity and avoiding protein hydrolysis, thus preventing the loss of protein functional properties. Therefore, PG is an ideal enzyme for catalyzing the deamidation of food proteins and has great potential for improving protein appearance, texture, and flavor.

[0003] Currently, the natural enzymatic properties of PG (such as catalytic activity and thermal stability) are insufficient to fully meet the needs of application scenarios. Therefore, targeted optimization of the enzymatic performance of PG through molecular modification has become the core content for expanding its applications.

[0004] Therefore, this invention is proposed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a protein glutaminase mutant, its gene, and a method for preparing engineered bacteria. Through rational design of a protein glutaminase derived from *Chryseobacterium proteolyticum*, a protein glutaminase with improved activity and thermostability was obtained. Subsequently, using expression systems of *Bacillus subtilis*, *Bacillus licheniformis*, and *Bacillus amyloliquefaciens*, a protein glutaminase mutant with even higher activity and stability was expressed and prepared.

[0006] In order to achieve the objective of this invention, the following technical solution is adopted:

[0007] This invention provides a protein glutaminase mutant, which is obtained by any one of the following mutations: S64T, V295G, or S64T / V295G, based on wild-type protein glutaminase; wherein the amino acid sequence of the wild-type protein glutaminase is shown in SEQ ID NO.1; and the amino acid sequence of the S64T mutant is shown in SEQ ID NO.2.

[0008] The amino acid sequence of the V295G mutant is shown in SEQ ID NO.3;

[0009] The amino acid sequence of the S64T / V295G mutant is shown in SEQ ID NO.4.

[0010] SEQ ID NO.1

[0011] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0012] SEQ ID NO.2

[0013] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYITMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0014] SEQ ID NO.3

[0015] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYISMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDGSSCGF

[0016] SEQ ID NO.4

[0017] MDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKENEQYITMLRQAVKNESPAHIFDKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVATLNSLFNQIKNQSCGTSTASSPCITF RYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDGSSCGF

[0018] The present invention also provides a polynucleotide encoding the above-mentioned mutants, wherein the polynucleotide sequence encoding the S64T / V295G mutant is shown in SEQ ID NO.6, the polynucleotide sequence encoding the S64T mutant is shown in SEQ ID NO.7, and the polynucleotide sequence encoding the V295G mutant is shown in SEQ ID NO.8.

[0019] The polynucleotide sequence encoding the wild type is shown in SEQ ID NO.5.

[0020] SEQ ID NO.5

[0021] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAAAATGAATCACCG GCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0022] SEQ ID NO.6

[0023] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTACTATGCTGAGACAAGCAGTTAAAAATGAATCACCGGCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGGTAGCTCATGCGGCTTCTAA

[0024] SEQ ID NO.7

[0025] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTACTATGCTGAGACAAGCAGTTAAAAATGAATCACCGGCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTA GATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCC GGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGTTAGCTCATGCGGCTTCTAA

[0026] SEQ ID NO.8

[0027] ATGGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTGTCAGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACAGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTTTCATTTATGCTGACAGCACAATTTTATGAAATTAAACCGACAAAAGAAAATGAACAATATATTAGCATGCTGAGACAAGCAGTTAAAAATGAATCACCGGCACATATTTTTGATAAACCGAATTCAAATGAAATTGGCAAAGTTGAATCAGCATCACCGGAAGATGTTAGATATTTTAAAACAATTCTGACAAAAGAAGTTAAAGGCCAAACAAATAAACTGGCATCAGTTATTCCGGATGTTGCAACACTGAATTCACTGTTTAATCAAATTAAAAATCAATCATGCGGCACATCAACAGCATCATCACCGTGCATTACATTTAGATATCCGGTTGATGGCTGCTATGCAAGAGCACATAAAATGAGACAAATTCTGATGAATAATGGCTATGATTGCGAAAAACAATTTGTTTATGGCAATCTGAAAGCATCAACGGGCACATGCTGCGTTGCATGGTCATATCATGTTGCAATTCTGGTTTCATATAAAAATGCAAGCGGCGTTACAGAAAAAAGAATTATTGATCCGTCACTGTTTTCAAGCGGCCCGGTTACAGATACAGCATGGAGAAATGCATGCGTTAATACATCATGCGGCTCAGCATCAGTTTCATCATATGCAAATACAGCGGGCAATGTGTACTATCGCTCACCTTCAAATAGCTATCTGTATGATAATAATCTTATCAATACAAATTGCGTTCTGACAAAATTTTCACTGCTGAGCGGCTGCTCACCGTCACCGGCACCGGATGGTAGCTCATGCGGCTTCTAA

[0028] The present invention further provides a recombinant plasmid comprising the above-mentioned polynucleotide.

[0029] Furthermore, the expression vector for the recombinant plasmid is pBSA43.

[0030] The present invention also provides a host cell comprising the above-mentioned polynucleotides or recombinant plasmids.

[0031] Furthermore, the host cell is a prokaryotic cell or a eukaryotic cell.

[0032] Furthermore, the host cell is any one of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus licheniformis.

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

[0034] This invention also provides the application of the above-mentioned protein glutaminase mutant in the preparation of catalytic deamidation products of food proteins.

[0035] The following definitions are used in this invention:

[0036] Nomenclature for amino acids and DNA nucleic acid sequences:

[0037] Amino acid residues are named according to the recognized IUPAC nomenclature, using single-letter or three-letter codes. DNA nucleic acid sequences are named according to the recognized IUPAC nomenclature.

[0038] The identification of protein glutaminase mutants uses "original amino acid + position + substituted amino acid" to represent the mutated amino acid in the PG mutant. For example, S64T / V295G indicates that the amino acid at position 64 is replaced by Thr (Ser in wild-type PG), and the amino acid at position 295 is replaced by Gly (Val in wild-type PG). The position number corresponds to the amino acid sequence number of the mature peptide of wild-type PG in SEQ ID NO.1.

[0039] The present invention has the following technical effects:

[0040] This invention utilizes molecular modification to modify the wild-type PG, obtaining a mutant S64T / V295G with improved activity and stability at 40℃ compared to the wild-type. The specific activity of mutant S64T / V295G is 3.10 U / mg, which is 6.1 times that of the wild-type (0.51 U / mg), and the half-life at 40℃ is 58.7 min, which is 2.3 times that of the wild-type (25.6 min). Attached Figure Description

[0041] 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.

[0042] Figure 1 Electrophoresis images of the upstream fragment P1 and downstream fragment P2 of the PG target gene;

[0043] Figure 2 Electrophoresis images of pBSA43 plasmid and the PG target gene after double enzyme digestion;

[0044] Figure 3 Electrophoresis image of the pBSA43-pg recombinant plasmid after double enzyme digestion;

[0045] Figure 4 Specific activity of PG mutants;

[0046] Figure 5 : Half-life of the PG mutant. Detailed Implementation

[0047] 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.

[0048] The following is a detailed explanation using specific embodiments:

[0049] The solutions and culture media used in the embodiments of this invention are as follows:

[0050] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride (15 g / L agar powder added to solid medium), autoclaved at 121 °C for 20 min.

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

[0052] Preparation of CBZ-Gln-Gly solution as substrate for enzyme-catalyzed 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] 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] 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] Preparation of colorimetric reagent B: Accurately weigh 49.94 g KOH, add pure water to dissolve and make up to 1 L, mix well and it is ready for use.

[0056] Preparation of colorimetric 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, mix well and it is ready for use.

[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, NcoI and XhoI. The amplification primers for the protein glutaminase gene pg of this invention are as follows:

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

[0060] R: 5'-CCGCTCGAGTTA 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 amplification products were subjected to 1.2% agarose gel electrophoresis and recovered using a small-volume DNA recovery kit. The recovered products were digested with NcoI and XhoI and ligated into the vector pET-28a(+). The ligation product was first transformed into the cloning host E. coli JM109, plated on LB solid medium, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformations were sequenced for verification. The correctly verified plasmid was then transformed into the expression host BL21, plated on LB solid medium, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain the pET28a-pg plasmid.

[0065] Bioinformatics prediction was used to determine the construction of single-point mutations S64T, V295G, and combined mutation S64T / V295G, and the mutants S64T, V295G and S64T / V295G were obtained by overlapping PCR.

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

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

[0068] f-S64T:TCTCAGCATAGTAATATATTGTTC

[0069] r-S64T: AACAATATATTACTATGCTGAGA

[0070] f-V295G: ACCGGATGGTAGCTCATG

[0071] r-V295G: CATGAGCTACCATCCGGT

[0072] The mutant S64T / V295G can be obtained by mutating the mutant S64T into the V295G variant.

[0073] Taking the mutant S64T as an example, in the first step of the overlap PCR reaction system, F and r-S64T were used as upstream and downstream primers, respectively, and f-S64T and R were used as upstream and downstream primers, respectively. Using pET28a-pg as a template, PCR1 reaction was performed to obtain the upstream and downstream fragments, respectively.

[0074] The amplification system for the upstream fragment is shown in Table 2.

[0075] Table 2: Amplification system of upstream fragment

[0076] F 2 μL r-S64T 2 μL pET28a-pg 2 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0077] The reaction system for downstream fragment amplification is shown in Table 3:

[0078] Table 3: Reaction system for downstream fragment amplification

[0079] f-S64T 2 μL R 2 μL pET28a-pg 2 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 19 μL

[0080] 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.

[0081] After gel extraction and recovery of upstream and downstream fragments, PCR 2 was performed. The reaction system is shown in Table 4.

[0082] Table 4: PCR 2 reaction system

[0083] Upstream segment 2.0 μL Downstream segments 2.0 μL Primer Star Max Enzyme 25 μL <![CDATA[ddH2O]]> 21 μL

[0084] 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.

[0085] 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.

[0086] The wild-type pg gene was ligated to the pET-28a vector using NcoI and XhoI. 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 transformants were sequenced to obtain the pET28a-pg wild-type plasmid. The successfully verified plasmid was then transformed into the expression host BL21, plated on LB agar, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain the pET28a-pg wild-type recombinant strain.

[0087] The pg mutant gene was ligated to the pET-28a vector using NcoI and XhoI. The ligation product was first transformed into the cloning host E. coli JM109, plated on LB agar, and plasmids were extracted from transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain pET28a-S64T, pET28a-V295G, and pET28a-S64 / V295G mutant plasmids. The correctly verified plasmids were then transformed into the expression host BL21, plated on LB agar, and plasmids were extracted from transformants for PCR and double enzyme digestion verification. Successful transformants were sequenced to obtain pET28a-S64T, pET28a-V295G, and pET28a-S64 / V295G mutant recombinant strains.

[0088] Example 3: Expression and Enzyme Activity Assay of Protein Glutaminase

[0089] The successfully constructed wild-type and mutant PG strains were inoculated into 5 mL LB tubes and cultured overnight at 37 °C and 220 r / min. Then, they were transferred to 50 mL LB medium at a 2% inoculation rate and fermented at 37 °C and 220 r / min until the OD600 reached approximately 0.6. Isopropyl thio-β-D-galactopyranoside (IPTG, final concentration 0.5 mM) was added for induction, and fermentation continued for 18 h at 16 °C and 150 r / min. After fermentation, the cells were collected, sonicated, and centrifuged at 10,000 ×g for 15 min. The crude extract (supernatant) was collected, and the wild-type (WT) and mutant PG strains were purified using a nickel column method. The purified protein concentration was determined using a BCA protein concentration kit.

[0090] 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.

[0091] The specific method is as follows:

[0092] Experimental group: Take 10 µL of enzyme solution, preheat at 40 ℃ for 1 min, then add 100 µL of substrate solution (preheated for 10 min beforehand), react the mixture at 40 ℃ 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 ).

[0093] Control group: Take 10 µL of enzyme solution, preheat at 40 °C for 1 min, then add 100 µL of 400 mM CCl3COOH solution preheated at 40 °C for 10 min. Incubate the mixture in a 40 °C 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 reagent A, 30 µL of chromogenic reagent B, and 60 µL of chromogenic reagent C sequentially, mix thoroughly by pipetting, and incubate at 37 °C for 20 min. Finally, measure the wavelength at 630 nm (OD) using a microplate reader. 630 The absorbance value of ).

[0094] The PG enzyme activity is defined as: under conditions of 40 ℃ 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 ).

[0095] The enzyme activity formula is: Enzyme activity (U) = (AB) × (2.1 / 0.1) / 17.03 / 30 / a,

[0096] A: Absorbance of the experimental group at a wavelength of 630 nm;

[0097] B: Absorbance of the control group at a wavelength of 630 nm;

[0098] a: NH4 + The slope of the standard curve.

[0099] The formula for calculating the specific enzyme activity of PG is: Specific enzyme activity (U / mg) = enzyme activity (U / mL) / enzyme protein concentration (mg / mL). The specific enzyme activity of PG is calculated according to this formula.

[0100] Experimental results are as follows Figure 4As shown, the results indicated that the specific activity of mutant S64T was 1.7 U / mg, which was 3.3 times that of WT (0.51 U / mg); the specific activity of mutant V295G was 1.10 U / mg, which was 2.16 times that of WT; and the specific activity of the combined mutant S64T / V295G was 3.10 U / mg, which was 6.1 times that of WT.

[0101] Experimental Example 4: Determination of the half-life of protein glutaminase

[0102] The half-life of purified PG, S64T, V295G and S64T / V295G at 40 °C (pH 6.8) was determined by placing the purified PG, S64T, V295G and S64T / V295G in a 40 °C water bath for a certain period of time and then measuring the residual enzyme activity. The enzyme activity detection method and reaction system were the same as those for the detection of the pure enzyme in Example 3.

[0103] Experimental results are as follows Figure 5 As shown, the half-life of mutant S64T at 40 °C is 46.1 min, which is 1.8 times that of WT (25.6 min); the half-life of mutant V295G at 40 °C is 38.0 min, which is 1.48 times that of WT. The half-life of the combined mutant S64T / V295G at 40 °C is 58.7 min, which is 2.3 times that of WT.

[0104] Experiment Example 5: Expression and preparation of protein glutaminase mutant in Bacillus subtilis

[0105] The wild-type WT and mutant glutaminase genes (S64T, V295G, and S64T / V295G) were ligated with the expression plasmid pBSA43. The ligation products were first transformed into the cloning host E. coli JM109 and plated on LB agar. Plasmids were extracted from transformants and subjected to PCR and double enzyme digestion verification. Successful verification was followed by sequencing to obtain wild-type and mutant plasmids pBSA43-pg, pBSA43-S64T, pBSA43-V295G, and pBSA43-S64 / V295G. The double enzyme digestion electrophoresis of the pBSA43-pg recombinant plasmid was also performed. Figure 3 As shown, the plasmid that was verified to be correct was transformed into Bacillus subtilis WB600, plated on LB solid medium, and the plasmid was extracted from the transformants for PCR and double enzyme digestion verification. The successfully verified transformants were sequenced to obtain wild-type and mutant recombinant strains of WB600 / pBSA43-pg, WB600 / pBSA43-S64T, WB600 / pBSA43-V295G and WB600 / pBSA43-S64T / V295G.

[0106] Wild-type and mutant recombinant strains of protein glutaminase were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 rpm. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 rpm. The fermentation medium (g / L) consisted of 64 g corn flour, 40 g soybean meal, 2.7 g amylase, 4 g Na₂HPO₄, 0.3 g KH₂PO₄, and the remainder water. The culture was incubated at 90 °C for 30 min and then sterilized at 121 °C for 20 min.

[0107] The glutaminase activity of the protein obtained from Bacillus subtilis fermentation was determined using the method in Example 3 (the supernatant of the fermentation broth was collected after centrifugation to determine the enzyme activity). The fermentation activities of WT and mutants S64T, V295G and S64T / V295G were 1.43 U / mL, 1.58 U / mL, 3.14 U / mL and 9.67 U / mL, respectively.

[0108] Example 6: Expression and preparation of protein glutaminase mutant in Bacillus licheniformis

[0109] The wild-type and mutant plasmids pBSA43-pg, pBSA43-S64T, pBSA43-V295G, and pBSA43-S64 / V295G constructed in Example 4 were transformed into Bacillus licheniformis TCCC2709, plated on LB agar plates, and transformed individuals were picked for colony PCR. The successfully verified strains were TCCC2709 / pBSA43-pg, TCCC2709 / pBSA43-S64T, TCCC2709 / pBSA43-V295G, and TCCC2709 / pBSA43-S64T / V295G recombinant strains.

[0110] Wild-type and mutant recombinant strains of protein glutaminase were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 rpm. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 rpm. The fermentation medium (g / L) consisted of 64 g corn flour, 40 g soybean meal, 2.7 g amylase, 4 g Na₂HPO₄, 0.3 g KH₂PO₄, and the remainder water. The culture was incubated at 90 °C for 30 min and then sterilized at 121 °C for 20 min.

[0111] The glutaminase activity of the protein obtained from Bacillus licheniformis fermentation was determined using the method in Example 3 (the supernatant of the fermentation broth was centrifuged and the enzyme activity was determined). The fermentation activities of WT and mutants S64T, V295G and S64T / V295G were 5.03 U / mL, 17.05 U / mL, 10.85 U / mL and 31.47 U / mL, respectively.

[0112] Example 7: Expression and preparation of protein glutaminase mutant in Bacillus amyloliquefaciens

[0113] The wild-type and mutant plasmids of the protein glutaminase successfully verified in Example 4 were transformed into B. amyloliquefaciens TCCC111018, plated on LB agar plates, and transformed individuals were picked for colony PCR verification. The successfully verified strains were TCCC111018 / pBSA43-pg, TCCC111018 / pBSA43-S64T, TCCC111018 / pBSA43-V295G, and TCCC111018 / pBSA43-S64T / V295G.

[0114] Wild-type and mutant recombinant strains of protein glutaminase were inoculated into 5 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured overnight at 37 °C and 220 rpm. The inoculum was then transferred at a 2% inoculation rate to 50 mL of fermentation medium (containing kanamycin, 50 µg / mL) and cultured for another 48 h at 37 °C and 220 rpm. The fermentation medium (g / L) consisted of 64 g corn flour, 40 g soybean meal, 2.7 g amylase, 4 g Na₂HPO₄, 0.3 g KH₂PO₄, and the remainder water. The culture was incubated at 90 °C for 30 min and then sterilized at 121 °C for 20 min.

[0115] The glutaminase activity of the protein obtained by fermentation of Bacillus amyloliquefaciens was determined using the method in Example 3 (the supernatant of the fermentation broth was taken by centrifugation to determine the enzyme activity). The fermentation activities of WT and mutants S64T, V295G and S64T / V295G were 3.51 U / mL, 12.16 U / mL, 7.65 U / mL and 22.35 U / mL, respectively.

[0116] 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 mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO. 2, 3 or 4.

2. A polynucleotide, characterized in that, The mutant according to claim 1, wherein the polynucleotide sequence encoding the amino acid sequence of the mutant as shown in SEQ ID NO.4 is shown in SEQ ID NO.6, the polynucleotide sequence encoding the amino acid sequence of the mutant as shown in SEQ ID NO.2 is shown in SEQ ID NO.7, and the polynucleotide sequence encoding the amino acid sequence of the mutant as shown in SEQ ID NO.3 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. The host cell according to claim 6, characterized in that, The host cell is any one of Escherichia coli, Bacillus subtilis, Bacillus amyloliquefaciens, or Bacillus licheniformis.

8. 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.

9. The application of a protein glutaminase mutant as described in claim 1 in the preparation of catalytic deamidation products of food proteins.

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