Protein glutaminase and application thereof

Abstract

The present invention belongs to the field of enzyme genetic engineering technology, and specifically relates to a protein glutaminase mutant with improved enzyme activity obtained by in vitro directed evolution using error-prone PCR technology and its preparation. The mutant is obtained by causing an A144G mutation at position 144 of a wild-type protein glutaminase from Chryseobacterium proteolyticum, and has an amino acid sequence shown in SEQ ID NO.3. The maximum enzyme activity of the high-activity protein glutaminase fermented in Bacillus subtilis and Bacillus amyloliquefaciens expression systems is 2.9 U / mL and 6.5 U / mL, respectively.

Description

Technical field:

[0001] The invention belongs to the technical field of enzyme genetic engineering, and specifically relates to a protein glutaminase mutant with improved enzyme activity obtained by in vitro directed evolution using error-prone PCR technology and its preparation. Background technology:

[0002] Protein glutaminase (EC 3.5.1.44, protein glutaminase, PG) is a deamidase that has attracted widespread attention in recent years and belongs to the class of amide hydrolases. PG can hydrolyze glutamine in the side chains of macromolecular proteins and some small peptides to generate protein L-glutamic acid and ammonia, thereby improving the functional properties of proteins and reducing the allergenicity of plant proteases. It has great application value in the food, biopharmaceutical and cosmetic industries.

[0003] PG was first discovered by Japanese scientists Yamaguchi S and Yokoe M in 2000. They isolated the PG-producing strain C.proteolyticium 9670 from the soil. Like most Chryseobacteria, this strain can produce a Flexirubin-type pigment, which turns red after KOH treatment and restores its original color after HCl neutralization. Subsequently, in 2001, Yamaguchi S et al. found that PG is expressed in the form of pro-PG and published its gene sequence. In 2001, Japanese scholars purified PG from Chryseobacteria, and since then, many scholars have used PG for application research. In 2001, YSGu et al. studied the deamidation effect of PG on α-lactoprotein. The results showed that after 24 hours of reaction in the natural state, the deamidation degree of α-lactoprotein reached 55%. When the α-lactoprotein was partially folded to form a molten globule state, the deamidation degree increased significantly, and it could increase to 66% after 24 hours. CD spectral analysis showed that the stability of the tertiary structure of α-lactoprotein was related to the degree of deamidation. In 2004, Yasuki Matsumura et al. studied the effect of PG on zein. The results showed that after 137 hours of reaction at 40°C, the deamidation degree of zein, which has extremely low solubility, reached 62%. After deamidation, the solubility and emulsification properties of zein were significantly improved, the number of β-folds between molecules was significantly reduced, and the secondary structure of the protein was changed.

[0004] Molecular modification of enzymes includes rational design and irrational design. Irrational design is generally achieved through directed evolution, and there is no need to understand the relationship between the high-level structure and function of the enzyme protein molecule. The key step is to select efficient methods and conditions for constructing a mutant library, and to select a convenient method for screening target mutants for the mutant library, so as to obtain mutants that meet the experimental requirements. At present, the methods for constructing mutant libraries mainly include error-prone PCR technology, DNA reorganization, staggered extension and in vitro random priming recombination technology. Irrational design has been reported to be used to improve the thermal stability of amylase, β-galactosidase and pullulanase. In addition, the mutagenesis of the target strain by physical (ultraviolet mutagenesis, α-rays, β-rays) and chemical (alkylating agents, antibiotics, nucleic acid base analogs) mutagens, and then directed screening to obtain strains with improved thermal stability is also a commonly used directed evolution method. In contrast, when the spatial structure, active site, catalytic mechanism and other factors of the protein are known, the targeted modification of the gene on this basis is site-directed mutagenesis, that is, rational design. Because it can only replace, delete or insert a few amino acids in natural enzyme proteins, the modification of enzyme functions is limited. Therefore, for enzymes with unknown structures and functions, directed evolution can make up for the shortcomings of rational design to a certain extent.

[0005] As a safe, efficient, multifunctional and highly potential microbial strain, the Bacillus expression system has been widely used in industry, agriculture, medicine, health, food, animal husbandry, aquatic products and scientific research. Compared with the commonly used Escherichia coli expression system, it has a unique advantage in that the product of the target gene expression can be secreted outside the cell, thereby reducing the cost and workload of further collecting, separating and purifying the gene expression product. Among the Bacillus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium and the like can all be used as expression host bacteria. In the field of microbial genetics, the background research of Bacillus is also very clear, with the advantages of no obvious codon preference, simple fermentation, rapid growth, no production of pathogenic toxins, and no special requirements for culture medium. With the development of molecular biology technology and the in-depth study of Bacillus, a large number of genes have been cloned and expressed using the Bacillus expression system, some of which have been put into large-scale industrial production. A variety of enzymes and clinically required chemical drugs or industrial products are expressed and produced by Bacillus.

[0006] In the present invention, the original protein glutaminase gene is directed to evolve to obtain a highly active protein glutaminase mutant gene, and its efficient expression in the Bacillus subtilis expression system and the Bacillus amyloliquefaciens expression system is achieved, thereby obtaining a mutant strain producing highly active protein glutaminase. Summary of the invention:

[0007] Based on the problems existing in the prior art, in order to further promote the application of protein glutaminase in the industrial field, its existing properties need to be further improved. The purpose of the present invention is to provide a mutant of high-activity protein glutaminase.

[0008] The technical route for achieving the purpose of the present invention is summarized as follows:

[0009] The wild-type protein glutaminase from Chryseobacterium proteolyticum was obtained through basic molecular biological techniques. After constructing a recombinant vector through enzyme digestion and ligation, the wild-type protein glutaminase encoding gene was obtained through sequencing. The wild-type gene was randomly mutated using error-prone PCR technology, and the mutants and their encoding genes were screened using an Escherichia coli expression system. The recombinant vector was reconstructed, and its efficient expression in Bacillus subtilis and Bacillus amyloliquefaciens was achieved. Mutants with improved enzyme activity were obtained through fermentation, extraction and other techniques.

[0010] One of the technical solutions provided by the present invention is a protein glutaminase mutant, wherein the mutant is obtained by causing an A144G mutation at position 144 of the wild-type protein glutaminase from Chryseobacterium proteolyticum, and the protein glutaminase mutant has an amino acid sequence shown in SEQ ID NO.3;

[0011] The present invention also provides a gene encoding the above protein glutaminase mutant;

[0012] Furthermore, the protein glutaminase mutant encoding gene has a nucleotide sequence shown in SEQ ID NO.4;

[0013] The present invention also provides a recombinant plasmid or a recombinant strain comprising the mutant encoding gene;

[0014] Furthermore, the expression vectors used in the recombinant plasmid are pET-28a and pBSA43;

[0015] Furthermore, the host cell used by the recombinant strain may be Escherichia coli BL21, Bacillus subtilis WB600, or Bacillus amyloliquefaciens CGMCC No.11218.

[0016] The present invention also provides the application of the above recombinant plasmid or recombinant strain, especially the application in producing the protein glutaminase mutant shown in SEQ ID NO.3.

[0017] The present invention also provides the application of the protein glutaminase mutant shown in SEQ ID NO.3, especially in improving the solubility, emulsification and foaming properties of protein foods, and more particularly in hydrolyzing the amide group of glutamine residues in proteins.

[0018] The experimental scheme of the present invention is as follows:

[0019] 1. The acquisition of the PG mutant encoding gene comprises the following steps:

[0020] (1) using the wild-type PG encoding gene pg (SEQ ID No. 2) of Chryseobacterium utilis as a template, random mutation of the wild-type PG encoding gene was performed by error-prone PCR;

[0021] (2) The randomly mutated PG coding gene was transformed into Escherichia coli BL21 by constructing a recombinant plasmid through enzyme digestion and ligation, and PG mutants with improved enzyme activity were screened. The PG mutant coding gene pgm was obtained by sequencing, and the plasmid pET-28a-pgm containing the PG mutant coding gene with improved enzyme activity was preserved.

[0022] 2. A recombinant Escherichia coli strain containing a protein glutaminase encoding gene and screening, comprising the following steps:

[0023] (1) Obtain the mutant encoding gene by error-prone PCR, and connect the PG mutant encoding gene with pET-28a to obtain a new recombinant plasmid;

[0024] (2) The recombinant plasmid was transferred into Escherichia coli BL21, and the recombinant strain was screened by kanamycin (Kan) resistance screening and enzyme activity testing to obtain protein glutaminase with high enzyme activity.

[0025] (3) The PG mutant encoding gene pgm was obtained by sequencing, and the plasmid pET-28a-pgm containing the PG mutant encoding gene with improved enzyme activity was preserved.

[0026] 3. A recombinant strain of Bacillus subtilis containing a gene encoding protein glutaminase and a process for preparing protein glutaminase with improved enzyme activity using the same, comprising the following steps:

[0027] (1) The PG mutant encoding gene was connected with the Escherichia coli-Bacillus subtilis shuttle plasmid pBSA43 to obtain a new recombinant plasmid pBSA43-pgm;

[0028] (2) The recombinant plasmid was transferred into Bacillus subtilis WB600, and the recombinant strain was obtained by kanamycin (Kan) resistance screening and enzyme digestion verification. The recombinant strain was then cultured and fermented to obtain protein glutaminase.

[0029] 4. A recombinant strain of Bacillus amyloliquefaciens containing a gene encoding protein glutaminase and a process for preparing protein glutaminase with improved enzyme activity using the strain, comprising the following steps:

[0030] (1) The recombinant plasmid pBSA43-pgm was transferred into Bacillus amyloliquefaciens CGMCC No.11218, and the obtained recombinant strain was subjected to Kan resistance screening and protein glutaminase enzyme activity assay to obtain a protein glutaminase high-yielding strain;

[0031] (2) Fermentation is then performed to prepare protein glutaminase.

[0032] The enzymatic properties of the PG wild type and mutant A144G are as follows:

[0033] (1) Specific activity: The specific activity of wild-type PG is 1.8 U / mg, and the specific activity of PG mutant A144G is 3.1 U / mg, which is about 72% higher than that of the wild-type.

[0034] (2) Optimum reaction temperature: 37°C.

[0035] (3) Optimum pH: 7.0.

[0036] Beneficial effects:

[0037] 1. The present invention uses error-prone PCR technology to randomly mutate wild-type PG to obtain a mutant A144G with improved enzyme activity. The maximum enzyme activity of high-activity protein glutaminase in the expression system of Bacillus subtilis and Bacillus amyloliquefaciens is 2.9U / mL and 6.5U / mL respectively.

[0038] 2. The present invention uses a Bacillus subtilis expression system and a Bacillus amyloliquefaciens expression system to achieve efficient expression of PG mutants with improved enzyme activity. Description of the drawings:

[0039] Figure 1 The electrophoresis diagram of the PCR amplification of the wild-type pg gene of the present invention is

[0040] Among them: M is DNAMarker, 1 is pg gene;

[0041] Figure 2 : is the restriction enzyme verification diagram of the recombinant plasmid pBSA43-pgm of the present invention, wherein: M is DNA Marker, 1 is the electrophoresis diagram of the recombinant plasmid pBSA43-pgm in Bacillus subtilis after double restriction enzyme digestion with BamHI and NotI, 2 is the electrophoresis diagram of the recombinant plasmid pBSA43-pgm in Bacillus amyloliquefaciens after double restriction enzyme digestion with BamHI and NotI;

[0042] Figure 3 The SDS-PAGE diagram of the purified samples of wild-type PG and mutant A144G protein of the present invention, wherein: M is Protein Marker, 1 is the purified sample of wild-type PG, and 2 is the purified sample of mutant A144G;

[0043] Figure 4 NH4 + Standard curve;

[0044] Figure 5 The optimum temperature curve of wild-type PG and mutant A144G of the present invention is

[0045] Wherein: WT is the wild-type PG of the present invention, and A144G is the mutant of the present invention;

[0046] Figure 6 The optimum pH curve of the wild-type PG and mutant A144G of the present invention is

[0047] Wherein: WT is the wild-type PG of the present invention, A144G is the mutant of the present invention;

[0048] Figure 7 This is the deamidation degree curve of casein treated with A144G of the present invention. Specific implementation method:

[0049] The technical content of the present invention is further described below in conjunction with the embodiments, but the present invention is not limited to these embodiments, and the protection scope of the present invention cannot be limited by the following embodiments.

[0050] The following definitions are used in the present invention:

[0051] 1. Nomenclature of amino acid and DNA sequences

[0052] The generally accepted IUPAC nomenclature for amino acid residues is used, either in the form of three-letter or one-letter codes. The generally accepted IUPAC nomenclature for DNA nucleic acid sequences is used.

[0053] 2. Identification of protein glutaminase mutants

[0054] The mutated amino acid in the mutant is represented by "amino acid replaced by the original amino acid position", such as Ala144Gly, which means that the amino acid at position 144 is replaced by Gly from wild-type Ala, and the position number corresponds to the amino acid sequence number of the wild-type in SEQ ID No. 1.

[0055] In the present invention, lowercase italic pg represents the coding gene of wild-type PG, and lowercase italic pgm represents the coding gene of mutant A144G, and the information is as shown in the following table.

[0056]

[0057]

[0058] Among them, the amino acid sequence of the A144G mutant SEQ ID NO.3 is as follows:

[0059] MKNLFLSMMAFVTVLTFNSCADSNGNQEINGKEKLSVNDSKLKDFGKTVPV

[0060] GIDEENGMIKVSFMLTAQFYEIKPTKENEQYIGMLRQAVKNESPVHIFLKPNSN

[0061] EIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVGTLNSLFNQIKNQSCG

[0062] TSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKASTGTC

[0063] CVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSA

[0064] SVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF*

[0065] The present invention is further explained below through specific implementation modes.

[0066] Example 1: Obtaining the wild-type PG proenzyme gene pg

[0067] 1. The wild-type PG encoding gene pg comes from Chryseobacterium utilis. The gene sequence was downloaded from the NCBI database, synthesized by GeneWeiZ, connected to the pUC57 plasmid, and stored in Escherichia coli JM109. In order to obtain the wild-type PG proenzyme gene, we first extracted the pUC57-pg plasmid as a template and amplified the wild-type pg. We performed the following operations:

[0068] (1) Strain activation: Use an inoculation loop to take the above-mentioned E. coli JM109 bacterial solution containing the target gene pg from the glycerol tube, inoculate it on an LB solid culture medium plate, draw three zones, and culture at 37°C for 12 h;

[0069] (2) Transfer: Pick a single colony with neat edges and smooth surface from the plate where the bacteria are cultured and inoculate it into 5 mL of liquid LB medium. Cultivate at 220 rpm and 37°C for 12 h.

[0070] (3) Collecting bacteria: Take an appropriate amount of culture medium and dispense it into a 1.5 mL EP tube, centrifuge at 12000 rpm for 2 min, and discard the supernatant;

[0071] (4) Add 250 μL ddH2O to resuspend the cells, pipette to mix, centrifuge at 12000 rpm for 1 min, and discard the supernatant;

[0072] (5) Add 250 μL Solution I, pipette and mix thoroughly, and let stand for 1-2 min;

[0073] (6) Add 250 μL Solution II and slowly invert 7-8 times. Mix thoroughly and open the lid to see if there is stringiness.

[0074] (7) Add 250 μL of Solution III within 5 min and immediately invert several times to mix. A white flocculent precipitate will appear.

[0075] (8) Centrifuge at 12000 rpm for 15 min, transfer the supernatant to an adsorption column, and repeat 3 times at 12000 rpm for 1 min, and discard the filtrate;

[0076] (9) Add 500 μL HBC Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0077] (10) Add 700 μL DNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0078] (11) Add 500 μL DNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate;

[0079] (12) 12000 rpm, air-free for 2 min, 55°C metal bath for 10 min, air-dried;

[0080] (13) Add 40 μL ddH2O to elute the plasmid and obtain pUC57-pg.

[0081] 2. Amplify the wild-type PG encoding gene pg

[0082] The primers for amplification of the wild-type PG encoding gene pg were designed, and the sequences were as follows:

[0083] Upstream P1 (SEQ ID No.5):

[0084] CATG CCATGG AAAAATCTTTTTTTATCAATGATGGCC (the underlined part is the NcoI restriction site)

[0085] Downstream P2 (SEQ ID No.6):

[0086] CCG CTCGAG TTAGTGGTGGTGGTGGTGGTGGTGAAATCCACAGCTGGATACAT (the underlined part is the XhoI restriction site)

[0087] The reaction system for PCR amplification is 50 μL, and its composition is:

[0088] PrimeSTAR Max 25μL Upstream primer P1 (20 μmol / L) 2μL Downstream primer P2 (20 μmol / L) 2μL pUC57-pg plasmid 2μL <![CDATA[ddH2O]]> 19μL Total volume 50μL

[0089] Note: The above reagents are from Takara Biotech Co., Ltd.

[0090] The settings for the amplification program were:

[0091] a. Pre-denaturation: 98℃30s;

[0092] b. Denaturation: 98℃ for 10s;

[0093] c. Annealing: 56°C for 45 seconds;

[0094] d. Extension: 72℃ for 10s;

[0095] EB-D reaction for 30 cycles;

[0096] f. Extension: 72℃ for 10 min.

[0097] The PCR product was subjected to agarose gel electrophoresis, and a band of the wild-type PG encoding gene pg was observed, which was about 900 bp (see Figure 1 ), and then the PCR product was recovered by a DNA gel excision recovery kit, and the recombinant plasmid pET-28a-pg was constructed by enzyme digestion and ligation with the pET-28a plasmid, and sent to a sequencing company for sequencing to obtain the wild-type pg gene sequence (shown in SEQ ID NO.2).

[0098] Example 2: Obtaining PG mutant A144G

[0099] 1. Error-prone PCR: Error-prone PCR was performed using the wild-type coding gene pg as a template. The reaction system is as follows:

[0100] <![CDATA[ddH2O]]> 10μL Recombinant plasmid pET28a-pg (5ng / μL) 2μL Upstream primer P1 (10 μmol / L) 2μL Downstream primer P2 (10 μmol / L) 2μL Taq DNA polymerase 0.5μL 10×Taq buffer 5μL dATP (10mmol / L) 1μL dGTP (10mmol / L) 1μL dTTP (10mmol / L) 5μL dCTP (10mmol / L) 5μL <![CDATA[MgCl2(25mmol / L)]]> 14μL <![CDATA[MnCl2(10mmol / L)]]> 2.5μL

[0101] Note: The above reagents are from Takara Biotech Co., Ltd.

[0102] After the system is completed, the error-prone PCR reaction is performed, and the program settings are as follows:

[0103] a. Pre-denaturation: 95℃5min;

[0104] b. Denaturation: 95℃ for 30s;

[0105] c. Annealing: 56°C for 45 seconds;

[0106] d. Extension: 72℃ 90s;

[0107] EB-D reaction for 35 cycles;

[0108] f. Extension: 72℃ for 10 min.

[0109] After the PCR reaction, the PCR product and the vector plasmid were double-digested with NcoI and XhoI, purified and recovered, and the error-prone PCR product was connected with the vector plasmid pET-28a that was also double-digested. Escherichia coli JM109 was transformed and coated on LB solid culture medium containing Kan (100 μg / mL), and cultured in a 37°C incubator for 12 hours to obtain transformants. The recombinant plasmid was extracted and the enzyme digestion was verified to be correct. The recombinant plasmid was transferred into Escherichia coli BL21 and fermented and expressed in a 96-well plate. The recombinant strain was screened for kanamycin (Kan) resistance to obtain protein glutaminase with high enzyme activity. The PG mutant encoding gene pgm was obtained by sequencing, and the plasmid pET-28a-pgm containing the PG mutant encoding gene with improved enzyme activity was preserved.

[0110] 3. Screening of mutant library: Add 200 μL of LB liquid medium containing Kan (100 μg / mL) to each well of the 96-well plate. Then, use a sterilized toothpick to pick up a single clone of each transformant into the 96-well plate. Try to pick up just a small amount of bacteria each time. Transfer the 96-well plate to a shaker for incubation at 160 rpm and 37°C for 48 hours. Then, a low-temperature centrifuge (4°C) was used to centrifuge at 4000rpm for 10 min, the supernatant was discarded, the bacteria were mixed by pipetting with 200 μL PBS buffer, 10 μL lysozyme was added, and dissolved for 2 h. After centrifugation, 10 μL of the supernatant was added to 96-well plate 1 containing 100 μL substrate solution, and reacted at 37°C for 30 min. 100 μL of CCl3COOH solution was added and mixed evenly. Then 12 μL of the mixed solution was added to the 96-well plate, and 48 μL of distilled water, 60 μL of color developer A, 30 μL of color developer B and 60 μL of color developer C were added in sequence, and mixed evenly. The mixture was placed in a constant temperature incubator at 37°C and incubated for 20 min. The absorbance at 630 nm was measured using an enzyme marker, and the enzyme activity was calculated according to the enzyme activity determination method of Example 7.

[0111] Note:

[0112] PBS (0.176 M phosphate buffer, pH = 7.0): PBS-1: weigh 0.176 mol of Na2HPO4·12H2O, add pure water to dissolve, and dilute to 1 L; PBS-2: weigh 0.176 mol of NaH2PO4·2H2O, add water to dissolve, and dilute to 1 L, and slowly add PBS-1 to PBS-2 until the pH is adjusted to 7.0.

[0113] CBZ-Gn-Gly (enzyme reaction substrate) solution: weigh 0.337 g CBZ-Gln-Gly, add pH 7.0 PBS to dissolve, dilute to 100 mL, mix well and use.

[0114] Trichloroacetic acid (TCA) solution: weigh 65.36 g of trichloroacetic acid, dissolve it in pure water, make up to 1 L, mix well and use.

[0115] Color developer A: Weigh 40.06g phenol (melted at 60℃, then weigh), 0.15g sodium nitroprusside, dissolve in pure water, make up to 1L, mix well before use, store at 4℃, away from light.

[0116] Color developer B: Weigh 49.94 g of KOH, dissolve it in pure water, make up to 1 L, mix well and use.

[0117] Color developer C: 200.4g anhydrous K2CO3, 8.33mL NaClO (food grade), dissolve in pure water, dilute to 1L (prepare immediately before use), mix well before use.

[0118] 5. Select mutants with improved enzyme activity. According to the situation in plate 1, select mutants with improved enzyme activity compared to the wild type, inoculate the mutants into the plate, and send bacterial samples for sequencing.

[0119] After the error-prone PCR in the above steps, a mutant with improved enzyme activity was selected, and after sequencing, a mutant containing an amino acid mutation, namely A144G (G C G→G G G), thereby obtaining the PG mutant A144G (SEQ ID NO.3) and its encoding gene pgm (SEQ ID NO.4).

[0120] Example 3: Construction of protein glutaminase recombinant Bacillus subtilis

[0121] 1. Construction of expression plasmid pBSA43-pgm

[0122] pgm and the Bacillus subtilis expression vector pBSA43 were double-digested with BamHI and NotI, and then ligated to construct the recombinant plasmid pBSA43-pgm, which was transformed into Escherichia coli DH5α competent cells, and the positive transformants were selected. The plasmid was extracted for restriction digestion verification and sequencing to confirm that the construction was successful, that is, the recombinant expression plasmid pBSA43-pgm was obtained.

[0123] 2. Transformation of expression plasmid pBSA43-pgm into Bacillus subtilis WB600

[0124] (1) Bacillus subtilis WB600 was activated, streaked in three zones on an antibiotic-free LB plate, and cultured for 12 h;

[0125] (2) Pick a single colony and inoculate it into a test tube containing 5 mL of LB medium and culture it at 37°C and 220 rpm for 12 h;

[0126] (3) At an inoculum size of 2%, 100 μL of seed solution was inoculated into a test tube containing 5 mL of SPI medium and cultured at 37°C, 220 rpm for 3-4 h until the OD 600 =1.2;

[0127] (4) Quickly take 200 μL and inoculate it into 2 mL SPII medium, incubate at 37°C, 100 rpm, and culture for 1.5 h;

[0128] (5) Add 20 μL of 10 mM EGTA, incubate at 37°C, 100 rpm, and incubate for 10 min;

[0129] (6) Add 1-2 μL of recombinant plasmid pBSA43-pgm, culture at 37°C, 100 rpm, and incubate for 30 min. Then adjust the speed to 220 rpm and continue culturing for 1-2 h.

[0130] (7) Transfer the bacterial solution to a sterilized 1.5 mL EP tube, centrifuge at 5000 rpm for 5 min, discard the supernatant, retain 50 μL of culture medium to resuspend the bacteria, and spread the bacterial solution on a plate containing Kan;

[0131] (8) Select transformants, extract plasmids, and perform enzyme digestion verification (see Figure 2 Band 1 in the sample was used to obtain the recombinant Bacillus subtilis strain WB600 / pBSA43-pgm.

[0132] Example 4: Construction of a recombinant strain of protein glutaminase Bacillus amylolyticus

[0133] (1) Preparation of competent cells of Bacillus amyloliquefaciens CGMCC No.11218

[0134] ① Activate the strain, streak three zones on antibiotic-free LB solid medium, and culture at 37℃ for 24h;

[0135] ② Pick a single colony and inoculate it into LBS medium, culture at 37℃, 220rpm for 12h;

[0136] ③Inoculate the seed solution into 100 mL LBS medium at 2% inoculation volume, and culture at 37°C, 220 rpm for 2-3 hours until OD 600 =0.4-0.6;

[0137] ④ Use a low-temperature centrifuge (4°C) at 5000 rpm for 10 min and discard the supernatant;

[0138] ⑤ Resuspend the cells in 30 mL of washing buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol), centrifuge at 5000 rpm for 10 min in a low-temperature centrifuge (4°C), and discard the supernatant;

[0139] ⑥Repeat step ⑤ for a total of 3 washes;

[0140] ⑦ Resuspend the cells in 10 mL buffer (0.5 M sorbitol, 0.5 M mannitol, 10% glycerol, 14% PEG6000);

[0141] ⑧ Aliquot the competent cells into 100 μL tubes and store at -80℃ for later use.

[0142] (2) Electrotransformation of Bacillus amyloliquefaciens

[0143] ① Clean the electric rotating cup with 75% alcohol;

[0144] ② Mix 10 ng of recombinant plasmid pBSA43-pgm and 100 μL of competent medium and transfer to an electroporation cup and place on ice for 2 minutes;

[0145] ③2100-2500V, after 4-6ms of electric shock, immediately add 1mL of recovery solution (LB+0.5M sorbitol+0.38M mannitol), recover at 37℃, 220rpm for 3h, and spread on a plate containing Kan resistance;

[0146] ④ Select transformants, extract plasmids, and perform enzyme digestion verification (see Figure 2 Band 2 in the sample was used to obtain the recombinant Bacillus amyloliquefaciens strain CGMCC No.11218 / pBSA43-pgm.

[0147] Example 5: Expression and preparation of protein glutaminase with improved enzyme activity in recombinant Bacillus subtilis

[0148] 1. The recombinant strain of Bacillus subtilis WB600 / pBSA43-pgm was inoculated into LB liquid medium containing kanamycin (50 μg / mL) and cultured overnight at 37°C and 220 r / min;

[0149] 2. Transfer the inoculum to 50 mL of LB medium at 1% inoculation volume, culture at 37°C, 220 r / min for 48 h, and collect the fermentation supernatant by centrifugation to obtain A144G crude enzyme solution with improved enzyme activity;

[0150] 3. The collected fermentation broth supernatant was first salted out with 25% saturation ammonium sulfate to remove impurities, and then the saturation was increased to 65% to precipitate the target protein. Dissolved with 0.02mol / L Tris-HCl (pH7.0), dialyzed for desalting, and then the active components obtained after dialysis and desalting were loaded onto a cellulose ion exchange chromatography column. The unadsorbed proteins were first eluted with the same buffer, and then gradient eluted with 0.02mol / L Tris-HCl (pH7.0) buffer containing 0.1-1mol / L NaCl to collect the target protein. The active components obtained by ion exchange were first equilibrated with 0.02mol / L Tris-HCl (pH7.0) buffer containing 0.15mol / L NaCl, loaded onto a sephadex g25 gel chromatography column, and eluted with the same buffer at a rate of 0.5mL / min to obtain a purified enzyme solution. The purified enzyme solution was taken for SDS-PAGE analysis to obtain a single band of 35kDa (see Figure 3 The purified enzyme solution was freeze-dried to obtain high-activity A144G enzyme powder.

[0151] Example 6: Expression and preparation of protein glutaminase with improved enzyme activity in Bacillus amyloliquefaciens

[0152] 1. Activate the recombinant strain CGMCC No.11218 / pBSA43-pgm by streaking three zones on a plate;

[0153] 2. Pick a single colony and inoculate it into 50 mL of LB medium containing Kan, and culture it at 37°C and 220 rpm for 12 hours;

[0154] 3. Transfer the inoculum to LB medium containing kanamycin resistance at a 2% inoculum volume and ferment at 37°C and 220 r / min for 48 h.

[0155] 4. Centrifuge at 12000 rpm for 10 min and collect the fermentation supernatant to obtain the crude enzyme solution of PG mutant A144G;

[0156] 5. The fermentation supernatant was collected, and the enzyme protein was precipitated by graded salting-out method using the method of Example 6. The protein precipitate was collected, dissolved, dialyzed for desalination, and then subjected to ion exchange chromatography and gel chromatography to obtain the eluted enzyme solution. The purified enzyme solution was subjected to SDS-PAGE analysis to obtain a single band of 35 kDa (see Figure 3The high-activity protein glutaminase A144G enzyme powder was obtained by vacuum freeze-drying.

[0157] Example 7: Determination of protein glutaminase activity

[0158] 1. Principle of protein glutaminase activity assay

[0159] Protein glutaminase can specifically hydrolyze the amide group on the glutamine residue in protein to release free NH4 + NH4 + Under the catalytic action of sodium nitroprusside, sodium hypochlorite is used as an oxidant to react with phenol to generate a blue-green indigo blue dye, which has a specific absorption effect on the spectrum with a wavelength of 630nm. 630 It can reflect the relative activity of protein glutaminase.

[0160] 2. Protein glutaminase activity determination method and steps used in the present invention

[0161] Experimental group: Take 10 μL of enzyme sample solution (adjust pH = 7.0), preheat in a 37°C water bath for 1 min, then add 100 μL of substrate solution that has been preheated at 37°C for 10 min, incubate the mixture at 37°C for 30 min, then add 100 μL of CCl3COOH solution, mix well, pipette 12 μL of the mixture into a 96-well plate, then add 48 μL ddH2O, 60 μL of color developer A, 30 μL of color developer B and 60 μL of color developer C in sequence, mix well, place in a 37°C constant temperature incubator and incubate for 20 min, and measure the absorbance at 630 nm with an enzyme marker.

[0162] Control group: Take 10μL of enzyme sample solution and add it to the test tube, preheat at 37℃ for 1 min; then add 100μL of 400mM CCl3COOH solution that has been preheated in a 37℃ water bath for 10min, and incubate the mixture in a 37℃ water bath for 30min. Then add 100μL of substrate solution and mix well. Pipette 12μL of the mixture into a 96-well plate, then add 48μL ddH2O, 60μL color developer A, 30μL color developer B and 60μL color developer C in sequence, mix well, place in a 37℃ constant temperature incubator and incubate for 20min, and measure the absorbance at 630nm with an enzyme marker.

[0163] Note:

[0164] PBS (0.176 M phosphate buffer, pH = 7.0): PBS-1: weigh 0.176 mol of Na2HPO4·12H2O, dissolve it in pure water, and make up to 1 L; PBS-2: weigh 0.176 mol of NaH2PO4·2H2O, dissolve it in water, and make up to 1 L. Slowly add PBS-1 into PBS-2 until the pH is adjusted to 7.0.

[0165] CBZ-Gln-Gly (enzyme reaction substrate) solution: weigh 0.337 g CBZ-Gln-Gly, add pH 7.0 PBS to dissolve, dilute to 100 mL, mix well and use.

[0166] Trichloroacetic acid (TCA) solution: weigh 65.36 g of trichloroacetic acid, dissolve it in pure water, make up to 1 L, mix well and use.

[0167] Color developer A: Weigh 40.06g phenol (melted at 60℃, then weigh), 0.15g sodium nitroprusside, dissolve in pure water, make up to 1L, mix well before use, store at 4℃, away from light.

[0168] Color developer B: Weigh 49.94 g of KOH, dissolve it in pure water, make up to 1 L, mix well and use.

[0169] Color developer C: 200.4g anhydrous K2CO3, 8.33mL NaClO (food grade), dissolve in pure water, dilute to 1L (prepare immediately before use), mix well before use.

[0170] 3. NH4 + Standard solution:

[0171] Weigh 0.314g NH4Cl (dried in an oven for 1h), add ddH2O to dissolve and dilute to 1L, as the ammonia standard mother solution, for later use. Dilute according to the following table to obtain NH4 with different concentration gradients. + Standard solution.

[0172]

[0173] 4.96-well plate determination of NH4 + Standard curve and calculate the slope:

[0174] Take 12 μL of each ammonia standard solution of different concentrations into a 96-well plate, then add 48 μL ddH2O, 60 μL color developer A, 30 μL color developer B and 60 μL color developer C in sequence, mix them evenly, place them in a 37℃ constant temperature incubator and incubate for 20 minutes. Use an enzyme marker to measure the absorbance at 630 nm and draw a graph of NH4 + Standard curve (such as Figure 4 ) and calculated the slope a of the curve to be 0.0073.

[0175] 5. Definition of protein glutaminase activity

[0176] Protein glutaminase activity definition: Unit volume of enzyme solution hydrolyzes dipeptide CBZ-GIn-Gly to release 1 μmol NH4 per minute at 37°C and pH 7.0 + One unit of enzyme activity (U·mL -1 ).

[0177] Enzyme activity formula: Enzyme activity (U) = (AB) × (2.1 / 0.1) / 17.03 / 30 / a

[0178] A: absorbance of the experimental group at 630 nm wavelength;

[0179] B: absorbance of the control group at 630 nm wavelength;

[0180] a: NH4 + The slope of the standard curve.

[0181] 6. The results of enzyme activity determination are shown in the following table (using the purified enzyme solution prepared in Example 5 or 6 as the sample):

[0182]

[0183] Note: 1. The wild-type sample is obtained by constructing a Bacillus subtilis / Bacillus amyloliquefaciens recombinant strain using the wild-type pg gene shown in SEQ ID NO.2 according to the method of Example 3 or 4, and preparing a purified enzyme solution of wild-type protein glutaminase according to the method of Example 5 or 6.

[0184] 2. The protein concentration of the purified enzyme solution was determined using a BCA protein concentration assay kit. The specific enzyme activity was calculated as follows (specific enzyme activity (U / mg) = enzyme activity (U / mL) / protein concentration (mg / mL)): the specific activity of the wild-type PG was 1.8 U / mg, and the specific activity of the PG mutant A144G was 3.1 U / mg (Bacillus subtilis expression system).

[0185] Example 8: Determination of enzymatic properties

[0186] The enzyme liquid purified by the Bacillus subtilis expression system in Example 5 was used as a sample, and the enzyme activity determination method in Example 7 was used to determine the enzymatic properties of the wild-type PG and the mutant A144G (e.g. Figure 5 and Figure 6 As shown, the wild-type sample is to construct a recombinant strain of Bacillus subtilis using the wild-type pg gene shown in SEQ ID NO.2 according to the method of Example 3, and prepare a wild-type protein glutaminase enzyme solution according to the method of Example 5), and the results are as follows:

[0187] (1) Specific activity: The specific activity of wild-type PG was 1.8 U / mg, and the specific activity of PG mutant A144G was 3.1 U / mg.

[0188] (2) Optimum reaction temperature: 37°C.

[0189] (3) Optimum pH: 7.0.

[0190] Example 9: Application of protein glutaminase

[0191] Protein glutaminase can specifically hydrolyze the amide group of glutamine residues in proteins, thereby improving the functional properties of proteins. Therefore, it is considered to have broad application prospects in the food industry.

[0192] In this example, protein glutaminase is used to deamidate casein:

[0193] First, weigh 1g of casein, dilute to 100mL with 200mM phosphate buffer (pH 7.0) to obtain a 1% casein suspension, add protein glutaminase to the 1% casein suspension to make the ratio of enzyme to substrate reach 30U / gram protein, then place the mixed solution in a 37°C water bath for 18h, take samples of different reaction times, take the supernatant after centrifugation, and measure the ammonia content therein (the test of ammonia content refers to Example 7), which is used as an experimental group. At the same time, sulfuric acid is added to 1M in the same volume of 1% casein suspension, reacted at 100°C for 18h, and then the ammonia content in the supernatant is measured, which is the total ammonia content (the test of ammonia content refers to Example 7). Ammonia content in sample / total ammonia content, the percentage obtained is the degree of deamidation.

[0194] The results show (such as Figure 7 ), during the reaction time of 0-18h, the deamidation degree of casein increased rapidly, reaching more than 40% at 2h, and the rate of increase of the deamidation degree became slow during the subsequent reaction time of 2-18h. Finally, at the reaction time of 18h, the deamidation degree reached 52.56%.

[0195] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent. It should be pointed out that, for ordinary technicians in this field, the above-mentioned implementation methods can also be modified, combined and improved without departing from the concept of this patent, which all belong to the protection scope of this patent. Therefore, the protection scope of this patent shall be based on the claims.

Claims

1. A protein glutaminase mutant, characterized in that: The amino acid sequence of the protein glutaminase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the protein glutaminase mutant according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

4.

4. A recombinant plasmid or recombinant strain comprising the coding gene according to claim 2.

5. The recombinant plasmid according to claim 4, characterized in that The expression vector used was pET-28a or pBSA43.

6. The recombinant strain according to claim 4, characterized in that The host cells used were Escherichia coli BL21, Bacillus subtilis WB600, or Bacillus amyloliquefaciens CGMCC No.11218.

7. Use of the recombinant plasmid or recombinant strain according to claim 4 in producing a glutaminase mutant of the protein shown in SEQ ID NO.

3.

8. Use of the protein glutaminase mutant according to claim 1 in hydrolyzing the amide group of glutamine residues in proteins.

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