A plant protein and its application in the preparation of liquid enzyme protectants

Abstract

The present invention discloses a plant protein and its application in the preparation of a liquid enzyme protective agent. The plant protein has the characteristics of improving the thermal stability and enzyme activity of liquid enzymes (especially citrate synthase), and has great advantages in solving the problems that enzyme preparations are easily inactivated at normal ambient temperatures and transportation must be carried out in a low-temperature environment. By mixing the plant protein with the liquid enzyme, the protective agent of the present invention can improve the stability of the enzyme, and further can maintain the activity of the enzyme at room temperature or even at a high temperature, thereby significantly improving the convenience of use and economic benefits. Plant protein can be expressed and purified in vitro by engineered bacteria to achieve simple, low-cost industrial production. The present invention provides a new liquid enzyme protective agent for biomedicine, food processing, environmental protection and related scientific research fields, which has broad application prospects.

Description

(I) Technical field

[0001] The invention relates to a plant protein and application of the plant protein in preparing a liquid enzyme protective agent. (II) Background technology

[0002] Protease preparations have been widely used in various industrial and scientific research fields due to their efficient catalytic ability and specific reaction selectivity. These fields include printing and dyeing, biomedicine, food, environmental protection and scientific research. However, the current problem is that most enzyme preparations are easily inactivated during long-term storage at normal ambient temperature, and the transportation process must also be carried out in a low-temperature environment. This brings troubles to use and increases the overall economic burden.

[0003] In the existing technology of protecting enzyme activity, such as freeze-drying the enzyme into powder and adding stabilizers, although this problem can be alleviated to some extent, it is still difficult to maintain the activity of the enzyme in the required liquid state by existing methods. There are also some methods that add high concentrations of salt in the liquid state of the enzyme and store at low temperatures to maintain the activity of the enzyme, but high concentrations of salt can seriously affect the activity of the enzyme in practical applications, and it is necessary to use physical methods to remove (such as ultrafiltration or dialysis operations) before the enzyme is used, and the operation is relatively cumbersome, which increases time and labor costs. In addition, although the enzyme is made into freeze-dried powder and its stability can be slightly improved, the process of dissolution and recovery may affect the final enzyme activity.

[0004] Therefore, it is an urgent need to develop an effective method to maintain enzyme activity for a long time in liquid, especially at room temperature or even high temperature. (III) Summary of the invention

[0005] The object of the present invention is to provide a plant protein and its application in the preparation of a liquid enzyme protective agent. The plant protein as a protective agent can improve the heat resistance and stability of proteins such as citrate synthase, and the preparation method of the protective agent is simple, low in cost, high in enzyme activity yield, and can be industrially produced.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides a plant protein for a liquid enzyme protectant (to improve the stability and / or enzyme activity of the liquid enzyme), wherein the amino acid sequence of the plant protein is shown in SEQ ID NO. 2 or SEQ ID NO. 4. The present invention obtains two plant proteins capable of improving the activity and stability of citrate synthase by screening, namely, plant protein NDP1 and plant protein NDP1C12, wherein the plant protein NDP1C12 is obtained by deleting nucleotides 1 to 279 of the plant protein NDP1.

[0008] The present invention also provides a gene encoding the plant protein, and the nucleotide sequence of the gene encoding is shown as SEQ ID NO.1 or SEQ ID NO.3.

[0009] The present invention also provides a recombinant vector constructed by the plant protein encoding gene and a recombinant genetic engineering bacterium constructed by the recombinant vector. The recombinant vector uses pET28a as a basic vector, and the recombinant genetic engineering bacterium uses Escherichia coli BL21 (DE3) as a host bacterium.

[0010] The present invention also provides an application of the plant protein in preparing a liquid enzyme protective agent, wherein the liquid enzyme comprises citrate synthase; and the protective agent comprises a thermal stability protective agent or an enzyme activity protective agent.

[0011] Furthermore, the protective agent is a pure enzyme solution extracted by ultrasonically crushing wet bacteria obtained by fermenting and culturing engineered bacteria containing plant protein encoding genes.

[0012] Furthermore, the application is to mix the pure enzyme solution with the liquid enzyme at a protein molar ratio of 0.01-2:1, preferably 1:1.

[0013] Further, the pure enzyme solution is prepared as follows:

[0014] (1) Inducing expression in engineered bacteria: Inoculate engineered bacteria containing plant protein encoding genes (preferably recombinant bacteria E. coli BL21 (DE3)-pET28a-NDP1 and E. coli BL21 (DE3)-pET28a-NDPC12) in LB liquid culture medium and culture at 37°C overnight to obtain a pre-cultured bacterial solution; take the pre-cultured bacterial solution and inoculate it into LB liquid culture medium at an inoculum concentration of 2% by volume, and culture at 37°C, 260 rpm until the OD reaches 0. 600 =0.6; then add IPTG to a final concentration of 0.4 mM, 16°C, 100 rpm for induction for 12 h;

[0015] (2) Ultrasonic disruption: The induced bacterial solution was centrifuged at 5000 rpm for 10 min and the supernatant was removed; the precipitate was resuspended in ddH2O and centrifuged at 5000 rpm for 10 min and the supernatant was removed; the precipitate was resuspended in protein lysis buffer and 100 mM PMSF (phenylmethylsulfonyl chloride) was added to the resuspended solution and allowed to stand on ice for 5 min; ultrasonic disruption was performed at 20 W power, 4 s working time, and 8 s interval for 15 min until the solution was clear; centrifuged at 13,000 g for 10 min and the supernatant was collected; protein lysis buffer: 50 mM Tris-Cl (pH 7.5), 0.1 M KCl, 10% glycerol;

[0016] (3) Protein purification: Use a Ni-NTA gravity purification column, load the supernatant collected in step (2), incubate at 4°C for 10 min, and allow the supernatant to flow out naturally by gravity; first use 1 column volume of Wash buffer to elute until there is no obvious solution residue on the resin, then add Wash buffer again, repeat this step 3-5 times to elute non-specifically bound proteins or impurities; use 1 column volume of Elution buffer to elute the target protein specifically bound to the purification resin, and collect the eluate; each 50 mL of Wash buffer consists of: 49 mL of protein lysis buffer, 1 mL of 1 M imidazole; each 50 mL of Elution buffer consists of: 37.5 mL of protein lysis buffer, 12.5 mL 1M imidazole; the Ni-NTA gravity purification column is preferably a Sangon NO.C006197 gravity protein purification empty column, 20ml, before loading, add an appropriate amount of 95% ethanol to rinse the column and the filter membrane, then add an appropriate amount of ddH2O to wash away the ethanol, and slowly add 3mL of purification resin (Company: Tiandirenhe Ni NTA Beads, Ni-NTA is used for the purification of His-tagged proteins);

[0017] (4) Dialysis: The eluate collected in step (3) was dialyzed overnight using a dialysis bag with a molecular weight cutoff of 14 kDa. The solution in the dialysis bag after overnight dialysis was collected as the pure enzyme solution; dialysis solution: 20 mM HEPES-KOH, pH = 7.5.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0019] The present invention screens out a plant protein that can improve the thermal stability and enzyme activity of liquid enzymes such as citrate synthase. The plant protein can be expressed and purified in vitro using Escherichia coli to achieve industrial production.

[0020] The plant protein and citrate synthase were heat denatured at 48°C, and it was found that the activity of the simple citrate synthase was lost by about 88.76% after heating for three minutes, but the activity of the citrate synthase with the addition of the plant protein NDP1 protective agent was only lost by 35.89%, and under high temperature conditions (48°C), the protective agent was beneficial to improving the stability of the citrate synthase. (IV) Description of the drawings

[0021] Figure 1 , Example 1 Simplified linear map of the recombinant plasmid.

[0022] Figure 2 , Gel electrophoresis of the recombinant plasmid of Example 1; Lane Maker represents TIANGEN MD116.

[0023] Figure 3, sequence alignment results of Example 1; A represents the alignment of the NDP1 database sequence (TUPU) with the actual sequencing (28aNOCUT-2); B represents the alignment of the NDP1C12 database sequence (TUPU) with the actual sequencing (28aCUT12-3); C represents the alignment of the NDP1C6 database sequence (TUPU) with the actual sequencing (28aCUT6-5); D represents the alignment of the NDP1C23 database sequence (TUPU) with the actual sequencing (28aCUT23-3); E represents the alignment of the NDP1C123 database sequence (TUPU) with the actual sequencing (28aCUT123-5).

[0024] Figure 4 , Example 1, 2 Gel electrophoresis of samples before and after plant protein induction and during separation and purification; A represents NDP1, B represents NDP1C12; C and D represent NDP1C6, NDP1C23 and NDP1C123. The red box is the target protein band.

[0025] Figure 5 , Gel electrophoresis diagram of the plant protein after dialysis in Example 2.

[0026] Figure 6 , the effect curve of plant protein on the stability of citrate synthase in Example 3.

[0027] Figure 7 , the effect curve of plant protein on citrate synthase activity in Example 4. (V) Specific implementation methods

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0029] Arabidopsis thaliana (L.) Heynh. used in the embodiments of the present invention is an annual weak herb of the family Cruciferae and the genus Arabidopsis.

[0030] LB liquid medium composition: 10g tryptone (OXOID product number: LP0042), 5g yeast extract (OXOID product number: LP0021), 10g sodium chloride, pH 7.0 (5mol / L sodium hydroxide to adjust pH). LB solid medium is LB liquid medium with 15g / L agar added.

[0031] Example 1. Extraction and identification of plant protein

[0032] 1. Plant total RNA extraction

[0033] Table 1 Kit composition

[0034]

[0035]

[0036] Arabidopsis thaliana total RNA was extracted using a plant total RNA extraction kit (FastPure Plant Total RNA Isolation from Vazyme), and the specific operation was as follows:

[0037] (1) Grind the pre-frozen plant samples into powder in liquid nitrogen, and transfer the powder into a centrifuge tube for later use.

[0038] (2) Pre-add 25 μL of β-mercaptoethanol to 475 μL of Buffer PRLPlus, add the mixed Buffer PRLPlus to the ground powder, shake to mix, centrifuge at 12,000 rpm for 5 min, and collect the supernatant.

[0039] (3) Transfer the supernatant to FastPure gDNA-Filter Column II, centrifuge at 12,000 rpm for 2 min, and collect the filtrate.

[0040] (4) Add 0.5 times the volume of anhydrous ethanol to the filtrate and mix gently by pipetting.

[0041] (5) Transfer the entire mixture to FastPure RNA Column IV, centrifuge at 12,000 rpm for 2 min, and discard the filtrate.

[0042] (6) Add 700 μL of Buffer PRW1 to the centrifuge column and centrifuge at 12,000 rpm for 30 s. Discard the filtrate.

[0043] (7) Add 500 μL of Buffer PRW2 (with anhydrous ethanol) to the centrifuge column, centrifuge at 12,000 rpm for 30 s, and discard the filtrate.

[0044] (8) Repeat step 7.

[0045] (9) Centrifuge at 12,000 rpm for 2 min to remove excess ethanol and previously added solution from the RNA.

[0046] (10) Preheat RNase-free ddH2O in a 65°C water bath for 5 min. Transfer the centrifuge column to a new centrifuge tube, add 40 μL of RNase-free ddH2O to the center of the filter to dissolve the RNA, and centrifuge at 12,000 rpm for 1 min.

[0047] (11) Store the samples at -80°C.

[0048] 2. Reverse transcription

[0049] The operation was performed according to the instructions of FastKing cDNA First-Strand Synthesis Kit of Tiangen Biochemical Technology Co., Ltd.

[0050] (1) Establish a 10 μL gDNA removal reaction system according to Table 2.

[0051] Table 2 gDNA removal reaction system

[0052]

[0053] (2) Prepare the reverse transcription reaction system according to Table 3.

[0054] Table 3 Reverse transcription reaction system

[0055]

[0056] (3) Add the mixed solution of step (2) to the reaction solution of step (1) and mix thoroughly.

[0057] (4) Incubate at 42°C for 15 min.

[0058] (5) Incubate at 95°C for 3 min and place on ice to store the resulting cDNA at low temperature.

[0059] 3. Cloning of plant protein

[0060] (1) Primer design for amplifying target fragments

[0061] Primers were designed according to the DNA sequence of NDP1 protein in the Ncbi database (AT5G02130) and the pet28a plasmid map. Primer design programs include but are not limited to CE Designer, NCBI / Primer-Blast, Snapgene, and Oligo7. The primer sequences are shown in Table 4.

[0062] Table 4. Primers

[0063]

[0064] (2) Cloning of plant proteins

[0065] Using the final product cDNA of reverse transcription as a template, PCR amplification was performed using primers 28a-NDP1-F / 28a-NDP1-R and 28a-NDP1(cut12)-F / 28a-NDP1-R, 28a-NDP1-F / 28a-NDP1(cut6)-R, 28a-NDP1-F / 28a-NDP1(cut23)-R, 28a-NDP1(cut23)-F / 28a-NDP1-R, 28a-NDP1(cut123)-F / 28a-NDP1-R, and the PCR reaction system in Table 5 under the reaction program in Table 6.

[0066] Table 5. PCR reaction system

[0067]

[0068] Table 6. PCR reaction program

[0069]

[0070] Note: The annealing temperature should refer to the Tm value of the upstream and downstream primers. This patent uses (Tm 上游 +Tm 下游 ) / 2, the Tm value data is returned together with the primer synthesis by Youkang Company.

[0071] (3) Recovery of target gene fragments

[0072] Use the TIANGEN-DP209-02 kit shown in Table 7 to recover the gel. The specific operation is as follows:

[0073] 1) Column equilibration step: add 500 μL of equilibration solution BL to the adsorption column CA2 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400 x g) for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0074] 2) Cut the single target DNA band from the PCR amplification product in step (2) from the agarose gel (try to remove the excess part) and put it into a clean centrifuge tube and weigh it.

[0075] 3) Add an equal volume of sol solution PN to the gel block (if the gel weighs 0.1 g, its volume can be regarded as 100 μL, then add 100 μL of sol solution PN), place in a 50°C water bath, and gently turn the centrifuge tube upside down until the gel block is completely dissolved.

[0076] 4) Add the solution obtained in step 3) to the adsorption column CA2 treated in step 1) (the adsorption column is placed in the collection tube), place at room temperature for 2 minutes, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CA2 in the collection tube.

[0077] 5) Add 600 μL of rinse solution PW to the adsorption column CA2, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and place the adsorption column CA2 in the collection tube.

[0078] 6) Repeat step 5).

[0079] 7) Place the adsorption column CA2 back into the collection tube and centrifuge at 12,000 rpm for 2 minutes to remove as much of the rinse solution as possible. Place the adsorption column CA2 at room temperature for 10 minutes.

[0080] 8) Place the adsorption column CA2 in a clean centrifuge tube, add 40 μL of elution buffer EB to the middle of the adsorption membrane, and place at room temperature for 2 minutes. Centrifuge at 12,000 rpm for 2 minutes to collect the DNA solution.

[0081] 9) The DNA solution was stored at -20°C, and the target gene fragments NDP1, NDP1C12, NDP1C6, NDP1C23 (for NDP1C23, the PCR products of two pairs of primers 28a-NDP1-F / 28a-NDP1(cut23)-R and 28a-NDP1(cut23)-F / 28a-NDP1-R were mixed in equal proportions of 1 ul, and PCR was performed again using primers 28a-NDP1-F / 28a-NDP1-R according to Tables 5 and 6, and the fragments were recovered by gel) and NDP1C123.

[0082] Table 7. Composition of TIANGEN-DP209-02 Kit

[0083]

[0084] (4) Connection Reorganization

[0085] The recombinant plasmids in the experiment were cloned using the one-step method. Figure 1 The linear map was used to construct the recombinant plasmid. The linear map of the recombinant plasmid included the promoter, fusion tag and insert fragment, the relative sequence length and position relationship of each element. Both expression vectors contained the lactose operon sequence, and protein expression was induced by IPTG.

[0086] 1) Vector linearization

[0087] pET28a was digested with BamH I using the enzyme digestion system in Table 8 and the reaction program in Table 9 to obtain the linearized vector pET28a.

[0088] Table 8, enzyme digestion system (10 μL):

[0089]

[0090] Table 9, reaction procedure:

[0091]

[0092] 2) Connection reorganization

[0093] The target gene fragment collected in step (2) was mixed with the linearized vector according to Table 10. The optimal amount of cloning vector used in the recombination reaction system was 0.03 pmol, and the optimal amount of insert fragment used was 0.06 pmol (the molar ratio of vector to insert fragment was 1:2). The reaction was carried out at 37°C for 30 min, and the product was stored on ice or at 4°C for standby use. Recombinant plasmids pET28a-His-NDP1, pET28a-His-NDP1C12, pET28a-His-NDP1C6, pET28a-His-NDP1C23 and pET28a-His-NDP1C123 were obtained respectively. For convenience of description, the protein expression products of the plasmids are abbreviated as NDP1, NDP1C12, NDP1C6, NDP1C23 and NDP1C123, respectively.

[0094] Table 10. Recombination system

[0095]

[0096] 4. Conversion

[0097] 1) Thaw the E. coli BL21 (DE3) competent cells (company: TIANGEN) on ice (transfer to ice when the competent cells are slightly thawed).

[0098] 2) According to V 重组产物 :V 感受态细胞 =1:10. Add the recombinant plasmid obtained by ligation and recombination in step 3 to the competent cells, flick the tube wall to mix, and let it stand on ice for 30 minutes. When dispensing competent cells, you need to cut off the front part of the pipette tip to avoid the tip of the pipette tip being too thin to squeeze the competent cells and break them.

[0099] 3) Heat shock at 42°C for 90 seconds and immediately cool on ice for 2-3 minutes.

[0100] 4) Add 1 mL of LB liquid medium without antibiotics and shake at 37°C, 230 rpm for 1 hour. Centrifuge at 12,000 g for 1 minute, remove 900 μL of supernatant, and gently mix the bacterial mass at the bottom of the centrifuge tube with the bacterial solution.

[0101] 5) Preheat the LB solid culture medium plate containing 50 μg / ml kanamycin sulfate (Kan) resistance in a 37°C incubator for 1 hour.

[0102] 6) Pipette the bacterial solution from step 4) onto the preheated plate from step 5) and spread it evenly with a clean and sterile spreader until the surface of the plate is not noticeably wet. Incubate the plate upside down in a 37°C incubator overnight.

[0103] 5. Screening of recombinant bacteria

[0104] Pick up a single colony with uniform shape on the plate after overnight culture in step 4 with a pipette tip, and draw a short line with consistent depth on a blank LB plate with 50μg / ml kanamycin sulfate (Kan) resistance to facilitate screening of the correct clone after culture. The single colony cultured after streaking is then inoculated into 50μg / ml kanamycin sulfate (Kan) resistance LB liquid culture medium as an experimental tube.

[0105] The control tube was added with an equal volume of double distilled water (1 μL) and LB liquid culture medium containing 50 μg / ml kanamycin sulfate (Kan) resistance.

[0106] Incubate overnight at 37°C and 240 rpm. Screen the experimental tubes with significantly weaker light transmittance than the control tubes, extract the bacterial liquid plasmid, and verify the recombinant bacteria.

[0107] 6. Gel electrophoresis detection

[0108] (1) Plasmid extraction

[0109] Alkaline lysis solution I: 50 mM glucose, 25 mM Tris-Cl (pH 8.0), 10 mM EDTA (pH 8.0).

[0110] Alkaline lysis solution II (prepared and used immediately): 0.2N NaOH, 1% SDS.

[0111] Alkaline lysis solution III: 3M potassium acetate, 2M glacial acetic acid.

[0112] TE solution: 10mM Tris-Cl (pH 8.0), 1mM EDTA (pH 8.0).

[0113] 1) Take 1.5 mL of the bacterial solution from the experimental tube and the control tube in step 5 into a 1.5 mL EP tube, centrifuge at 13,000 g, 1 min, 4°C, and remove the supernatant.

[0114] 2) Add 100 μL of alkaline lysis solution I to the precipitate and resuspend by shaking until the suspension becomes milky white and there are no obvious bacterial lumps in the solution.

[0115] 3) Add 200 μL of alkaline lysis solution II and gently invert the tube until the solution becomes slightly clear.

[0116] 3) Add 150 μL of alkaline lysis solution III, invert until the solution is transparent and the protein is agglutinated without flocs, and place the centrifuge tube on ice for 15 min.

[0117] 4) Centrifuge at 13,000g for 2 min and transfer the supernatant to a new clean EP tube.

[0118] 5) Add 2 volumes of anhydrous ethanol, let stand at room temperature for 5 min, centrifuge at 13,000 g for 2 min, discard the supernatant, and leave it open for 1-2 min to allow the ethanol to evaporate.

[0119] 6) Add 1 mL of 70% ethanol to the precipitate, invert it, let it stand for 1 min, centrifuge it at 13,000 g for 2 min, discard the supernatant, and leave it open for 1-2 min to allow the ethanol to evaporate.

[0120] 7) Add 30-50 μL TE solution to dissolve the plasmid, and store the plasmid at -20°C as a sample for nucleic acid electrophoresis.

[0121] (2) Agarose gel electrophoresis detection

[0122] 6×DNA loading buffer: 0.5M EDTA (pH 8.0), 40% glycerol, 0.05g bromophenol blue, 0.05g xylene cyanol. After fully dissolved, add sterile water to make up to 100ml and store at 4℃.

[0123] 50×TAE: 242 g Tris, 57.1 mL glacial acetic acid, 37.2 g EDTA, 800 mL deionized water.

[0124] 1) Dissolve 0.6 g of agarose in 60 mL of 1×TAE, mix the gel solution in a conical flask, heat it in a microwave oven for 1 min-2 min until there is no obvious gel-like solid in the solution, then let it stand and cool to 50-60°C, add 6 μL of nucleic acid dye RedGel to the gel solution, mix well, pour into a gel casting machine, and gel for 45 min.

[0125] 2) Place the gel block in the electrophoresis tank, pour TAE until the gel is submerged, fill the gel loading wells with TAE, and rinse the gel wells with TAE.

[0126] 3) Take 10 μL of sample, add 1 μL of 6× DNA loading buffer, mix well and load. DNA Maker uses TianGen's 15000+2000bp maker, take 5 μL of sample, and run electrophoresis at 120 V for 40 min.

[0127] 4) Put the finished gel into the gel imaging instrument to observe the bands. The results are shown in Figure 2 As shown, the results showed that the plasmid extracted from the screened single colony was roughly the correct size and constructed correctly, and the positive bacteria were sent for sequencing.

[0128] 7. Sequencing and alignment

[0129] The bacterial solution of the recombinant bacteria whose band positions were preliminarily verified to be in accordance with the expectations by gel electrophoresis in step 6 was sequenced (sequencing was completed by Hangzhou Youkang Technology Co., Ltd.), and the nucleotide sequence of NDP1 was shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein was shown in SEQ ID NO.2; the nucleotide sequence of NDP1C12 was shown in SEQ ID NO.3 (SEQ ID NO.3 was obtained by deleting 1-279bp in SEQ ID NO.1), and the amino acid sequence of the encoded protein was shown in SEQ ID NO.4; the nucleotide sequence of NDP1C6 was shown in SEQ ID NO.5 (SEQ ID NO.5 was obtained by deleting 670-1155bp in SEQ ID NO.1), and the amino acid sequence of the encoded protein was shown in SEQ ID NO.6; the nucleotide sequence of NDP1C23 was shown in SEQ ID NO.7 (SEQ ID NO.7 was obtained by deleting 178-393bp in SEQ ID NO.1), and the amino acid sequence of the encoded protein was shown in SEQ ID NO.8; the nucleotide sequence of NDP1C123 was shown in SEQ ID NO.9 (SEQ ID NO.9 was obtained by deleting SEQ ID NO.1 The amino acid sequence of the encoded protein is shown in SEQ ID NO.10. The returned sequencing results were compared with the nucleotide sequence information of NDP1 on Ncbi (AT5G02130) using CLUSTALW and ESPript 3.0. The comparison results are shown in Figure 3 As shown, the sequence is correct, with no base mismatch or deletion.

[0130] After identification, the recombinant bacteria E. coli BL21 (DE3)-pET28a-NDP1 and E. coli BL21 (DE3)-pET28a-NDPC12, E. coli BL21 (DE3)-pET28a-NDP1C6, E. coli BL21 (DE3)-pET28a-NDP1C23, and E. coli BL21 (DE3)-pET28a-NDP1C123 were finally obtained.

[0131] Example 2: Induced expression and purification of plant protein

[0132] The experiment used E. coli BL21 (DE3) (BL21 (DE3) competent cells purchased from TIANGEN) for prokaryotic expression of proteins. The preliminary experiment was a small-scale expression of 50 mL to determine the induction conditions and induction status of the protein. The volume for purification was 250 mL, and the purified protein was used to prepare a liquid enzyme protection preparation.

[0133] 1. Inducible expression of plant proteins

[0134] (1) 10 μL of bacterial solution (or single colony inoculation from a plate) of the recombinant bacteria E. coli BL21 (DE3) -pET28a-NDP1 and E. coli BL21 (DE3) -pET28a-NDPC12, E. coli BL21 (DE3) -pET28a-NDP1C6, E. coli BL21 (DE3) -pET28a-NDP1C23, and E. coli BL21 (DE3) -pET28a-NDP1C123 screened in Example 1 were inoculated into 15 mL of LB liquid culture medium and cultured overnight at 37° C. to obtain pre-cultured bacterial solution.

[0135] (2) Take 5 mL of the pre-cultured bacterial solution in step (1) and inoculate it into 250 mL of LB liquid medium. Cultivate at 37°C and 260 rpm until the OD 600 =0.6, 1 mL of bacterial solution was taken out as the protein control before induction for gel electrophoresis. Figure 4 Middle lane before induction.

[0136] Treatment of bacterial solution: centrifuge at 13,000g for 1 min, remove the supernatant, add 75μL ddH2O and 25μL 4×SDS loading buffer to resuspend, incubate in a 95℃ water bath for 5 min, and place the protein on ice for later use.

[0137] (3) After removing 1 mL of bacterial solution in step (2), add IPTG with a final concentration of 0.4 mM (IPTG is added in the form of a 1 M aqueous solution) to the culture medium, and induce at 16°C and 100 rpm for 12 h.

[0138] Measure the absorbance of the bacterial solution after induction. To make the electrophoresis bands of the protein before and after induction comparable, the sample amount needs to be unified. Considering that the ratio of protein absorbance is approximately equal to the ratio of protein amount, dilute in proportion to obtain 1 mL of bacterial solution and treat the bacterial solution as in step (2). The gel electrophoresis diagram after induction is shown in Figure 4 The middle lane is induced. Figure 4 From A and B, we can see that NDP and NDPC12 protein induction effects are obvious, and the protein is in the supernatant, so subsequent experiments can be carried out. Figure 4As shown in C and D, the induction effect of NDP1C6, NDP1C23 and NDP1C123 proteins was obvious, but the proteins were all expressed in the precipitate and subsequent experiments could not be performed.

[0139] 2. Purification of plant protein

[0140] Protein lysis buffer: 50 mM Tris-Cl (pH 7.5), 0.1 M KCl, 10% glycerol.

[0141] Wash buffer (50 mL): 49 mL protein lysis buffer, 1 mL 1 M imidazole.

[0142] Elution buffer (50 mL): 37.5 mL protein lysis buffer, 12.5 mL 1 M imidazole.

[0143] (1) Dispense 250 mL of the bacterial solution induced in step 1 into six 50 mL centrifuge tubes, balance them, centrifuge at 5000 rpm for 10 min, and remove the supernatant.

[0144] If multiple cumulative subpackaging centrifugation is performed, the subsequent centrifugation conditions are 8000 rpm, 15 min.

[0145] (2) Add 10 mL of ddH2O to the precipitate from step (1) and resuspend. Centrifuge at 5000 rpm for 10 min and remove the supernatant.

[0146] (3) Add 10 mL of protein lysis buffer to the precipitate from step (2) to resuspend it, add 200 μL of 100 mM PMSF (company: Beyotime) to the resuspended solution, and let it stand on ice for 5 min.

[0147] (4) The resuspended solution from step (3) was placed in ice (or an ice-water mixture) for ultrasonic crushing, with the parameters set to a power of 20 W, a working time of 4 s, a rest interval of 8 s, and a total time of 15 min (working time of 5 min) until the solution was clear.

[0148] (5) Centrifuge at 13,000 g for 10 min. Transfer the supernatant to a clean centrifuge tube and store at 4 °C for later use. See the gel electrophoresis diagram for details. Figure 4 The supernatant in the middle lane was collected, and 10 mL of protein lysis buffer was added to the precipitate to resuspend it. The precipitate was stored at -20 °C for future use. The gel electrophoresis diagram is shown in Figure 4 Medium precipitation.

[0149] (6) Take a clean Ni-NTA gravity purification column (Shenggong NO.C006197 gravity protein purification empty column, 20ml), add an appropriate amount of 95% ethanol to rinse the column and filter membrane. Add an appropriate amount of ddH2O to the purification column to wash away the ethanol. Slowly add 3mL of purification resin (Company: Tiandirenhe Ni NTA Beads, Ni-NTA is used for purification of His-tagged proteins) to the purification column, and let the outflowing liquid phase stand to stabilize the bed volume (about 1.5mL).

[0150] (7) Evenly and slowly add 2 times the volume of the resin into the purification column. Repeat 4-6 times.

[0151] (8) Place a bottom stopper on the bottom of the purification column, slowly add 10 mL of the supernatant obtained in step (5) into the purification column, and place the purification column on a shaker and incubate at 4°C for 10 min.

[0152] (9) Remove the bottom plug and allow the supernatant to flow out naturally by gravity. Collect the naturally flowing solution and store it at 4°C for later use. This is called flow-through. The gel electrophoresis diagram is shown in Figure 4 The middle lane flows through.

[0153] *In order to obtain a higher purification rate, the naturally eluted solution can be added back into the column and passed through the column again to increase the binding rate between the purification resin and the target protein.

[0154] (10) Slowly add 2 mL of Wash buffer to the purification column until no obvious solution remains on the resin, then add Wash buffer again. Repeat this step 3-5 times to elute nonspecifically bound proteins or impurities. Collect the effluent from the last wash and store it at 4°C for later use.

[0155] (11) Add 2 mL of elution buffer to the purification column to elute the target protein that is specifically bound to the purification resin. Collect the elution solution in a pre-cooled 2 mL centrifuge tube.

[0156] (12) Repeat step (11) 3 times. There are 3 tubes of eluted products, recorded as elution 1, elution 2, and elution 3. The gel electrophoresis diagram is shown in Figure 4 The elutions 1, 2 and 3 in the middle lanes are combined to obtain the purified NDP1 protein solution and the purified NDPC12 protein solution.

[0157] (13) Add 10% glycerol to the eluted product and store the product at -20°C.

[0158] *During the protein purification process, all buffers must be placed on ice or at a low temperature to reduce protein degradation. The volume of buffer used for washing medium is 2-4 times the volume of the medium. Since we use a gravity purification column, we need to ensure that each added liquid resuspends the purification medium during purification, so that the washing is sufficient and the target protein can be effectively bound to the medium.

[0159] 3. SDS-PAGE

[0160] (1) Preparation of electrophoresis gel

[0161] The gel was prepared using Vazyme's One-Step PAGE Gel Fast Preparation Kit (12%).

[0162] Table 11, lower layer 12% separation gel

[0163]

[0164] Table 12, upper concentrated gel

[0165]

[0166] 1) Mix all components by inversion 6-8 times before use.

[0167] 2) Preparation of lower layer gel: Take equal volumes of Resolver A and Resolver B, 2.7 ml each, and mix well.

[0168] 3) Preparation of the top layer: Take equal volumes of Stacker A and Stacker B, 0.75 ml each, and mix well.

[0169] 4) Add 60 μL of APS to the mixed solution of step 2), mix thoroughly immediately, and then inject into the glass plate so that the liquid level is about 1.5 cm from the upper edge of the short glass plate.

[0170] 5) Add 15 μL of APS to the mixed solution in step 3) and mix thoroughly immediately. Without waiting for the lower layer of gel to solidify, the mixed solution can be gently injected into the glass plate for making gel and the comb teeth are gently inserted.

[0171] 6) After the gel solidifies (about 15 minutes at room temperature), remove the comb teeth and proceed with electrophoresis.

[0172] (2) Reagent preparation

[0173] 1) 10× Tris-glycine electrophoresis buffer: 15 g Tris-HCl, 72 g glycine, 1 g SDS, add water to make up to 500 mL.

[0174] 2) Protein 4× loading buffer (10 mL): 200 mM Tris-HCl (pH 6.8), 40% glycerol, 0.8 g SDS, 400 mM DTT, 0.04 g bromophenol blue.

[0175] 3) Protein decolorizing solution: 500 mL methanol, 100 mL glacial acetic acid, add water to make up to 1 L.

[0176] 4) Coomassie staining solution: 0.25 g Coomassie brilliant blue, 100 mL protein destaining solution.

[0177] (3) Processing of protein samples

[0178] 1) The loading control group is the total bacterial solution before induction and the total bacterial solution after induction. 600 = 0.6, aspirate 1 mL of the uninduced bacterial solution, centrifuge at 1,2000 g, 4°C for 1 min, and discard the supernatant.

[0179] 2) Add 75μL ddH2O, 25μL 4×SDS loading buffer, vortex and resuspend, place in a 95℃ water bath for 5min, let stand at room temperature for 1-2min, and then store at -20℃. (You can load the sample directly, centrifuge at 1,2000g for 30s before loading, and take the supernatant when loading the sample).

[0180] 3) The eluted protein is compared with the protein before elution (the concentration of the induced band after the column is compared to roughly determine the gel binding specific protein), take 12μL of protein solution and add 4μL of 4×SDS loading buffer, mix well, bathe at 95℃ for 5min, stand at room temperature for 1-2min, and then store at -20℃. (Can be loaded directly)

[0181] (4) Rubber sheet assembly and electrophoresis

[0182] 1) Place the thin glass plate and thick plate for glue making close together and press them tightly with a glue making machine. Add distilled water between the glass plates and check for leaks for 10 minutes.

[0183] 2) Use a paper towel to absorb the moisture between the glass plates, prepare the separation gel and concentrated gel according to the recipe, use a pipette to slowly add the separation gel between the glass plates to 1-2 cm from the top of the glass plate, add concentrated gel until the liquid surface is saturated, slowly insert a suitable gel comb between the glass plates, and gel for 1 hour.

[0184] 3) Place the prepared gel plate in a vertical electrophoresis tank (if there is only one protein gel, use another plastic plate to assist in clamping it), and pour protein electrophoresis buffer into the electrophoresis tank to the top of the glass plate. Slowly pull out the comb and rinse the sample tank with buffer until there is no obvious gel residue in the tank.

[0185] 4) Electrophoresis time and voltage: 180V for 1 hour. After electrophoresis, the gel sheet was pried open with a plastic scraper, cut and stained with 10mL Coomassie Brilliant Blue. After staining for 2-3 hours, the stain on the gel surface was washed off with protein destaining solution, and photographed for observation.

[0186] 5) The electrophoresis results are as follows Figure 4 As shown, the protein size is correct, the concentration of the purified target protein is high, there are fewer impurity bands, and the effect is good.

[0187] 4. Protein dialysis and protein quantification

[0188] (1) Reagent preparation and purchase

[0189] Dialysate: 4 L 20 mM HEPES-KOH, pH = 7.5.

[0190] Citrate synthase enzyme solution: Abbreviation: CS, from pig heart, sigma, product number: C3260.

[0191] (2) Dialysis bag treatment

[0192] 1) Cut three 10-20 cm economical dialysis bags (14 kDa company: BBI), boil the dialysis bags in 500 mL of an aqueous solution containing 20 mg / L sodium bicarbonate and 1 mmol / L EDTA-2Na (pH 8.0) for 10 min, and rinse thoroughly with ddH2O.

[0193] 2) Place it in 500 mL of 1 mmol / L EDTA-2Na (pH 8.0) aqueous solution and boil it for 10 min. Take it out and thoroughly wash the dialysis bag with ddH2O.

[0194] (3) Dialysis

[0195] 1) Fold the bottom 1 cm of the dialysis bag in half and clamp it with a dialysis bag sealing clip. Use a pipette to add an appropriate amount of ddH2O to the dialysis bag and shake it slightly to check for leaks. If there is no obvious liquid leakage from the seal, suck out the ddH2O in the dialysis bag and exhaust the air in the dialysis bag.

[0196] 2) Use a pipette to add the citrate synthase solution and the NDP1 and NDPC12 protein solutions purified in step 2 into the dialysis bag respectively, clamp the upper end with a sealing clip, and slightly turn it upside down to ensure that no liquid leaks.

[0197] 3) Place 1500 mL of dialysate in a 2000 mL beaker and place it on a magnetic stirrer for dialysis (dialysis temperature 4°C). Replace the dialysate after 3 hours of dialysis, pour 1500 mL of dialysate into the beaker again, stir overnight and replace the dialysate again. At noon on the second day, the liquid in the bag was sucked out separately, stored in a 10 ml centrifuge tube, and stored at -20 degrees, and recorded as plant protein NDP1 pure enzyme solution, plant protein NDP1C12 pure enzyme solution and citrate synthase (CS) pure enzyme solution.

[0198] Note: The purpose of dialysis of NDP1 and NDP1C12 is to dialyze away imidazole, glycerol and small molecular weight impurities; the purpose of dialysis of citrate synthase is to remove high concentration of (NH4)2SO4 to avoid affecting subsequent experimental results.

[0199] The dialyzed protein (10 μL of protein solution was taken from each dialysis bag, 2.5 μL of 5xSDS loading buffer was added, and SDS-PAGE electrophoresis was performed. Maker (Vazyme MP102) and COL protein (lane 2) were used as controls. The results are shown in Figure 5 shown.

[0200] (4) Protein quantification

[0201] The BSA protein concentration of the pure enzyme solution was determined using the BCA protein quantification kit (PC0020). The results are shown in Table 13.

[0202] Table 13. Protein concentration

[0203]

[0204] Example 3: Effects of plant proteins NDP1 and NDP1C12 on the stability of citrate synthase (CS)

[0205] (1) Experimental materials and reagent preparation

[0206] 2×buffer_2: 40 mM HEPES-KOH, 2.8 mM β-mercaptoethanol, pH=7.5.

[0207] NDP1: the plant protein NDP1 pure enzyme solution prepared in Example 2, with a protein concentration of 0.3580 μg / μL.

[0208] NDP1C12: the pure enzyme solution of plant protein NDP1C12 prepared in Example 2, with a protein concentration of 0.2321 μg / μL.

[0209] CS: pure citrate synthase (CS) enzyme solution prepared in Example 2, with a protein concentration of 3.0080 μg / μL.

[0210] (2) Experimental groups and procedures

[0211] Add 75 μL 2×buffer_2 to each well of a 96-well plate, and then add components of each final concentration according to the groups in Table 14, and then add ddH2O to 150 μL. React at 48°C for 90 minutes, and use a multifunctional microplate reader to detect the absorbance at a wavelength of 340 nm every 1 minute, for a total of 91 times, and calculate the ΔOD value (the difference between the absorbance at the corresponding time and the minimum absorbance of the group). The results are shown in Figure 6 As shown, the stability of citrate synthase alone at 48°C is poor; the addition of NDP1C12 improves the stability of citrate synthase to a certain extent; after adding NDP1, the stability of citrate synthase is greatly improved.

[0212] Table 14. Experimental groups and final concentrations

[0213]

[0214] Note: Because different proteins have different sizes, the same mass contains different molar numbers of protein. We believe that the protective effect should be related to the ratio of protein moles. Therefore, the final molar concentration should be noted here, and the final volume, mass, molar number and molecular weight should be converted.

[0215] Example 4: Effects of plant proteins NDP1 and NDP1C12 on citrate synthase (CS) activity

[0216] (1) Experimental materials and reagent preparation

[0217] 2×buffer_2, NDP1, NDP1C12, CS are the same as in Example 3. 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB, sigma D8130, 500 mg), acetyl coenzyme A (sigma A2056, 5 mg), oxaloacetate (sigma O4126, 1 g).

[0218] (2) Experimental groups

[0219] Table 15. Experimental groups and final concentration of protein added to each component

[0220]

[0221] Table 16. Final molar concentration of proteins added in different experimental groups (kDa refers to the corresponding enzyme molecular weight)

[0222]

[0223] (3) Experimental methods

[0224] Add 15 μL of 2×buffer_2 to each of the 5 1.5 mL EP tubes, then add the components of each final concentration according to Table 15, and finally add ddH2O to 30 μL. Take out 3 μL from each EP tube as the protein samples of different groups at 0 min, repeat three times, and reduce 9 μL in total from each EP tube.

[0225] The remaining samples in each EP tube were incubated together in a metal incubator at 48°C for 3 min, and recorded as 3 min protein samples of different groups.

[0226] Add 1mM DTNB, 0.5mM acetyl-CoA, 1μL of 0 or 3min different groups of protein samples to each well of a 96-well plate, and make up to 90μL with 100mM Tris-HCl (pH 7.5) (Note: the molar concentration here should be calculated based on the final system of 100μL). Vibrate the plate to mix the reaction mixture thoroughly. Measure the absorbance at 412nm every 13s for 2min. This is to exclude background activity. Finally, add 5mM oxaloacetate to make the final system 100μL. At this time, the molar concentration of each component protein in the final system is shown in Table 16, and the reaction is started. DTNB (ε412=14.2mM -1 cm -1 ) was used as the reagent, and the absorbance at 412 nm was measured every 13 s for 5 min using a multifunctional microplate reader preheated to 25 °C.

[0227] According to the Lambert-Beer law, A = εbc (A: absorbance; ε: extinction coefficient; b: path length difference; c: substrate concentration), the specific activity (nmol min) of citrate synthase in different groups at different times when oxaloacetate was used as substrate -1 mg -1 protein), such as Figure 7 As shown, it can be seen that NDP1 itself has no obvious enzyme activity and will not interfere with the reaction of citrate synthase. On the contrary, it is very helpful for the activity of citrate synthase at high temperature. When heated for three minutes, the activity of pure citrate synthase is lost by about 88.76%, but the activity of citrate synthase with the addition of plant protein NDP1 is only lost by 35.89%. The protective effect of NDP1C12 is not very obvious under this condition.

Claims

1. A plant protein for a liquid enzyme protectant, characterized in that: The amino acid sequence of the plant protein is shown as SEQ ID NO.2 or SEQ ID NO.

4.

2. A recombinant genetically engineered bacterium constructed with the gene encoding the plant protein according to claim 1.

3. Use of the plant protein according to claim 1 in preparing a liquid enzyme protective agent, characterized in that: The liquid enzyme is citrate synthase; and the protective agent is a heat stability protective agent.

4. The use according to claim 3, characterized in that The protective agent is a pure enzyme solution extracted by ultrasonically crushing wet bacteria obtained by fermentation and culture of engineered bacteria containing plant protein coding genes.

5. The use according to claim 4, characterized in that The application is to mix the pure enzyme solution with the liquid enzyme at a protein molar ratio of 0.01-2:

1.

6. The use according to claim 4, characterized in that The pure enzyme solution was prepared as follows: (1) Induced expression of engineered bacteria: The engineered bacteria containing the plant protein encoding gene were inoculated into LB liquid medium and cultured at 37°C overnight to obtain a pre-cultured bacterial solution; the pre-cultured bacterial solution was inoculated into LB liquid medium at an inoculum concentration of 2% by volume, and cultured at 37°C and 260 rpm until the OD 600 =0.6; then add IPTG to a final concentration of 0.4 mM, 16°C, 100 rpm for induction for 12 h; (2) Ultrasonic disruption: The induced bacterial solution was centrifuged at 5000 rpm for 10 min and the supernatant was removed; the precipitate was resuspended in ddH2O and centrifuged at 5000 rpm for 10 min and the supernatant was removed; the precipitate was resuspended in protein lysis buffer and 100 mM phenylmethylsulfonyl chloride was added to the resuspended solution and the solution was placed on ice for 5 min; ultrasonic disruption was performed at a power of 20 W, a working time of 4 s, and an interval of 8 s for 15 min until the solution was clear; centrifuged at 13,000 g for 10 min and the supernatant was collected; protein lysis buffer: pH 7.5, 50 mM Tris-Cl, 0.1 M KCl, 10% glycerol; (3) Protein purification: Use a Ni-NTA gravity purification column, load the supernatant collected in step (2), incubate at 4°C for 10 min, and allow the supernatant to flow out naturally by gravity; first elute with 1 column volume of wash buffer until no obvious solution remains on the resin, then add wash buffer again, and repeat this step 3-5 times to elute non-specifically bound proteins or impurities; use 1 column volume of elution buffer to elute the target protein specifically bound to the purification resin, and collect the eluate; each 50 mL of wash buffer consists of: 49 mL of protein lysis buffer, 1 mL of 1 M imidazole; each 50 mL of elution buffer consists of: 37.5 mL of protein lysis buffer, 12.5 mL of 1 M imidazole; (4) Dialysis: The eluate collected in step (3) is dialyzed overnight using a dialysis bag with a molecular weight cutoff of 14 kDa, and the solution in the dialysis bag after overnight dialysis is collected as the pure enzyme solution; Dialysate: 20 mM HEPES-KOH, pH=7.5.

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