A method for purifying a wheat-derived protein disulfide isomerase and its application in processing of plant proteins
Patent Information
- Application Number
- CN202510293081.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2026-09-15
Smart Images
Figure CN122750656A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biochemistry, and in particular to a method for purifying wheat-derived protein disulfide isomerase and its application in plant protein processing. Background Technology
[0002] Protein disulfide isomerases (PDIs), members of the thioredoxin family, are widely distributed in the endoplasmic reticulum of various eukaryotes, playing multiple enzymatic roles. They exhibit diverse biological functions, including: promoting disulfide bond formation during protein synthesis through oxidation; recognizing and correcting mispaired disulfide bonds, performing isomerase rearrangement activity; and acting as molecular chaperones, guiding protein molecules along the correct folding pathways to ensure the precision and functionality of protein structure. Wheat protein disulfide isomerase (wPDI) is mainly composed of four domains (a, b, b', a') and a c-terminus. Its a and a' domains contain CXXC active sites, which interact with the sulfhydryl groups of the substrate, promoting substrate cross-linking.
[0003] Currently, the market potential for plant-based meat analogues is growing due to increasing consumer interest in sustainable alternatives to meat products. The success of plant-based meat depends on its ability to mimic various characteristics of real meat. Soy protein isolate is considered an ideal choice for producing plant-based meat due to its good processing properties, balanced nutritional value, and relatively low price. Specifically, soy protein isolate has a predominantly β-sheet secondary structure and a spherical shape; therefore, its structural properties can be altered through texturization techniques to obtain plant-based meat products with a fibrous structure similar to traditional meat products.
[0004] Current strategies for improving the quality of plant-based meat products primarily focus on controlling processing parameters (such as moisture content, thermomechanical processing temperature, and shear stress) to improve product texture by optimizing the fibrosis process. However, simply relying on physical parameter adjustments cannot fundamentally overcome the limitations of the functional properties of plant proteins. In contrast, protease-based biomodification technology offers a new path for upgrading the quality of plant-based meat products. Enzymatic modification, through precise control of protein molecular structure, not only holds promise for solving various problems related to texture simulation and nutritional fortification in plant-based meat products, but also aligns with the needs of green manufacturing and industrialization.
[0005] Currently, the most common enzyme cross-linking agent on the market is transglutaminase (TG), which promotes the formation of a stable protein network in soybean protein through ε-(γ-glutamyl)lysine covalent cross-linking between glutamine and lysine residues, thereby improving the texture of protein products. Summary of the Invention
[0006] The purpose of this invention is to provide a method for purifying wheat-derived protein disulfide isomerase and its application in plant protein processing.
[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0008] This invention provides a method for purifying wheat-derived protein disulfide isomerases, comprising the following steps:
[0009] (1) Transform the pET-30b-wPDI recombinant plasmid with His tag into Escherichia coli and induce the expression of recombinant wPDI protein in Escherichia coli with an inducer.
[0010] (2) The induced Escherichia coli obtained in step (1) was broken up, and the supernatant containing recombinant wPDI protein was collected after solid-liquid separation.
[0011] (3) Mix the supernatant obtained in step (2) with the column material of the Ni-NTA affinity chromatography column, transfer the mixture to the gravity column, and after the sample flow through the liquid has finished, add the equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D and elution buffer E in sequence for elution.
[0012] The equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D, and elution buffer E contain imidazole at concentrations of 8–12 mmol / L, 15–25 mmol / L, 40–60 mmol / L, 80–120 mmol / L, 130–170 mmol / L, and 200–300 mmol / L, respectively.
[0013] (4) After concentrating the eluent obtained in step (3), it is purified by anion exchange column to obtain purified wheat-derived protein disulfide isomerase.
[0014] The anion exchange buffer A used contains phosphate at a concentration of 15–25 mmol / L and NaCl at a concentration of 40–60 mmol / L, while the anion exchange buffer B contains phosphate at a concentration of 15–25 mmol / L and NaCl at a concentration of 800–1200 mmol / L.
[0015] Preferably, the Escherichia coli in step (1) is Escherichia coli BL21, and the inducer is IPTG.
[0016] Preferably, the crushing in step (2) is ultrasonic crushing, the ultrasonic crushing power is 400-800W, the ultrasonic crushing is paused for 3-7 seconds every 2-4 seconds, and the ultrasonic time is 25-35 minutes;
[0017] The solid-liquid separation process is carried out at a temperature of 3–5°C, a speed of 10,000–15,000 rpm, and a time of 25–35 min.
[0018] Preferably, the supernatant is filtered through a 0.22 μm filter before mixing in step (3), and the mixing temperature is 3-5 °C and the time is 20-40 min.
[0019] Preferably, the equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D and elution buffer E mentioned in step (3) also contain Tris-HCl with a concentration of 40-60 mmol / L and NaCl with a concentration of 300-600 mmol / L, respectively;
[0020] During elution, the amounts of equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D, and elution buffer E added are 50–70 mL, 40–60 mL, 40–60 mL, 30–50 mL, 20–40 mL, and 20–40 mL, respectively.
[0021] Preferably, the concentration in step (4) is achieved by centrifugation using an ultrafiltration tube, wherein the ultrafiltration tube has a molecular weight cutoff of 8 to 12 kDa.
[0022] Before purification via an anion exchange column, the concentrated eluent is mixed with anion exchange buffer A and centrifuged 1–3 times, then filtered through a 0.22 μm filter. The centrifugation concentration is performed at a temperature of 3–5 °C, a speed of 4000–8000 rpm, and a time of 20–30 min.
[0023] Preferably, in step (4), anion exchange buffer A is eluted at a flow rate of 1.5 to 2.5 mL / min for 10 to 20 CVs, and anion exchange buffer B is slowly increased from 0% to 100% and eluted in a linear elution manner for 15 to 25 CVs.
[0024] This invention provides an application of wheat-derived protein disulfide isomerase purified according to the method in plant protein processing. By adding the wheat-derived protein disulfide isomerase during the plant protein production process, the fiber formation rate of plant protein can be improved.
[0025] Preferably, the plant protein is soybean protein, and the amount of wheat-derived protein disulfide isomerase added is 0.05-0.2%.
[0026] Preferably, soybean protein is produced by high-moisture extrusion. During production, soybean protein and wheat starch are mixed at a mass ratio of 8.5–9.5:0.5–1.5 and extruded at a feed rate of 6–10 kg / h (dry basis), a moisture content of 50–70% (dry basis), and a screw speed of 200–300 rpm. The extruder barrel temperature in zones one through five is 50–70°C, 70–90°C, 140–150°C, 145–155°C, and 125–135°C, respectively.
[0027] Compared with the prior art, the present invention has the following beneficial effects:
[0028] This invention provides a method for purifying wheat-derived protein disulfide isomerase. High-purity wheat-derived protein disulfide isomerase is purified using a combination of Ni column affinity chromatography and anion exchange chromatography. The purified wheat-derived protein disulfide isomerase is then added to soy protein isolate, and protein fibers are produced through high-moisture extrusion. Results show that the addition of the wheat-derived protein disulfide isomerase effectively enhances the fiber content of the protein, thereby improving the texture and nutritional value of the extrudate, with a superior effect compared to traditional Tg enzymes.
[0029] Wheat-derived protein disulfide isomerase, as a natural protein derived from wheat, can reduce the safety risks in the production of enzyme products and is expected to be developed into a natural enzyme preparation. Attached Figure Description
[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0031] Figure 1 The result is the purified wPDI by Ni column affinity chromatography in Example 2;
[0032] Figure 2 The results of anion exchange chromatography using wPDI in Example 2;
[0033] Figure 3 The results of SDS-PAGE analysis of the eluent near the peak position in Example 2;
[0034] Figure 4 The results of the study on the optimal addition amount of wPDI and Tg enzyme in the production of soy protein isolate in Example 3;
[0035] Figure 5The results of observation under a laser confocal microscope show the soybean protein isolates obtained from different treatments in Example 3. Detailed Implementation
[0036] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0037] Example 1
[0038] Based on the pET-30b-wpdi recombinant plasmid provided by Professor Hu Songqing's research group at South China University of Technology, a pET-30b-wPDI recombinant plasmid with a His tag was constructed. The construction process involved the following modifications to the template provided by Professor Hu Songqing (Zhang Tingting. Expression and Properties of Wheat Protein Disulfide Isomerases and Their Effects on Flour Quality [D]. South China University of Technology, 2014):
[0039] Insert “MHHHHHHGSG(SEQ ID NO.1)” before P50-L538, and amplify the target fragment using PCR:
[0040] Upstream primer: gtggtggtggtggtgctcgagCTAGAGCTCGTCCTTCAGAGGCTC (SEQ ID NO.2)
[0041] Downstream primer: taagaaggagatatacatatgCACCACCACCACCACCACGGTTCTG GTCCA (SEQ ID NO.3)
[0042] PCR amplification program: After pre-denaturation at 95℃ for 3 min, perform 34 cycles of the following settings: denaturation at 95℃ for 15 s, annealing at melting temperature (Tm) for 15 s, and extension at 72℃ for 90 s. Finally, proceed to the total extension stage: extension at 75℃ for 5 min. After the PCR program is complete, store the product at -20℃ for later use.
[0043] Expression vector construction process: Constructed using overlap extension PCR. 0.5 U of DpnI digestive enzyme was added to the obtained PCR product, and digestion was performed at 37℃ for 60 min to completely digest the methylated His-tagged pET-30b-wPDI plasmid template. For the His-tagged pET-30b-wPDI recombinant plasmid, homologous recombination was constructed using a kit. PCR amplification products were detected by 2% agarose gel electrophoresis at 140V for 30 min. After electrophoresis, the target gene band was excised using a scalpel and the target fragment was recovered using an agarose gel extraction kit. The expression vector pET-30b(+) was treated with double restriction endonucleases to linearize the vector. The double restriction sites were NdeI and XhoI, and the digestion conditions were 37℃ for 30 min. Immediately after double digestion, the restriction endonucleases were heat-inactivated at 65℃ for 30 min. The concentration of the target fragment in the recovered product was measured using the double-stranded DNA concentration measurement mode of an ultra-micro spectrophotometer. The amounts of linearized vector and target fragment added to the recombinant reaction system were calculated using CEDesign software. 55 ng of the amplified target fragment and 110 ng of the linearized vector were added to the recombinant reaction buffer, along with 2 μL of recombinant ligase (Exnase II), 4 μL of reaction buffer, and sterile deionized water, to construct a 20 μL recombinant reaction system. After reacting at 37°C for 30 min, the centrifuge tube containing the reaction product was immediately placed on ice to obtain the His-tagged pET-30b-wPDI recombinant plasmid.
[0044] Example 2
[0045] A method for purifying a wheat-derived protein disulfide isomerase, comprising the following steps:
[0046] 1. Transformation of recombinant plasmids
[0047] Take 50 μL of E. coli BL21(DE3) competent cells and thaw them on ice. Add 2 μL of the His-tagged pET-30b-wPDI recombinant plasmid constructed in Example 1 to the thawed competent cells, mix well, and incubate on ice for 30 min. Then heat shock in a 42°C water bath for 45 s, followed by an ice bath for 3 min.
[0048] Add 200 μL of LB liquid medium to the above mixed solution in a clean bench and incubate at 200 rpm and 37°C for 30 min. Spread the resulting bacterial culture evenly onto LB solid medium containing 50 mg / L kanamycin sulfate, invert the medium, and incubate overnight at 37°C.
[0049] 2. Induction of recombinant wPDI protein expression in Escherichia coli
[0050] This invention selects Escherichia coli BL21(DE3) to express the target protein. This bacterium contains a gene encoding T7 RNA polymerase. IPTG is used to induce the expression of T7 RNA polymerase. The polymerase acts on the T7 promoter, thereby promoting the expression of downstream exogenous target fragments. This method is suitable for the expression of non-toxic proteins.
[0051] Pick a single colony and add it to 20 mL of LB liquid medium. Add 10 μL of Kan stock solution and incubate overnight at 37°C and 200 rpm. Take 1 mL of the bacterial culture and add it to 400 mL of LB liquid medium. Add 200 μL of Kan stock solution and incubate at 37°C and 220 rpm until the OD600 is about 0.6 to 0.8. Add 200 μL of LPTG stock solution to the bacterial culture and incubate overnight at 18°C and 200 rpm for 12 h.
[0052] 3. Ultrasonic disruption of fermentation broth
[0053] Centrifuge the overnight culture broth obtained above at 4000×g for 10 min, discard the supernatant, and repeat this step until all cells are collected. Add 40 mL of equilibration buffer (50 mmol / L Tris-HCl, 500 mmol / L NaCl, 10 mmol / L imidazole, pH 7.5) to the centrifuge flask, resuspend the cell pellet on a vortex mixer until no cell clumps are observed in the solution, and add protease inhibitor PMSF at a ratio of 100:1 for the bacterial culture to the protease inhibitor stock solution. Pay attention to the timing of the addition; before the purification step, 500 μL of protease inhibitor needs to be added every 1 hour.
[0054] The bacterial culture was sonicated in an ice-water mixture to dissolve the recombinant proteins in the bacterial cells in the equilibration buffer. The sonicator power was adjusted to 600W, with a 3-second sonication interval followed by a 5-second pause, for a total of 30 minutes. The completely sonicated bacterial culture was then removed and aliquoted into 2 mL centrifuge tubes. The tubes were centrifuged at 12000 rpm for 30 minutes at 4°C. The supernatant was collected in a 50 mL beaker and filtered through a 0.22 μm filter. All operations were performed with the beakers on ice.
[0055] 4. Ni column affinity chromatography
[0056] The recombinant wPDI protein was initially purified using a Ni-NTA affinity chromatography column, utilizing the metal ion Ni 2+ The target protein is purified by specific coordination binding with His.
[0057] First, mix the filtered supernatant with the column material at 4°C for 30 min. Then, transfer the mixture to a gravity column. After the sample flow through the column has completely finished, add 60 mL of equilibration buffer, 50 mL of elution buffer A, 50 mL of elution buffer B, 40 mL of elution buffer C, 30 mL of elution buffer D, and 30 mL of elution buffer E in sequence. The components of the equilibration buffer and elution buffer are as follows:
[0058] Equilibration buffer: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 10 mmol / L imidazole, pH 7.5;
[0059] Elution buffer A: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 20 mmol / L imidazole, pH 7.5.
[0060] Elution buffer B: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 50 mmol / L imidazole, pH 7.5;
[0061] Elution buffer C: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 100 mmol / L imidazole, pH 7.5;
[0062] Elution buffer D: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 150 mmol / L imidazole, pH 7.5;
[0063] Elution buffer E: 50 mmol / L Tris-HCl, 500 mmol / L NaCl, 250 mmol / L imidazole, pH 7.5.
[0064] Collect the elution fraction in a 2 mL centrifuge tube and label it. Incubate at 4 °C.
[0065] 5. SDS-PAGE analysis
[0066] SDS-PAGE utilizes the porosity of polyacrylamide gels to separate proteins of different sizes, thereby identifying the purity and content of proteins. The specific procedure is as follows:
[0067] (1) Prepare SDS-PAGE stacking gel and separating gel according to the following formula (single gel formula):
[0068] 5% Stacking Gel: 1.375 mL water, 325 μL 30% acrylamide, 250 μL Tris-HCl (pH 6.8), 20 μL 10% APS, 20 μL 10% SDS, 8 μL LTEMED.
[0069] 15% separating gel: 1.15 mL water, 2.5 mL 30% acrylamide, 1.25 mL Tris-HCl (pH 8.8), 50 μL 10% APS, 50 μL 10% SDS, 8 μL LTEMED.
[0070] (2) Preparation of samples for gel running:
[0071] Mix 10 μL of protein sample with 3 μL of loading buffer, boil for 10 min, and centrifuge for 10 s on a handheld centrifuge to allow the sample to gather at the bottom of the centrifuge tube for easy aspiration and loading.
[0072] (3) Clean the electrophoresis tank and glass plate in advance and let them dry.
[0073] (4) First, prepare a 15% separating gel according to the formula in (1), mix it evenly, pour the gel along the glass plate wall until it is about 1.5 cm from the top of the gel tank, add distilled water to seal it, and press it flat. After the separating gel solidifies, pour off the upper layer of water and blot it dry with filter paper.
[0074] (5) According to the formula of the stacking gel in (1), add each component and mix evenly, fill the gel tank, and immediately insert the electrophoresis comb. After the stacking gel solidifies, slowly pull out the electrophoresis comb and put the glass plate with gel into the electrophoresis tank.
[0075] (6) Sample loading. Take 10 μL of the protein sample prepared in (2) and slowly inject it into the sample well along the wall of the thin glass plate to avoid sample overflow and affecting the experimental results.
[0076] (7) Electrophoresis. Turn on the power of the electrophoresis apparatus and adjust the voltage to 80V. After the sample has finished running the stacking gel, adjust the voltage to 120V. When the bromophenol blue indicator is 0.5cm away from the bottom edge of the glass plate, stop the electrophoresis and turn off the power.
[0077] (8) Staining, Destaining, and Photography. After electrophoresis, slowly remove the gel and place it in Coomassie Brilliant Blue staining solution, ensuring the gel is completely submerged. Gently shake on a shaker for approximately 45 minutes. After staining, recover the staining solution for reuse. Then, place the gel in destaining solution and gently shake to destain, changing the destaining solution twice during this process, until the protein bands are washed away. Remove the gel from the destaining solution, rinse it thoroughly with distilled water, and place it on a white background plate of a gel imaging system for imaging. After the process, clean and wipe the background plate with 75% alcohol solution.
[0078] The above steps can be used to identify the purity of protein samples after initial purification using Ni columns, and proteins with higher purity can be selected for further purification.
[0079] Figure 1The results of wPDI purification by Ni column affinity chromatography show that wPDI mainly eluted during elution of eluents A, B, C, and D, with a purity of over 95%. The protein eluents corresponding to lanes 1-18 were concentrated to 2 mL by ultrafiltration and then subjected to further anion exchange chromatography.
[0080] 6. Anion exchange chromatography
[0081] The highly purified eluent obtained after Ni column purification was concentrated to 2 mL by centrifugation using a 10 kDa ultrafiltration tube. An appropriate amount of anion exchange buffer A (20 mmol / L phosphate, 50 mmol / L NaCl, pH 6.5) was added, and the protein aggregated at the bottom of the ultrafiltration tube was gently aspirated with a pipette. The mixture was then centrifuged at 6000 rpm and 4°C for 25 min. This process was repeated three times to remove the salt components from the protein and concentrate the protein solution to a final volume of 2 mL. The concentrated wPDI sample was filtered through a 0.22 μm filter, and the protein concentration was determined using a micro-spectrophotometer for loading.
[0082] The specific steps for purifying proteins using anion exchange chromatography are as follows:
[0083] (1) Turn on the instrument, turn on the chromatography system and computer, wait for the instrument to self-test and display that the connection is successful.
[0084] (2) Place pump A in distilled water that has been filtered through a 0.22 μm filter membrane and ultrasonically degassed for 30 min. Place pump B in a similarly treated NaOH solution. Run the program to clean the anion exchange column, washing the column with distilled water to remove the 20% ethanol until the A280 curve on the display stabilizes. Start the pump washing program to wash away any residual NaOH in pump B to prevent precipitation and blockage of the chromatography system.
[0085] (3) Equilibrate the column. Place pump A into prepared anion exchange buffer A (20 mmol / L phosphate, 50 mmol / L NaCl, pH 6.5), and pump B into prepared anion exchange buffer B (20 mmol / L phosphate, 1000 mmol / L NaCl, pH 6.5). Run the anion exchange column equilibration program, first wash with 100% B solution for 5 CVs, and then equilibrate with 100% A solution for 5 CVs.
[0086] (4) Sample loading. Slowly draw up the prepared protein sample with a 2mL syringe, remove any air bubbles, and slowly inject the protein sample into the loading loop drop by drop. Set the flow rate to 0.5mL / min and the loading time to 8min.
[0087] (5) Protein separation. Run the protein purification program. First, elute with anion exchange buffer A at a flow rate of 2 mL / min for 15 CVs to remove impurities and proteins not bound to the packing material, until the UV absorbance is close to 0 and the curve is stable. Next, slowly increase anion exchange buffer B from 0% to 100% and elute linearly for 20 CVs. Collect the eluted sample fractions using an automated separator. Perform SDS-PAGE analysis on the collected fractions using the same method as above to determine the purity of the target protein. Concentrate the proteins that meet the required purity for subsequent experiments.
[0088] (6) Column cleaning. Place pump A in deionized water that has passed through a 0.22 μm filter membrane and been ultrasonically degassed, and place pump B in a NaOH solution that has been treated in the same way. Run the molecular sieve column cleaning program. First, clean 5 CVs with 0.5 mol / L NaOH solution, then clean 5 CVs with deionized water, and then rinse 5 CVs with 20% ethanol solution. Store the column in 20% ethanol solution.
[0089] After protein purification, the concentration of the target protein was determined using an ultra-micro spectrophotometer, and the molar extinction coefficient ε of wPDI was 43320 L / (mol·cm).
[0090] Figure 2 The results of anion exchange chromatography for wPDI are shown. The black line represents the change in absorbance of the eluent at 280 nm during the elution process, and the red line corresponds to the change in the content of anion exchange buffer B during the elution process. It can be seen that wPDI is mainly eluted when the elution volume is 94 mL.
[0091] Figure 3 The results are obtained from SDS-PAGE analysis of the elution buffer near the peak position. The proteins corresponding to lanes 1-8 are concentrated and their concentrations are measured for subsequent experiments.
[0092] Example 3
[0093] 1. Study on the optimal addition amount of wPDI and Tg enzyme in the production of soy protein isolate
[0094] Soybean protein fiber was produced using a high-moisture extrusion method. The extrusion process and parameter settings were as follows: A twin-screw extruder (FMHE36-24, FUMACH, China) with a screw diameter of 36 mm and an L / D ratio of 24 was used for high-moisture extrusion experiments. A long cooling die measuring 40 × 5 mm (width × height) was connected to the end of the extruder barrel. The extrusion conditions were set as follows: feed rate 8 kg / h (dry basis); moisture content 60% (dry basis); screw speed 240 rpm; extruder barrel temperatures from zone 1 to zone 5 were 60, 80, 145, 150, and 130 °C, respectively; and the die was cooled at 70 °C.
[0095] To ensure the extrusion of soy protein isolate during production, a soy protein isolate sample was first mixed with wheat starch (purchased from Beijing Sola Biotechnology Co., Ltd.) at a mass ratio of 9:1. During extrusion, different concentrations of wPDI and Tg enzyme were added to the soy protein raw material. By weighing the mass of the formed protein fibers, the yield of the protein fibers was calculated according to the following formula, thus obtaining the optimal addition amount of wPDI and Tg enzyme.
[0096]
[0097] The results of the study on the optimal addition levels of wPDI and Tg enzyme in the production of soy protein isolate are as follows: Figure 4 As shown, the highest fiber formation was observed when the wPDI addition was 0.15% and the Tg enzyme addition was 0.5%. Therefore, when performing microscopic examination of the fiber structure using laser confocal microscopy, wPDI addition of 0.15% and Tg enzyme addition of 0.5% were selected.
[0098] 2. Microscopic results of protein fibers observed using laser confocal microscopy.
[0099] The microstructure changes of soy protein isolate (SPI) fibers under four treatment conditions (soy protein isolate without enzyme, soy protein isolate with 0.5% Tg enzyme, soy protein isolate with 0.15% wPDI, and soy protein isolate with both 0.5% Tg enzyme and 0.15% wPDI) were observed using a laser confocal microscope (CLSM) FV1200 to investigate the regulatory effects of transglutaminase (TG enzyme) and wheat protein disulfide isomerase (wPDI) on protein fiber morphology.
[0100] The fibers were stained with Rhodamine B fluorescent dye, and their microstructure at an excitation wavelength of 488 nm was observed using a laser confocal microscope (CLSM). The specific staining procedure was as follows: the protein fibers were incubated in 1% Rhodamine B dye solution in the dark for 5 hours. After rinsing with deionized water to remove free dye, fluorescence images were acquired using a laser confocal microscope (CLSM) at an excitation wavelength of 488 nm.
[0101] The results are as follows Figure 5 As shown, the protein fibers formed by SPI alone exhibit an ordered filamentous arrangement, a smooth surface, few branches, and a typical linear structure. The fiber structure formed after adding Tg enzyme to SPI is loose, showing local fusion and forming a disordered network structure in some areas. The fiber structure formed after adding wPDI to SPI shows a significant increase in branching and the formation of complex porous structures, possibly due to wPDI promoting the directional arrangement of disulfide bonds. Adding both Tg enzyme and wPDI to SPI also forms a dense network structure, but the fiber structure is finer and less strong than that formed with wPDI alone. Therefore, adding wPDI can effectively enhance the fiberity of the protein, thereby improving the texture and nutritional value of the extrudate, and the improvement effect is superior to that of traditional Tg enzyme.
[0102] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for purifying a wheat-derived protein disulfide isomerase, characterized in that, Includes the following steps: (1) Transform the pET-30b-wPDI recombinant plasmid with His tag into Escherichia coli and induce the expression of recombinant wPDI protein in Escherichia coli with an inducer. (2) The induced Escherichia coli obtained in step (1) was broken up, and the supernatant containing recombinant wPDI protein was collected after solid-liquid separation. (3) Mix the supernatant obtained in step (2) with the column material of the Ni-NTA affinity chromatography column, transfer the mixture to the gravity column, and after the sample flow through the liquid has finished, add the equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D and elution buffer E in sequence for elution. The equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D, and elution buffer E contain imidazole at concentrations of 8–12 mmol / L, 15–25 mmol / L, 40–60 mmol / L, 80–120 mmol / L, 130–170 mmol / L, and 200–300 mmol / L, respectively. (4) After concentrating the eluent obtained in step (3), it is purified by anion exchange column to obtain purified wheat-derived protein disulfide isomerase. The anion exchange buffer A used contains phosphate at a concentration of 15–25 mmol / L and NaCl at a concentration of 40–60 mmol / L, while the anion exchange buffer B contains phosphate at a concentration of 15–25 mmol / L and NaCl at a concentration of 800–1200 mmol / L.
2. The method as described in claim 1, characterized in that, The Escherichia coli mentioned in step (1) is Escherichia coli BL21, and the inducer is IPTG.
3. The method as described in claim 1, characterized in that, The crushing in step (2) is ultrasonic crushing, the power of which is 400-800W, with a pause of 3-7 seconds every 2-4 seconds of ultrasonic crushing, and the ultrasonic time is 25-35 minutes. The solid-liquid separation process is carried out at a temperature of 3–5°C, a speed of 10,000–15,000 rpm, and a time of 25–35 min.
4. The method as described in claim 1, characterized in that, Before mixing in step (3), the supernatant is filtered through a 0.22 μm filter. The mixing temperature is 3-5 °C and the time is 20-40 min.
5. The method as described in claim 1, characterized in that, The equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D and elution buffer E mentioned in step (3) also contain Tris-HCl with a concentration of 40-60 mmol / L and NaCl with a concentration of 300-600 mmol / L, respectively. During elution, the amounts of equilibration buffer, elution buffer A, elution buffer B, elution buffer C, elution buffer D, and elution buffer E added are 50–70 mL, 40–60 mL, 40–60 mL, 30–50 mL, 20–40 mL, and 20–40 mL, respectively.
6. The method as described in claim 1, characterized in that, In step (4), the concentration is achieved by centrifugation using an ultrafiltration tube, wherein the molecular weight cutoff of the ultrafiltration tube is 8-12 kDa. Before purification via an anion exchange column, the concentrated eluent is mixed with anion exchange buffer A and centrifuged 1–3 times, then filtered through a 0.22 μm filter. The centrifugation concentration is performed at a temperature of 3–5 °C, a speed of 4000–8000 rpm, and a time of 20–30 min.
7. The method as described in claim 1, characterized in that, In step (4), anion exchange buffer A is eluted at a flow rate of 1.5 to 2.5 mL / min for 10 to 20 CVs, and anion exchange buffer B is slowly increased from 0% to 100% and eluted linearly for 15 to 25 CVs.
8. The application of a wheat-derived protein disulfide isomerase purified according to any one of claims 1 to 7 in plant protein processing, characterized in that, Adding the wheat-derived protein disulfide isomerase during the plant protein production process can improve the fiber formation rate of the plant protein.
9. The application as described in claim 8, characterized in that, The plant protein is soybean protein, and the amount of wheat-derived protein disulfide isomerase added is 0.05-0.2%.
10. The application as described in claim 9, characterized in that, Soy protein is produced by high-moisture extrusion. During production, soybean protein and wheat starch are mixed at a mass ratio of 8.5–9.5:0.5–1.5 and extruded at a feed rate of 6–10 kg / h (dry basis), a moisture content of 50–70% (dry basis), and a screw speed of 200–300 rpm. The extruder barrel temperatures in zones one through five are 50–70℃, 70–90℃, 140–150℃, 145–155℃, and 125–135℃, respectively.