A method for preparing a morel active protein having alpha-glucosidase activity

Abstract

The present application relates to a kind of active protein with alpha-glucosidase activity of morchella and its preparation method.Active protein of morchella includes the amino acid sequence shown in SEQ ID No.1.The present application adopts the combination process of wall breaking pulverization-supercritical extraction-ultrasonic assisted low temperature extraction-ion exchange chromatography, realizes the efficient directional extraction and purification of target protein with alpha-glucosidase activity in morchella.The active protein of morchella prepared in the present application is high in purity, stable in enzyme activity, and the preparation process is mild, no toxic reagent residue, suitable for industrial production, fill the blank of the directional preparation technology of alpha-glucosidase from morchella.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a morel active protein with α-glucosidase activity and its preparation method. Background Technology

[0002] α-Glucosidase is a key enzyme that catalyzes the hydrolysis of α-1,4 glycosidic bonds in substrates such as oligosaccharides and polysaccharides to produce glucose. It is widely used in food fermentation, glucose preparation industry, and pharmaceutical hypoglycemic agents, and has a large market demand.

[0003] Currently, the main sources of α-glucosidase preparation are microbial fermentation and plant extraction. Although enzymes from microorganisms have high yields, they suffer from problems such as complex expression and purification processes, the need to add inducers, and the products being prone to containing impurities. Plant-derived α-glucosidases are mostly extracted from legumes, fruits, and vegetables, but they have drawbacks such as low enzyme activity, poor stability, and limited extraction rates, making it difficult to meet the needs of industrial applications.

[0004] Morel mushrooms, a rare edible and medicinal fungus, are rich in various bioactive components such as active proteins and polysaccharides. Studies have shown that morel mushrooms contain glycoside hydrolases, but no publicly reported methods have been developed for the targeted extraction and purification of proteins with α-glucosidase activity from morel mushrooms. Existing methods for extracting morel mushroom proteins mostly employ single water or alcohol extraction, which suffers from problems such as incomplete removal of lipid-soluble impurities, easy denaturation of target proteins, and low purification efficiency. These methods cannot achieve the targeted separation of highly active α-glucosidases, and the extracted active proteins have not been accurately identified or characterized enzymatically.

[0005] To address the shortcomings of the existing technologies, there is an urgent need to develop a method for the targeted separation of proteins containing highly active α-glucosidase, so as to achieve efficient targeted extraction and purification of target proteins with α-glucosidase activity from morel mushrooms. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention provides a morel mushroom active protein with α-glucosidase activity and a method for preparing the same. The morel mushroom active protein provided by the present invention has α-glucosidase activity and is suitable for industrial production.

[0007] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: The present invention provides a morel active protein with α-glucosidase activity, wherein the morel active protein comprises the amino acid sequence shown in SEQ ID No. 1.

[0008] The relative molecular mass of this morel mushroom active protein is 121.5 kDa. Testing showed that the α-glucosidase activity of this morel mushroom active protein is ≥300 U / g, with an optimal reaction pH of 6 and an optimal reaction temperature of 55℃. This active protein was isolated from morel mushroom fruiting bodies, possesses α-glucosidase activity, and is a natural extract. It has advantages such as good safety and stable enzyme activity, and can be applied in the glucose production industry.

[0009] This invention provides a method for preparing the above-mentioned morel active protein with α-glucosidase activity, comprising the following steps: (1) The coarse powder of morel fruiting bodies was processed by cell wall breaking and pulverization to obtain cell wall broken powder of fruiting bodies; (2) Mix the fruiting body cell wall-breaking powder with anhydrous ethanol, perform supercritical carbon dioxide extraction, dry, and obtain residue; (3) The residue was subjected to ultrasonic-assisted low-temperature extraction to obtain fruiting body extract; (4) Desalt the fruiting body extract and collect the elution peaks; (5) Remove impurities from the elution peak using ion exchange chromatography and collect the flow-through solution; (6) Dialyze the flow-through solution to obtain the dialyzed solution; (7) Ultrafiltration concentration.

[0010] The preparation method provided by this invention fills the gap in the technology for the targeted preparation of α-glucosidase from morel mushrooms. The morel mushroom active protein prepared by the above method has α-glucosidase activity and is suitable for industrial production.

[0011] Furthermore, in step (1), the temperature for the cell wall breaking and pulverizing treatment is 4-15℃, and the treatment time is 15-30 min. Preferably, the temperature for the cell wall breaking and pulverizing treatment is 4℃, and the treatment time is 20 min.

[0012] Experimental results showed that using the above conditions is beneficial to improving the extraction yield and enzyme activity of morel active proteins.

[0013] Furthermore, in step (2), the pressure of supercritical carbon dioxide extraction is 300-340 bar, and the temperature of the extraction vessel is 35-45°C. Preferably, the pressure of supercritical carbon dioxide extraction is 320 bar, and the temperature of the extraction vessel is 40°C.

[0014] Furthermore, in step (2), during the supercritical carbon dioxide extraction process, the pressure relief valve temperature is 78-85℃, and the treatment time is 2-4 h. Preferably, the pressure relief valve temperature is 80℃, and the treatment time is 2 h.

[0015] Experimental results have shown that using the above conditions is beneficial for improving the enzyme activity of morel active proteins.

[0016] Furthermore, in step (3), the temperature of the ultrasonic-assisted low-temperature extraction is 15-25℃. Preferably, the temperature is 15℃.

[0017] Furthermore, in step (3), the ultrasonic-assisted low-temperature extraction time is 30-60 min, followed by centrifugation at 6000-10000 rpm for 10-30 min, and collection of the supernatant, which is the fruiting body extract.

[0018] Preferably, the ultrasonic-assisted low-temperature extraction time is 40 min.

[0019] Experimental results have shown that using the above conditions is beneficial for improving the enzyme activity of morel active proteins.

[0020] Furthermore, in step (4), a desalination column is used for desalination treatment.

[0021] Furthermore, in step (5), the ion exchange chromatography is performed using a QHP anion exchange chromatography column.

[0022] Furthermore, the process after ultrafiltration concentration also includes a freeze-drying step.

[0023] Furthermore, after freeze-drying, the process also includes a step of identifying the active protein profile of morel mushrooms.

[0024] The advantages and beneficial effects of this invention include: The preparation method provided by this invention employs a combined process of supercritical fluid extraction, ultrasonic-assisted low-temperature extraction, and ion exchange chromatography, achieving efficient and targeted extraction and purification of the target protein with α-glucosidase activity from morel mushrooms. The obtained protein has high purity and stable enzyme activity, and the preparation process is mild, with no toxic reagent residues, making it suitable for industrial production. This fills the gap in the targeted preparation technology of morel mushroom-derived α-glucosidase. The morel mushroom-derived α-glucosidase prepared by this invention has an enzyme activity ≥300 U / g, significantly higher than that of common plant and fungal crude extracts (typically 20–180 U / g), exhibiting a higher enzyme activity advantage among natural sources. Attached Figure Description

[0025] Figure 1 This is an SDS-PAGE electrophoresis image of the active proteins from morel mushrooms.

[0026] Figure 2 The results show the optimal pH for the reaction of morel active proteins.

[0027] Figure 3 The results show the optimal reaction temperature for morel active proteins.

[0028] Figure 4 The results show the pH stability of active proteins in morel mushrooms.

[0029] Figure 5 The results show the temperature stability of active proteins from morel mushrooms.

[0030] Figure 6 The protein yield is relative to that of Comparative Example 1 compared to Example 1.

[0031] Figure 7 The value represents the relative enzyme activity of Comparative Example 2 compared to Example 1.

[0032] Figure 8 The value represents the relative enzyme activity of Comparative Example 3 compared to Example 1.

[0033] Figure 9 The value represents the relative enzyme activity of Comparative Example 4 compared to Example 1. Detailed Implementation

[0034] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0035] The present invention provides a morel active protein with α-glucosidase activity, wherein the morel active protein comprises the amino acid sequence shown in SEQ ID No. 1.

[0036] The relative molecular mass of the morel active protein is 121.5 kDa. Testing showed that the α-glucosidase activity of this morel active protein is ≥300 U / g, the optimal reaction pH is 6, and the optimal reaction temperature is 55℃.

[0037] The preparation method of the above-mentioned morel active protein includes the following steps: (1) Put the dried morel fruiting bodies into a coarse powder mill and crush them through a 5-60 mesh sieve to obtain coarse powder of the fruiting bodies; (2) Put the coarse powder of the fruiting body into a low temperature cell wall breaking pulverizer, control the temperature at 4-15℃, break the cell wall for 15-30 minutes, and perform cell wall breaking treatment. Pass it through a 200-500 mesh sieve to obtain the broken cell wall powder of the fruiting body. (3) Mix the fruiting body cell wall-breaking powder with anhydrous ethanol at a ratio of 1 g: (0.5-1.5) mL and stir evenly. Place the mixture in a supercritical carbon dioxide extraction vessel, control the supercritical pressure at 300-340 bar, the extraction vessel temperature at 35-45℃, the pressure relief valve temperature at 78-85℃, process for 2-4 h, collect the residue powder and dry it at 28-40℃ for later use. (4) Dissolve the residue powder in pure water at a ratio of (1 g: 10 mL) - (1 g: 50 mL), place it in an extractor for ultrasonic-assisted low-temperature extraction, treat at 15-25℃ for 30-60 min, then centrifuge at 6000-10000 rpm for 10-30 min, collect the supernatant, which is the fruiting body extract. (5) Desalting and buffer replacement: The fruiting body extract obtained in step (4) is loaded onto a desalting column, which is a HiTrap Desalting column with a column volume of 5 mL. It is eluted with 20 mM Na2HPO4–NaH2PO4 buffer (the concentration of Na2HPO4 is 20 mM, the concentration of NaH2PO4 is 20 mM, the pH of the buffer is 7.0, and the buffer also contains 50 mM NaCl) at a flow rate of 1-2 mL / min. The elution peak containing the target protein is collected (i.e., the molecular weight corresponding to the elution sample band detected by SDS-PAGE electrophoresis is 121.5 KDa), and the buffer replacement is completed. (6) Ion exchange chromatography to remove impurities: The elution peak collected in step (5) is loaded onto a QHP anion exchange chromatography column equilibrated with the same buffer solution, and the flow-through is collected. The flow-through is the component rich in the target active protein. (7) Dialysis desalination: Place the flow-through solution obtained in step (6) into a dialysis bag with a molecular weight cutoff of 10-14 KDa, and perform dialysis at 2-8℃ using ultrapure water as the external solution. The external solution is changed no less than 3 times during the process, and the solution is changed once every 4 hours. (8) Concentration: The solution after dialysis in step (7) is concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to obtain concentrated protein solution; (9) Dispensing and freeze-drying: Dispense the concentrated protein solution obtained in step (8) and freeze-dry it at -40°C to obtain freeze-dried powder, which is the morel active protein with α-glucosidase activity.

[0038] Furthermore, in step (1), the morel fruiting bodies are crushed and passed through a 10-40 mesh sieve.

[0039] Furthermore, in step (3), the mixing ratio of the fruiting body powder to anhydrous ethanol is 1 g: 1 mL (W / V), the supercritical pressure is 320 bar, the extraction vessel temperature is 40℃, the pressure relief valve temperature is 80℃, the processing time is 2 h, and the residue powder drying temperature is 35℃.

[0040] Furthermore, in step (4), the mixing ratio of the residue powder and pure water is 1 g: 20 mL (W / V), the extraction temperature is 15℃, the extraction time is 40 min, the centrifugation speed is 8000 rpm, and the centrifugation time is 15 min.

[0041] Furthermore, in step (7), the molecular weight cutoff of the dialysis bag is 10 kDa, and the dialysis temperature is 5°C.

[0042] Furthermore, the QHP anion exchange chromatography column mentioned in step (6) is a HiTrap QHP anion exchange chromatography column.

[0043] Furthermore, the desalting column mentioned in step (5) is a HiTrap Desalting column with a column volume of 5 mL and an elution flow rate of 1-2 mL / min.

[0044] Furthermore, it also includes step (10) mass spectrometry identification, the specific method of which is as follows: the protein in step (9) is subjected to SDS-PAGE electrophoresis detection, the target cosmotic protein band after SDS-PAGE electrophoresis is subjected to in-gel enzyme digestion to obtain a peptide mixture; the peptide mixture is subjected to liquid chromatography-tandem mass spectrometry analysis to obtain the mass spectrometry data of the peptides; the mass spectrometry data is compared with the protein database, and based on the matching peptide sequence evidence, the identity of a high-confidence protein constituting the target cosmotic band is identified.

[0045] This invention provides the application of the morel mushroom active protein prepared by the above method in the glucose preparation industry.

[0046] In this embodiment, the morel fruiting body is *Morchella esculenta* (also known as *Morchella esculenta*). Morchella sextelata ) Sub-entity.

[0047] In the examples, the decolorizing solution was prepared in the following proportions: 500 mL of ethanol, 100 mL of glacial acetic acid, and 400 mL of deionized water.

[0048] Unless otherwise specified, all techniques or conditions used in the examples are conventional methods or performed according to techniques or conditions described in the literature in this field, or according to product instructions. Reagents used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels or prepared according to conventional methods in this field. Instruments used, unless otherwise specified, are all conventional products that can be purchased from legitimate channels.

[0049] Unless otherwise specified, all solutions in this invention are prepared using water as the solvent.

[0050] The following is a description through specific embodiments. Example 1

[0051] This invention provides a method for preparing morel active protein with α-glucosidase activity, comprising the following steps: (1) Put the dried morel fruiting bodies into a coarse powder mill and grind them for about 1-5 minutes. The particle size should be <3mm. Pass the powder through a 10-mesh sieve to obtain coarse powder of the fruiting bodies. (2) Put the coarse powder of the fruiting body into a low temperature cell wall breaking pulverizer, control the temperature at 4℃, break the cell wall for 20 min, and set the shaking time to 120 s for cell wall breaking treatment. Pass it through a 200 mesh sieve to obtain the broken cell wall powder of the fruiting body. (3) Mix the fruiting body cell wall-breaking powder with anhydrous ethanol at a ratio of 1 g: 1 mL (W / V), stir evenly, place in a supercritical carbon dioxide extraction vessel, control the supercritical pressure at 320 bar, the extraction vessel temperature at 40℃, the pressure relief valve temperature at 80℃, process for 2 h, collect the residue powder, and dry at 35℃ for later use. (4) Dissolve the residue powder in pure water at a ratio of 1 g: 20 mL (W / V), place it in an ultrasonic extractor for ultrasonic-assisted low-temperature extraction, control the ultrasonic frequency at 600 W, treat at 15℃ for 40 min, centrifuge at 8000 rpm for 15 min, collect the supernatant, which is the fruiting body extract. (5) Desalting and buffer replacement: The fruiting body extract obtained in step (4) is loaded onto a desalting column, which is a HiTrap Desalting column with a column volume of 5 mL. It is eluted with 20 mM Na2HPO4–NaH2PO4 buffer (the concentration of Na2HPO4 is 20 mM, the concentration of NaH2PO4 is 20 mM, the pH of the buffer is 7.0, and the buffer also contains 50 mM NaCl) at a flow rate of 1-2 mL / min. The elution peak containing the target protein is collected (i.e., the molecular weight corresponding to the elution sample band detected by SDS-PAGE electrophoresis is 121.5 KDa), and the buffer replacement is completed. (6) Removal of impurities by ion exchange chromatography: The elution peak collected in step (5) is loaded onto a QHP anion exchange chromatography column equilibrated with the same buffer. The QHP anion exchange chromatography column is a HiTrap Q HP anion exchange chromatography column. The loading flow rate is 10-20 mL / min and the column volume is 150 mL. The flow-through liquid is collected. The flow-through liquid is the component rich in the target active protein. This experiment used an AKTA Pure 25 chromatography system with a QHP anion exchange column. The entire process was performed at room temperature with a flow rate of 10-20 mL / min, and the maximum system pressure was set to 0.5 MPa to protect the column. All buffers and samples were filtered through a 0.22 μm filter and degassed by sonication. First, the desalting column was equilibrated with approximately 450 mL of buffer A for three column volumes. Then, approximately 450 mL of filtered sample was loaded at a flow rate of 10-20 mL / min. After loading, elution was performed with buffer A, and the protein peak was collected. The flow-through sample was then loaded with buffer A to equilibrate the QHP anion exchange column for five column volumes. After loading, equilibration was performed with approximately 750 mL of buffer A for five column volumes, followed by elution with approximately 750 mL of buffer B for five column volumes, and the eluted sample was collected. Throughout the process, 280 nm UV absorbance and conductivity were monitored, and the target fraction was collected in 5 mL volumes per tube based on peak shape. After the experiment, the system tubing and chromatography column were rinsed sequentially with ultrapure water and 20% ethanol. After completion, the system and chromatography column were sealed with 20% ethanol.

[0052] Buffer A: 20 mM Na2HPO4–NaH2PO4 buffer (Na2HPO4 concentration is 20 mM, NaH2PO4 concentration is 20 mM), pH 7.0, containing 50 mM NaCl.

[0053] Buffer B: 20 ​​mM Na2HPO4–NaH2PO4 buffer (Na2HPO4 concentration is 20 mM, NaH2PO4 concentration is 20 mM), pH 7.0, containing 0.4 M NaCl.

[0054] (7) Dialysis desalination: Place the flow-through solution obtained in step (6) into a dialysis bag with a molecular weight cutoff of 10 kDa, and perform dialysis at 5°C using ultrapure water as the external solution. Replace the external solution at least 3 times during the process, and change the solution once every 4 hours to completely remove the salt. (8) Concentration: The solution after dialysis in step (7) is concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa to obtain concentrated protein solution; (9) Dispensing and freeze-drying: The concentrated protein solution obtained in step (8) is dispensed and freeze-dried at -40°C to obtain freeze-dried powder, which is the high-purity morel active protein with α-glucosidase activity. Example 2

[0055] The morel mushroom active protein prepared in Example 1 was detected and bands were identified by SDS-PAGE electrophoresis. The electrophoresis image is shown below. Figure 1 As shown. Then, the following method was used for identification: (1) The target coumarin protein bands after SDS-PAGE electrophoresis were subjected to in-gel enzyme digestion to obtain a peptide mixture: the protein bands were washed 3 times with ddH2O, destained with 300 μL of destaining solution, washed 4 times with water, and then washed twice with 300 μL of 25 mM ammonium bicarbonate solution, 300 μL of 50% (v / v) acetonitrile solution and 300 μL of 100% acetonitrile solution respectively. Finally, the gel was dehydrated until the gel block turned white. 50 μL of 10 mM DTT solution was added and the gel was reduced in a water bath at 37℃ for 2 h. After the temperature dropped to room temperature, an equal volume of 50 mM IAA solution was added for alkylation in the dark for 30 min. Then, the gel was washed twice with 300 μL of 25 mM ammonium bicarbonate solution, 300 μL of 50% (v / v) acetonitrile solution and 300 μL of 100% acetonitrile solution respectively. Finally, the gel was dehydrated until the gel block turned white. Add 15-40 μL of 0.01 μg / μL proteomics-grade trypsin, allow it to fully swell and become transparent on ice, then add 30-40 μL of 50 mM NH4HCO3 solution to cover it. The NH4HCO3 solution contains acetonitrile, with a volume percentage of acetonitrile to NH4HCO3 solution of 10%. Digest overnight in a water bath at 37°C. After digestion, centrifuge and collect the supernatant ①. Transfer supernatant ① to another new EP tube, add 100 µL of extraction buffer to the remaining gel block. The extraction buffer is a mixture of acetonitrile and formic acid, with a volume percentage of acetonitrile of 67% and formic acid of 2%. Incubate at 37°C for 30 min, then sonicate for 15 min, centrifuge, and collect the supernatant ②. Combine supernatant ① and supernatant ②, concentrate using an ultrafiltration tube, and then freeze-dry at -35°C to obtain a peptide mixture for subsequent mass spectrometry analysis.

[0056] (2) The peptide mixture was analyzed by liquid chromatography-tandem mass spectrometry to obtain the mass spectrometry data of the peptides: After centrifugation and drying, the enzyme-digested peptide samples were redissolved in Nano LC mobile phase A and bottled for loading. Mobile phase A was a 0.1% (v / v) formic acid aqueous solution. Online LCMS analysis was performed. The dissolved sample was loaded into a nanoViper C18 pre-column (3 μm 100 Å) at a volume of 10-20 μL, and then washed with 20 μL for desalting. Liquid chromatography was performed using an MLtiMate 3000 RSLC nano-liquid chromatography system (ThermoFisher, USA). Samples were desalted and retained on a pre-column before separation by an analytical column. The analytical column was a C18 reversed-phase column (Acclaim PepMap RSLC, 75 μm × 25 cm C18 2 μm 100 Å). The gradient used was a 15-minute increase of mobile phase B from 5% to 40%. Mobile phase B was a mixed solution of acetonitrile and formic acid, with acetonitrile comprising 80% by volume and formic acid 0.1% by volume. Mass spectrometry was performed using a ThermoFisher Q Exactive plus system (ThermoFisher, USA) combined with a nano-spray ion source (ThermoFisher, USA). The spray voltage was 1.9 kV, and the ion transfer tube heating temperature was 320 °C. The mass spectrometry scan was performed in information-dependent acquisition mode (DDA / Data Dependent Analysis). The primary mass spectrometry scan resolution was 70,000 m / z, the scan range was 350–1500 m / z, and the maximum injection time was 100 ms. A maximum of 20 secondary mass spectra with charges 2+ to 5+ were acquired per DDA cycle. The maximum ion injection time for secondary mass spectrometry was 50 ms, the collision chamber energy (high-energy collision-induced dissociation, HCD) was set to 28 eV, applicable to all precursor ions, and the dynamic exclusion time was set to 25 s. Mass spectrometry data were obtained using the above method.

[0057] (3) The mass spectrometry data was compared with the protein database. Based on the matching peptide sequence evidence, the identity of a high-confidence protein constituting the target coumarin band was identified. The raw data files acquired by mass spectrometry were processed and analyzed using Proteome Discoverer 2.5 software. The protein database in the database folder was used, and the search parameters were set as follows: trypsin digestion, mass tolerance of 10 ppm for primary mass spectrometry, 0.02 Da for secondary mass spectrometry, and a maximum number of missed digestions of 2; fixed modification: cysteine ​​iodoacetamide; variable modification: protein N-term acetylation. The search parameter methionine oxidation (M) was set to compensate for possible chemical modifications introduced during sample processing to ensure the reliability of the identification results.

[0058] The mass spectrometry data were compared with a protein database. The false positive rate (FDR, strict) was <0.01, indicating high confidence. The sum of the negative logarithms of the PEP values ​​of all PSMs (scoring the secondary spectra of the matched peptides) was 273.941. The protein matching score was 1650.82, indicating very high confidence. The amino acid sequence of the morel active protein obtained by the above method is shown in SEQ ID No. 1.

[0059] Example 3

[0060] The α-glucosidase enzymatic properties of the morel active protein prepared in Example 1 were detected.

[0061] (1) Optimal reaction pH Mix 10 μL of 10 mmol / L pNPG substrate solution with 140 μL of BR buffer at different pH values ​​(pH 3-10), preheat at 40℃ for 5 min, then add 50 μL of morel active protein solution (the mass percentage of morel active protein to morel active protein solution is 10%) to react. Timing is precise at 15 min. Immediately add 600 μL of 1 mol / L Na2CO3 solution to terminate the reaction and develop color. After mixing, centrifuge, collect the supernatant, and measure the absorbance at 405 nm. Calculate enzyme activity according to the standard curve equation. Take the highest measured enzyme activity as 100% and calculate the relative enzyme activity of each group to determine the optimal reaction pH.

[0062] In the above, the standard curve equation is Y = 0.3809X - 0.0055, R0 2 =0.9996, where X is the OD 405nm Y represents the pNPG concentration, in mmol / L.

[0063] Experimental results are from Figure 2 As shown in the figure, it can be seen that the active protein of morel mushrooms exhibits the highest relative enzyme activity at a pH of 6, and the optimal pH is 6.

[0064] (2) Optimal reaction temperature Prepare a buffer solution (pH 6) containing Na2HPO4 and KH2PO4, with Na2HPO4 concentrations of 1 / 15 mol / L and KH2PO4 concentrations of 1 / 15 mol / L.

[0065] Take 50 μL of morel active protein solution (the mass percentage of morel active protein to morel active protein solution is 10%), add 10 μL of 10 mmol / L pNPG substrate solution and 140 μL of preheated buffer (pH 6). The preheating temperatures are 20℃, 30℃, 40℃, 50℃, 55℃, 60℃, 65℃, 70℃, 80℃ and 90℃ respectively. After mixing, place them at different temperatures for reaction. The reaction temperature is the same as the preheating temperature. Precisely time for 15 min, quickly add 600 μL of 1mol / L Na2CO3 solution to terminate the reaction and develop color. After mixing, centrifuge, take the supernatant, and measure the absorbance at 405 nm. Calculate the enzyme activity according to the standard curve equation. The standard curve equation is the same as in Example (1). Take the highest measured enzyme activity as 100%, calculate the relative enzyme activity of each group, and determine the optimal reaction temperature.

[0066] Experimental results are as follows Figure 3 As shown, the optimal temperature for the active protein in morel mushrooms is 55℃, classifying it as a mesophilic enzyme.

[0067] (3) pH stability 140 μL of BR buffer at different pH values ​​was mixed with 50 μL of morel active protein solution (the mass percentage of morel active protein to morel active protein solution was 10%) and incubated at 4℃ for 0-6 h (specific values ​​were 0 h, 1 h, 2 h, 3 h, 4 h, 5 h, and 6 h). Then, 10 μL of 10 mmol / L pNPG substrate solution was added, mixed, and reacted at pH 6 and 55℃ for 15 min. The reaction was terminated and colorimetric was developed by rapidly adding 600 μL of 1 mol / L Na2CO3 solution. After mixing, the mixture was centrifuged, the supernatant was collected, and the absorbance at 405 nm was measured. The enzyme activity was calculated according to the standard curve equation, which is the same as in Example (1). The enzyme activity of the untreated group (i.e., incubation time 0 h, pH 6, reaction at 55℃) was taken as 100%, and the remaining relative enzyme activity after incubation with BR buffer at different pH values ​​was calculated to determine its pH stability.

[0068] Experimental results are as follows Figure 4 As shown, the morel active protein can still maintain 97%, 92%, 76%, 75%, 88%, and 78% of its activity after 6 hours of incubation in buffer solutions at pH 5, 6, 7, 8, 9, and 10, respectively. This indicates that the enzyme has the strongest activity and stability under neutral conditions and also has good stability under alkaline conditions.

[0069] (4) Temperature stability Morel mushroom active protein solution (morel mushroom active protein to morel mushroom active protein solution mass percentage 10%) was placed in water baths at 40℃, 50℃, 55℃, 60℃ and 70℃ for 0-4 h. Take 50 μL of morel mushroom active protein solution, add 140 μL of BR buffer (pH 6, temperature 55℃), add 10 μL of 10 mmol / L pNPG substrate solution, mix well, react at pH 6 and 55℃, time precisely 15 min, then quickly add 600 μL of 1 mol / L Na2CO3 solution to terminate the reaction and develop color, mix well, centrifuge, collect the supernatant, and measure the absorbance at 405 nm. Calculate enzyme activity according to the standard curve equation, which is the same as in Example (1). Using the enzyme activity of the group without water bath treatment (i.e., water bath time 0 h) as 100%, calculate the remaining relative enzyme activity of each group to determine its stability at different temperatures.

[0070] Experimental results are as follows Figure 5 As shown, the morel active protein can still retain 73%, 72%, and 66% of its activity after incubation at 40℃, 50℃, and 55℃ for 4 hours, respectively. After incubation at 60℃ and 70℃ for 4 hours, its activity is still retained at 23% and 19%, respectively, indicating that the enzyme can tolerate temperatures not exceeding 60℃.

[0071] In summary, the optimal temperature for the α-glucosidase prepared by this invention is 55℃, the optimal pH value is 6, and the enzyme is relatively stable under neutral and alkaline conditions and in environments not exceeding 60℃. Example 4

[0072] This invention provides a method for preparing morel active protein with α-glucosidase activity, comprising the following steps: (1) Put the dried morel fruiting bodies into a coarse powder mill and grind them for about 1-5 minutes. The particle size should be <3mm. Pass the powder through a 5-mesh sieve to obtain coarse powder of the fruiting bodies. (2) Put the coarse powder of the fruiting body into a low temperature cell wall breaking pulverizer, control the temperature at 10℃, break the cell wall for 15 min, set the shaking time to 120 s, and carry out cell wall breaking treatment. Pass it through a 300 mesh sieve to obtain the broken cell wall powder of the fruiting body. (3) Mix the fruiting body cell wall-breaking powder with anhydrous ethanol at a ratio of 1 g: 0.5 mL (W / V), stir evenly, place in a supercritical carbon dioxide extraction vessel, control the supercritical pressure at 300 bar, the extraction vessel temperature at 35℃, the pressure relief valve temperature at 78℃, process for 3 h, collect the residue powder, and dry at 28℃ for later use. (4) Dissolve the residue powder in pure water at a ratio of 1 g: 10 mL (W / V), place it in an ultrasonic extractor for ultrasonic-assisted low-temperature extraction, control the ultrasonic frequency at 600 W, treat at 20℃ for 60 min, centrifuge at 6000 rpm for 30 min, collect the supernatant, which is the fruiting body extract. (5) Desalting and buffer replacement: The fruiting body extract obtained in step (4) is loaded onto a desalting column, which is a HiTrap Desalting column with a column volume of 5 mL. It is eluted with 20 mM Na2HPO4–NaH2PO4 buffer (the concentration of Na2HPO4 is 20 mM, the concentration of NaH2PO4 is 20 mM, the pH of the buffer is 7.0, and the buffer also contains 50 mM NaCl) at a flow rate of 1-2 mL / min. The elution peak containing the target protein is collected (i.e., the molecular weight corresponding to the elution sample band detected by SDS-PAGE electrophoresis is 121.5 KDa), and the buffer replacement is completed. (6) Removal of impurities by ion exchange chromatography: The elution peak collected in step (5) is loaded onto a QHP anion exchange chromatography column equilibrated with the same buffer. The QHP anion exchange chromatography column is a HiTrap Q HP anion exchange chromatography column. The loading flow rate is 10-20 mL / min and the column volume is 150 mL. The flow-through liquid is collected. The flow-through liquid is the component rich in the target active protein. This experiment used an AKTA Pure 25 chromatography system with a QHP anion exchange column. The entire process was performed at room temperature with a flow rate of 10-20 mL / min, and the maximum system pressure was set to 0.5 MPa to protect the column. All buffers and samples were filtered through a 0.22 μm filter and degassed by sonication. First, the desalting column was equilibrated with approximately 450 mL of buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl) for three column volumes. Then, approximately 450 mL of filtered sample was loaded at a flow rate of 10-20 mL / min. After loading, elution was performed with buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl), and the protein peak was collected and flowed through the sample. The flow-through sample was then loaded with buffer A to equilibrate five column volumes of the QHP anion exchange column. After sample loading, equilibrate with approximately 5 column volumes (750 mL) of buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl). Then elute with approximately 5 column volumes (750 mL) of buffer B (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, 0.4 M NaCl), and collect the eluted sample. Monitor the 280 nm UV absorbance and conductivity throughout the process, and collect the target fraction at 5 mL / tube based on peak shape. After the experiment, rinse the system tubing and column sequentially with ultrapure water and 20% ethanol, and then seal the system and column with 20% ethanol.

[0073] (7) Dialysis desalination: Place the flow-through solution obtained in step (6) into a dialysis bag with a molecular weight cutoff of 12 kDa, and perform dialysis at 2°C with ultrapure water as the external solution. The external solution is changed no less than 3 times during the process, and the solution is changed once every 4 hours to completely remove the salt. (8) Concentration: The solution after dialysis in step (7) is concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 12 kDa to obtain concentrated protein solution; (9) Dispensing and freeze-drying: Dispense the concentrated protein solution obtained in step (8) and freeze it at -40°C or below, and then freeze-dry it to obtain freeze-dried powder, which is the high-purity morel active protein with α-glucosidase activity. Example 5

[0074] This invention provides a method for preparing morel active protein with α-glucosidase activity, comprising the following steps: (1) Put the dried morel fruiting bodies into a coarse powder mill and grind them for about 1-5 minutes. The particle size should be <3mm. Pass the powder through a 60-mesh sieve to obtain coarse powder of the fruiting bodies. (2) Put the coarse powder of the fruiting body into a low temperature cell wall breaking pulverizer, control the temperature at 15℃, break the cell wall for 30 min, and set the shaking time to 120 s for cell wall breaking treatment. Pass it through a 500 mesh sieve to obtain the broken cell wall powder of the fruiting body. (3) Mix the fruiting body cell wall breakage powder with anhydrous ethanol at a ratio of 1 g: 1.5 mL (W / V), stir evenly, place in a supercritical carbon dioxide extraction vessel, control the supercritical pressure at 340 bar, the extraction vessel temperature at 45℃, the pressure relief valve temperature at 85℃, process for 4 h, collect the residue powder, and dry at 40℃ for later use. (4) Dissolve the residue powder in pure water at a ratio of 1 g: 50 mL (W / V), place it in an ultrasonic extractor for ultrasonic-assisted low-temperature extraction, control the ultrasonic frequency at 600 W, treat at 25℃ for 30 min, centrifuge at 10000 rpm for 10 min, collect the supernatant, which is the fruiting body extract. (5) Desalting and buffer replacement: The fruiting body extract obtained in step (4) is loaded onto a desalting column, which is a HiTrap Desalting column with a column volume of 5 mL. It is eluted with 20 mM Na2HPO4–NaH2PO4 buffer (the concentration of Na2HPO4 is 20 mM, the concentration of NaH2PO4 is 20 mM, the pH of the buffer is 7.0, and the buffer also contains 50 mM NaCl) at a flow rate of 1-2 mL / min. The elution peak containing the target protein is collected (i.e., the molecular weight corresponding to the elution sample band detected by SDS-PAGE electrophoresis is 121.5 KDa), and the buffer replacement is completed. (6) Removal of impurities by ion exchange chromatography: The elution peak collected in step (5) is loaded onto a QHP anion exchange chromatography column equilibrated with the same buffer. The QHP anion exchange chromatography column is a HiTrap Q HP anion exchange chromatography column. The loading flow rate is 10-20 mL / min and the column volume is 150 mL. The flow-through liquid is collected. The flow-through liquid is the component rich in the target active protein. This experiment used an AKTA Pure 25 chromatography system with a QHP anion exchange column. The entire process was performed at room temperature with a flow rate of 10-20 mL / min, and the maximum system pressure was set to 0.5 MPa to protect the column. All buffers and samples were filtered through a 0.22 μm filter and degassed by sonication. First, the desalting column was equilibrated with approximately 450 mL of buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl) for three column volumes. Then, approximately 450 mL of filtered sample was loaded at a flow rate of 10-20 mL / min. After loading, elution was performed with buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl), and the protein peak was collected and flowed through the sample. The flow-through sample was then loaded with buffer A to equilibrate five column volumes of the QHP anion exchange column. After sample loading, equilibrate with approximately 5 column volumes (750 mL) of buffer A (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, containing 50 mM NaCl). Then elute with approximately 5 column volumes (750 mL) of buffer B (20 mM Na₂HPO₄–NaH₂PO₄ buffer, pH 7.0, 0.4 M NaCl), and collect the eluted sample. Monitor the 280 nm UV absorbance and conductivity throughout the process, and collect the target fraction at 5 mL / tube based on peak shape. After the experiment, rinse the system tubing and column sequentially with ultrapure water and 20% ethanol, and then seal the system and column with 20% ethanol.

[0075] (7) Dialysis desalination: Place the flow-through solution obtained in step (6) into a dialysis bag with a molecular weight cutoff of 14 kDa, and perform dialysis at 8°C using ultrapure water as the external solution. Replace the external solution at least 3 times during the process, and change the solution once every 4 hours to completely remove the salt. (8) Concentration: The solution after dialysis in step (7) is concentrated by centrifugation using an ultrafiltration centrifuge tube with a molecular weight cutoff of 14 kDa to obtain concentrated protein solution; (9) Dispensing and freeze-drying: Dispense the concentrated protein solution obtained in step (8) and freeze it at -40°C or below, and then freeze-dry it to obtain freeze-dried powder, which is the high-purity morel active protein with α-glucosidase activity.

[0076] Comparative Example 1 Based on Example 1, Comparative Example 1 omits step (2) of low-temperature cell wall disruption and pulverization. The method for preparing morel active protein provided in Comparative Example 1 includes the following steps: (1) Put the dried morel fruiting bodies into a coarse powder mill and crush them through a 10-mesh sieve to obtain coarse powder of the fruiting bodies; (2) Mix the fruiting body powder with anhydrous ethanol at a ratio of 1:1 (W / V), stir evenly, place in a supercritical carbon dioxide extraction vessel, control the supercritical pressure at 320 bar, the extraction vessel temperature at 40℃, the pressure relief valve temperature at 80℃, process for 2 h, collect the residue powder and dry it at 35℃ for later use. (3) Dissolve the residue powder in pure water at a ratio of 1:20 (W / V), place it in an ultrasonic extractor, control the ultrasonic frequency at 600 W, treat at 15℃ for 40 min, centrifuge at 8000 rpm for 15 min, collect the supernatant, which is the fruiting body extract; use the Bradford protein concentration assay kit to determine the protein content, and calculate the protein extraction yield (the percentage of protein mass to the mass of residue powder). Subsequent steps are the same as steps (5) to (9) in Example 1.

[0077] Using the protein extraction yield measured in Example 1 as 100%, the relative protein yield of Comparative Example 1 compared to Example 1 was calculated. The experimental results are as follows: Figure 6 As shown, the relative protein yield of Comparative Example 1 compared to Example 1 is 29.17%. This indicates that the lack of low-temperature cell wall disruption treatment resulted in a significant decrease in protein yield.

[0078] Comparative Example 2 Based on Example 1, Comparative Example 2 adjusted the temperature of the low-temperature cell wall breaking and pulverizing step (2) of Example 1 to 25°C and the cell wall breaking time to 40 min, while the rest were the same as in Example 1.

[0079] The α-glucosidase activity of the morel active protein prepared in Comparative Example 2 was detected, as was the α-glucosidase activity of the morel active protein prepared in Example 1. Using the α-glucosidase activity of the morel active protein prepared in Example 1 as 100%, the relative enzyme activity value of the morel active protein prepared in Comparative Example 2 was calculated relative to that prepared in Example 1.

[0080] The method for detecting the α-glucosidase activity of morel active protein includes the following steps: Take 10 μL of 10 mmol / L pNPG substrate solution and mix it with 140 μL of BR buffer (pH=6.0), preheat it at 55℃ for 5 min, then add 50 μL of morel active protein solution (the mass percentage of morel active protein to morel active protein solution is 10%), react at 55℃, time it precisely for 15 min, quickly add 600 μL of 1 mol / L Na2CO3 solution to terminate the reaction and develop color, mix well and centrifuge, take the supernatant, measure the absorbance at 405 nm, calculate the enzyme activity according to the standard curve equation, the standard curve equation is the same as (1) in Example 3, and determine the enzyme activity.

[0081] Experimental results are as follows Figure 7 As shown, it can be seen that due to the adjustment of the cell wall breaking temperature and time, the relative enzyme activity value of Comparative Example 2 is only 23.17% of that of Example 1. This indicates that the cell wall breaking conditions selected in Example 1 of the present invention are more conducive to improving the α-glucosidase activity of morel active protein.

[0082] Comparative Example 3 Based on Example 1, Comparative Example 3 adjusted the supercritical pressure of step (3) of Example 1 to 350 bar and the extraction vessel temperature to 50°C, while the rest were the same as in Example 1.

[0083] The α-glucosidase activity of the morel active protein prepared in Comparative Example 3 was detected, as was the α-glucosidase activity of the morel active protein prepared in Example 1. Using the α-glucosidase activity of the morel active protein prepared in Example 1 as 100%, the relative enzyme activity value of the morel active protein prepared in Comparative Example 3 was calculated relative to that prepared in Example 1. The method for determining the α-glucosidase activity of the morel active protein was the same as that for Comparative Example 2.

[0084] Experimental results are as follows Figure 8 As shown, the relative enzyme activity of Comparative Example 3 compared to Example 1 was 4.26%. This indicates that the supercritical pressure and extraction vessel temperature selected in Example 1 of this invention are more conducive to improving the α-glucosidase activity of morel active proteins.

[0085] Comparative Example 4 Based on Example 1, Comparative Example 4 adjusts step (4) of Example 1 as follows: Dissolve the residue powder in pure water at a ratio of 1 g: 40 mL (W / V), place it in an ultrasonic extractor for ultrasonic-assisted low-temperature extraction, control the ultrasonic frequency at 600 W, treat at 40℃ for 60 min, centrifuge at 7800 rpm for 15 min, and collect the supernatant, which is the fruiting body extract. The rest is the same as in Example 1.

[0086] The α-glucosidase activity of the morel active protein prepared in Comparative Example 4 was detected, as was the α-glucosidase activity of the morel active protein prepared in Example 1. Using the α-glucosidase activity of the morel active protein prepared in Example 1 as 100%, the relative enzyme activity value of the morel active protein prepared in Comparative Example 4 was calculated relative to that prepared in Example 1. The method for determining the α-glucosidase activity of the morel active protein was the same as that for Comparative Example 2.

[0087] Experimental results are as follows Figure 9As shown, the relative enzyme activity of Comparative Example 4 compared to Example 1 was 7.83%. This indicates that the ultrasonic low-temperature extraction temperature selected in Example 1 of this invention is more conducive to improving the α-glucosidase activity of morel active proteins.

[0088] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A morel active protein with α-glucosidase activity, characterized in that, Includes the amino acid sequence shown in SEQ ID No.

1.

2. The method for preparing morel active protein with α-glucosidase activity as described in claim 1, characterized in that, Includes the following steps: (1) The coarse powder of morel fruiting bodies was processed by cell wall breaking and pulverization to obtain cell wall broken powder of fruiting bodies; (2) Mix the fruiting body cell wall-breaking powder with anhydrous ethanol, perform supercritical carbon dioxide extraction, dry, and obtain residue; (3) The residue was subjected to ultrasonic-assisted low-temperature extraction to obtain fruiting body extract; (4) Desalt the fruiting body extract and collect the elution peaks; (5) Remove impurities from the elution peak using ion exchange chromatography and collect the flow-through solution; (6) Dialyze the flow-through solution to obtain the dialyzed solution; (7) Ultrafiltration concentration.

3. The method for preparing morel active protein with α-glucosidase activity according to claim 2, characterized in that, In step (1), the temperature for cell wall breaking and pulverizing is 4-15℃, and the time for cell wall breaking is 15-30 min.

4. The method for preparing morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, In step (2), the pressure of supercritical carbon dioxide extraction is 300-340 bar, the temperature of the extraction vessel is 35-45℃, the temperature of the pressure relief valve is 78-85℃, and the processing time is 2-4 h.

5. The method for preparing morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, In step (3), the temperature for ultrasonic-assisted low-temperature extraction is 15-25℃.

6. The method for preparing morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, In step (3), the ultrasonic-assisted low-temperature extraction time is 30-60 min, followed by centrifugation at 6000-10000 rpm for 10-30 min, and collection of the supernatant, which is the fruiting body extract.

7. The method for preparing morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, In step (4), a desalination column is used for desalination treatment.

8. The method for preparing morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, In step (5), the ion exchange chromatography is QHP anion exchange chromatography column chromatography.

9. A method for preparing a morel active protein with α-glucosidase activity according to claim 2 or 3, characterized in that, Ultrafiltration concentration is followed by a freeze-drying step.

10. The method for preparing morel active protein with α-glucosidase activity according to claim 9, characterized in that, The freeze-drying process also includes a step of identifying the active protein profile of morel mushrooms.

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