A method for purifying nattokinase

Abstract

The present invention belongs to the technical field of compound purification, and relates to a method for purifying nattokinase, which includes enzymatically hydrolyzing a nattokinase fermentation broth and then purifying the obtained enzymatically hydrolyzed solution; the method for enzymatic hydrolysis includes diluting the nattokinase fermentation broth with water to a viscosity of 180-250 mPa·s, and then enzymatically hydrolyzing the obtained nattokinase dilution with an enzyme preparation into an enzymatically hydrolyzed solution. The enzyme preparation is selected from at least one of trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme, and β-lactamase. The addition amount of the enzyme preparation is 0.1%-5% of the volume of the nattokinase fermentation broth. The conditions for enzymatic hydrolysis are such that the viscosity of the enzymatically hydrolyzed solution is reduced to below 100 mPa·s. Using the method provided by the present invention can not only obtain a high yield and purity of enzyme protein, but also reduce the loss of enzyme activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of compound purification, and particularly relates to a method for purifying nattokinase. Background Art

[0002] Thrombotic diseases are a class of diseases that seriously endanger human health and life, and are currently the second largest disease after cancer. In China, the improvement of people's living standards and the change of lifestyle have led to excessive intake of high-fat and high-protein in the daily diet. With the advent of an aging society, thrombotic diseases have become the number one health hazard. The number of people who need thrombolytic therapy each year exceeds 3 million, and the number of people who die from cardiovascular and cerebrovascular diseases each year is about 2 million, accounting for 40.7% of the total number of deaths from diseases. Therefore, the development of highly efficient and specific thrombolytic drugs is an important means for the prevention and treatment of cardiovascular and cerebrovascular diseases. However, the adverse reactions caused by thrombolytic drugs have also attracted people's attention, such as internal bleeding, re-infarction, allergic reactions, etc. Therefore, searching for and developing new thrombolytic agents is an important part of the treatment of cardiovascular and cerebrovascular diseases.

[0003] Research shows that the metabolites of microorganisms are an important source of thrombolytic enzymes. Among them, nattokinase (NK) produced by fermentation of Bacillus subtilis subsp. natto is a serine protease with high fibrinolytic activity. Compared with urokinase (UK) and streptokinase (SK) currently used in clinical practice, it has the advantages of high safety, good thrombolytic effect, low bleeding tendency, long half-life in vivo, easy absorption, and low cost. Nattokinase itself has relatively stable properties in a frozen environment and has a high enzyme activity retention rate within 24 - 30 months.

[0004] Nattokinase is a secondary metabolite produced by Bacillus natto during the fermentation process and is well-known to consumers for its good thrombolytic effect. In recent years, research on the extraction, purification, and development of related products of nattokinase has gradually become a research hotspot in this field. For example, CN103589706B discloses a method for purifying nattokinase. This method pre-treats the nattokinase fermentation broth by centrifugation and ultrafiltration to obtain an ultrafiltration concentrate, and then obtains a purified concentrate through preliminary chromatography on Sephadex, step-by-step chromatography on cation resin and anion resin. After that, crystallization and drying are carried out at 4°C to obtain nattokinase pure product. Although this method can obtain a relatively pure nattokinase product, dialysis for desalting after column chromatography and multiple passes through the column make the whole purification process time-consuming and cumbersome, and frequent column chromatography increases the risk of enzyme protein loss, which is not suitable for industrial scale-up production. Another example is that CN1690196A discloses a method for separating and purifying nattokinase by reverse micelle method. This method first centrifuges the nattokinase fermentation broth at low speed to obtain a crude enzyme solution, mixes the crude enzyme solution with an extractant in proportion for extraction, and then mixes the extraction phase with a stripping agent in proportion for stripping, and centrifuges at high speed to obtain a purified nattokinase solution. After crystallization and drying, nattokinase product is obtained. Although this method has a simple process and is easy to operate, the extractant and stripping agent contain organic solvents such as ethanol, propanol, and isopropanol, which have a certain destructive effect on enzyme proteins. Moreover, the purification method by extraction has problems such as low purification multiple, incomplete extraction, and solvent residue, and the purity is relatively low. Generally speaking, this method is also not suitable for industrial production after scale-up. Summary of the Invention

[0005] The object of the present invention is to overcome the problems of low enzyme protein yield or purity and large loss of enzyme activity when separating and purifying nattokinase fermentation broth by existing methods, and to provide a new method for purifying nattokinase. By using the method provided by the present invention, not only can a high enzyme protein yield and purity be obtained, but also the loss of enzyme activity can be reduced.

[0006] In view of the existing nattokinase production process, the existing purification methods generally involve first performing solid-liquid separation on the fermentation broth (usually centrifugal separation), then selectively performing salting out or ultrafiltration, and finally purifying through a chromatography medium one or more times to obtain pure nattokinase. Therefore, current research on nattokinase purification focuses on the purification process parameters and purification media after solid-liquid separation. However, after in-depth and extensive research, the inventors of the present invention found that due to the high viscosity of the nattokinase fermentation broth, general solid-liquid separation methods not only make it difficult to effectively separate nattokinase from impurities, but also easily cause loss of enzyme protein, which has become a major difficulty in the nattokinase purification process. Trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme, and β-lactamase are all proteases with the ability to cleave amide bonds and have strong hydrolysis ability for animal and plant proteins, polypeptides, amides, etc. Therefore, in order to avoid the hydrolysis effect on nattokinase, those skilled in the art generally do not use the above proteases for enzymatic hydrolysis to reduce the viscosity of the fermentation broth. However, after in-depth and extensive research, the inventors of the present invention also found that enzymatic hydrolysis of the nattokinase fermentation broth with enzyme preparations such as trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme, and β-lactamase in a specific dosage (0.1%-5% of the volume of the nattokinase fermentation broth) can significantly reduce the viscosity of the fermentation broth on the basis of avoiding hydrolysis of nattokinase. Subsequently, combined with conventional purification methods, the entire separation process can operate stably and conveniently, which is more conducive to improving the yield and purity of nattokinase and reducing the loss of enzyme activity. In view of this, the present invention was completed.

[0007] Specifically, the present invention provides a method for purifying nattokinase, which includes enzymatically hydrolyzing the nattokinase fermentation broth and then purifying the obtained enzymatically hydrolyzed solution; the method of enzymatic hydrolysis includes diluting the nattokinase fermentation broth with water to a viscosity of 180-250 mPa·s, and then enzymatically hydrolyzing the obtained nattokinase dilution with an enzyme preparation into an enzymatically hydrolyzed solution. The enzyme preparation is selected from at least one of trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme, and β-lactamase. The addition amount of the enzyme preparation is 0.1%-5% of the volume of the nattokinase fermentation broth, and the conditions of enzymatic hydrolysis reduce the viscosity of the enzymatically hydrolyzed solution to below 100 mPa·s.

[0008] In a preferred embodiment, the conditions of enzymatic hydrolysis reduce the viscosity of the enzymatically hydrolyzed solution to 7-10 mPa·s.

[0009] In a preferred embodiment, the conditions of enzymatic hydrolysis include a temperature of 30-50°C and a time of 15-120 min.

[0010] In a preferred embodiment, the method of purification includes the following steps carried out in sequence:

[0011] S1. Microfiltration: The enzymolysis solution is microfiltered using a ceramic membrane with a pore size of 50 - 500 nm, and the obtained permeate is nattokinase enzyme clarified solution;

[0012] S2. Ultrafiltration: The nattokinase enzyme clarified solution is ultrafiltered using an ultrafiltration membrane with a molecular weight cut-off of 2 - 25 kDa, and the obtained retentate is nattokinase concentrate;

[0013] S3. Salting out: The crude nattokinase enzyme solution is salted out and then subjected to solid-liquid separation, and the obtained solid product is crude nattokinase;

[0014] S4. Refinement: The crude nattokinase is purified by column chromatography and electrophoresis in sequence to obtain pure nattokinase.

[0015] In a preferred embodiment, in step S1, the conditions of the microfiltration include that the temperature of the enzymolysis solution is 10 - 50 °C, the membrane pressure is 0.1 - 0.6 MPa, and the microfiltration time is based on the time when the volume of the permeate reaches 85% - 90% of the volume of the enzymolysis solution.

[0016] In a preferred embodiment, in step S2, the conditions of the ultrafiltration include that the temperature of the nattokinase enzyme clarified solution is 8 - 40 °C, the membrane pressure is 0.01 - 0.5 MPa, and the ultrafiltration time is based on the time when the volume ratio of the retentate to the nattokinase fermentation broth reaches 1:(3 - 20).

[0017] In a preferred embodiment, in step S3, the salting-out method is to add metal salt I to the crude nattokinase enzyme solution, stir until metal salt I is basically dissolved and then let it stand to complete the first-stage salting out. Then, the obtained first-stage salting-out solution is subjected to first-stage solid-liquid separation. Add metal salt II to the obtained liquid product, stir until metal salt II is basically dissolved, adjust the pH value of the solution to 7 - 10, and then let it stand at 4 - 10 °C to complete the second-stage salting out. Then, the obtained second-stage salting-out solution is subjected to second-stage solid-liquid separation, and the obtained solid product is crude nattokinase.

[0018] In a preferred embodiment, the metal salt I and the metal salt II are each independently selected from at least one of sodium sulfate, potassium sulfate, ammonium sulfate, sodium chloride, and potassium chloride.

[0019] In a preferred embodiment, the salt saturation of the first-stage salting out is 5 - 50%, and the salt saturation of the second-stage salting out is 30 - 80%.

[0020] In a preferred embodiment, the time of the first-stage salting out is 1 - 25 h, and the time of the second-stage salting out is 1 - 48 h.

[0021] In a preferred embodiment, in step S4, the methods of column chromatography purification and electrophoresis purification are as follows: after activating the packing material, it is loaded into a chromatography column, balanced with phosphate buffer solution, the crude nattokinase is redissolved with water to prepare a sample loading solution and then loaded onto the column, and then eluted with phosphate buffer solution containing sodium chloride. The eluate rich in nattokinase is collected and subjected to electrophoresis. The chromatography solution with a bright band of nattokinase is vacuum dried to obtain pure nattokinase product.

[0022] In a preferred embodiment, the pH values of the phosphate buffer solution and the phosphate buffer solution containing sodium chloride are independently 6 - 10, and the concentrations are independently 0.1 - 10.0 mol / L.

[0023] In a preferred embodiment, the concentration of sodium chloride in the phosphate buffer solution containing sodium chloride is 0.05 - 5 mol / L.

[0024] In a preferred embodiment, the concentration of nattokinase in the sample loading solution is 5 - 100 mg / mL.

[0025] In a preferred embodiment, the volume of the sample loading solution is 1% - 50% of the volume of the chromatography column.

[0026] In a preferred embodiment, the elution flow rate is 0.2 - 5 BV / h.

[0027] In a preferred embodiment, in step S4, the packing material used for column chromatography purification is selected from at least one of dextran gel, cation exchange resin, anion exchange resin and macroporous adsorption resin.

[0028] The key of the present invention lies in that before purification by conventional methods, the nattokinase fermentation broth is enzymatically hydrolyzed with enzyme preparations of specific types and specific dosages until the viscosity is reduced to below 100 mPa·s, thereby significantly improving the yield and purity of nattokinase and reducing the loss of enzyme activity. In addition, the method provided by the present invention also has the advantages of simple operation, environmental protection, energy saving and high efficiency, short cycle, continuous operation, etc., and has a wide application prospect. Detailed embodiments

[0029] According to the present invention, the nattokinase fermentation broth can be obtained by various existing methods. In a preferred embodiment, the nattokinase fermentation broth is prepared according to the following method:

[0030] S1. Strain activation: Under aseptic conditions, the Bacillus natto strain is inoculated into a plate medium with a pH value of 6.5 - 7.5 according to the gradient dilution method, and cultured at 30 - 40 °C for 24 - 36 h for activation to obtain mature single colonies; the formula of the plate medium is as follows: tryptone 2 - 20 g / L, beef extract 1 - 10 g / L, yeast extract 1 - 10 g / L, sodium chloride 5 - 15 g / L, agar 10 - 20 g / L, and glucose 1 - 10 g / L;

[0031] S2. Shake flask culture: Pick any single colony from the cultured plate medium, inoculate it into a shake flask medium with a pH value of 6.5 - 7.5 at an inoculation amount of 0.5% - 10%, then place it in a shaker and culture it at a temperature of 30 - 40 °C and a rotation speed of 150 - 200 rpm for 20 - 30 h; the formula of the shake flask medium is as follows: tryptone 35 - 45 g / L, yeast extract 1 - 10 g / L, sodium chloride 5 - 15 g / L, glucose 30 - 40 g / L, and beef extract 5 - 15 g / L;

[0032] S3. Seed tank culture: The strain after shake flask culture is inoculated into a seed tank medium at an inoculation amount of 0.1% - 10%, and then cultured at a temperature of 30 - 40 °C, a tank pressure of 0.02 - 0.08 MPa, an aeration ratio of 0.3 - 2.5 VVM, and a rotation speed of 50 - 800 rpm for 10 - 40 h. Continue to culture for 2 - 3 generations under the above conditions for strain rejuvenation to obtain a seed solution; the formula of the seed tank medium is as follows: peptone 2 - 20 g / L, sodium chloride 2 - 20 g / L, and soybean cake powder 2 - 20 g / L;

[0033] S4. Fermentation culture: The cultured seed solution is inoculated into a fermentation tank medium with a pH value of 7 - 7.5 at an inoculation amount of 0.1% - 20%, and fermentation culture is carried out at a temperature of 30 - 40 °C. Monitor the changes in the OD value and pH value of the fermentation broth throughout the fermentation process, and adjust the pH value in real time to keep it within the range of 7.0 - 7.5. Continue to culture until 24 - 25 h and then discharge the tank.

[0034] According to the present invention, the viscosity of the nattokinase fermentation broth is generally 280 - 320 mPa·s, such as 280, 290, 300, 310, 320 mPa·s or any value therebetween. The viscosity of the nattokinase dilution obtained by diluting the nattokinase fermentation broth is 180 - 250 mPa·s, such as 180, 190, 200, 210, 220, 230, 240, 250 mPa·s or any value therebetween. The viscosity of the enzymatic hydrolysate obtained by enzymatically hydrolyzing the nattokinase dilution is below 100 mPa·s, such as 1, 2, 5, 7, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mPa·s or any value therebetween, preferably 1 - 100 mPa·s, and more preferably 7 - 10 mPa·s.

[0035] According to the present invention, the enzyme preparation is selected from at least one of trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme and β-lactamase. Fermenting the fermentation broth with the above several enzyme preparations can not only effectively reduce the viscosity of the fermentation broth, improve the product yield, but also reduce the dilution volume of the material, with advantages such as simple operation, environmental friendliness, energy saving and high efficiency, and small loss of enzyme activity. While solving the problem of difficult treatment of the nattokinase fermentation broth due to high viscosity, it also significantly reduces the production cost. The addition amount of the enzyme preparation is 0.1% - 5% of the volume of the nattokinase fermentation broth, such as 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc. Trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme and β-lactamase are all proteases and have the function of shearing amide bonds, and have strong hydrolysis ability for animal and plant proteins, polypeptides, amides, etc. Therefore, in order to avoid the hydrolysis effect on nattokinase, those skilled in the art generally do not use the above proteases to enzymatically hydrolyze and reduce the viscosity of the fermentation broth. However, the inventor of the present invention unexpectedly found during the experiment that when the dosage of the enzyme preparation is controlled within a certain range, the viscosity of the fermentation broth can be perfectly reduced, and basically no hydrolysis effect is produced on nattokinase, and the effect is very ideal. In addition, although trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme and β-lactamase can all reduce the viscosity of the fermentation broth without substantially affecting nattokinase, when papain and / or polyglutamic acid polypeptide enzyme is selected, the effect is better, and the yield and purity of nattokinase can reach a higher level, and the loss of enzyme activity is also smaller.

[0036] According to the present invention, the conditions for enzymatic hydrolysis are such that the viscosity of the enzymatic hydrolysate is reduced to less than 100 mPa·s, preferably reduced to 7 - 10 mPa·s. Specifically, the conditions for enzymatic hydrolysis include that the temperature can be 30 - 50 °C, such as 30 °C, 32 °C, 35 °C, 37 °C, 40 °C, 42 °C, 45 °C, 48 °C, 50 °C or any value between them; the time can be 15 - 120 min, such as 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, 100 min, 110 min, 120 min or any value between them.

[0037] The key of the present invention lies in enzymatically hydrolyzing the nattokinase fermentation broth to less than 100 mPa·s with an enzyme preparation of a specific type and dosage before purification. In this way, not only can the loss of nattokinase be reduced during the subsequent purification process, but also impurities can be better removed, thereby improving the yield and purity of nattokinase. Among them, the purification method can be a conventional choice in the art. In a preferred embodiment, the purification method includes the following steps carried out in sequence: S1. Microfiltration: The enzymatic hydrolysate is microfiltered with a ceramic membrane having a pore size of 50 - 500 nm, and the obtained permeate is the nattokinase enzyme clear solution; S2. Ultrafiltration: The nattokinase enzyme clear solution is ultrafiltered with an ultrafiltration membrane having a molecular weight cut-off of 2 - 25 kDa, and the obtained retentate is the nattokinase concentrate; S3. Salting out: The crude nattokinase solution is subjected to salting out and then solid-liquid separation, and the obtained solid product is the crude nattokinase; S4. Refining: The crude nattokinase is purified by column chromatography and electrophoresis in sequence to obtain the nattokinase pure product.

[0038] According to the present invention, in step S1, the purpose of microfiltration with a ceramic membrane is to remove most of the bacteria, fermentation broth residues and macromolecular proteins, and obtain a clear nattokinase enzyme clear solution. Utilizing the characteristics of high microfiltration efficiency, large mechanical strength, acid and alkali resistance and resistance to microorganisms of the ceramic membrane, nattokinase is permeated together with the permeate, while impurities, bacteria and macromolecular proteins in the fermentation broth are retained by the membrane tube. After passing the fermentation broth through the ceramic membrane, the separation of the permeate and the retentate realizes the preliminary extraction of nattokinase. This operation has the advantages of high separation degree of enzyme protein, low energy consumption, short cycle and continuous operation. The pore size of the ceramic membrane used is 50 - 500 nm, such as 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm or any value between them. The ceramic membrane can be a single ceramic membrane or two or more ceramic membranes can be used in series. The ceramic membrane is equipped with a ceramic membrane core with a filtering function, which is well-known to those skilled in the art and will not be elaborated here.

[0039] According to the present invention, in step S1, the conditions for microfiltration generally include that the temperature of the enzymatic hydrolysate can be 10 - 50 °C, such as 10 °C, 20 °C, 30 °C, 40 °C, 50 °C or any value between them; the membrane pressure can be 0.1 - 0.6 MPa, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6 MPa or any value between them; the time for microfiltration is based on when the volume of the permeate reaches 85% - 90% (such as 85%, 86%, 87%, 88%, 89%, 90% or any value between them) of the volume of the enzymatic hydrolysate. In the present invention, the pressure refers to the gauge pressure.

[0040] According to the present invention, in step S2, the purpose of ultrafiltration using an ultrafiltration membrane is to remove small molecular weight impurity proteins and obtain a concentrated solution with a relatively high content of the target enzyme protein. The molecular weight cut-off of the ultrafiltration membrane is 2 - 25 kDa, such as 2 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa or any value between them. During ultrafiltration, by controlling the temperature of the feed liquid and the membrane pressure within a suitable range, ultrafiltration is stopped when the volume ratio of the concentrated solution to the fermentation broth reaches an appropriate ratio. Specifically, during ultrafiltration, the temperature of the nattokinase enzyme supernatant is preferably controlled at 8 - 40 °C, such as 8 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C or any value between them; the membrane pressure is preferably controlled at 0.01 - 0.5 MPa, such as 0.01, 0.05, 0.06, 0.1, 0.2, 0.3, 0.4, 0.5 MPa or any value between them; the time for ultrafiltration is preferably based on when the volume ratio of the retentate to the nattokinase fermentation broth reaches 1:(3 - 20) (such as 1:3, 1:5, 1:7, 1:10, 1:12, 1:15, 1:18, 1:20 or any value between them).

[0041] According to the present invention, in step S3, the crude nattokinase enzyme solution is subjected to salting out and then solid-liquid separation, and the obtained solid product is the crude nattokinase. In a preferred embodiment, the salting out is carried out by a two-stage salting out method. Specifically, metal salt I is added to the crude nattokinase enzyme solution, and after stirring until metal salt I is basically dissolved, it is left standing at room temperature to complete the first-stage salting out. Then, the obtained first-stage salting out solution is subjected to first-stage solid-liquid separation. Metal salt II is added to the obtained liquid product, and after stirring until metal salt II is basically dissolved, the pH value of the solution is adjusted to 7-10. Then, it is left standing at 4-10 °C to complete the second-stage salting out. Then, the obtained second-stage salting out solution is subjected to second-stage solid-liquid separation, and the obtained solid product is the crude nattokinase. The inventors of the present invention found during the experiment that in the two-stage salting out operation, if the pH value of the solution is adjusted to a specific range of 7-10 (such as 7, 7.5, 8, 8.5, 9, 9.5, 10 or any value between them), preferably adjusted to 8.3-8.9 (such as 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or any value between them), compared with not adjusting the pH value, the unit yield of the crude nattokinase can be greatly increased. Through experiments, it can be increased by more than 20%. The inventors of the present invention found through experiments that as the pH value of the salting out solution continuously increases, the yield of the crude product also increases. The yield reaches the maximum at pH 8.5, and there is no obvious change in the yield when the pH value is changed again. On the contrary, the protein content of the second-stage salting out filtrate gradually decreases with the increase of the pH value, and the protein content of the filtrate reaches the lowest when the yield of the crude product is the highest, indicating that most of the nattokinase proteins are precipitated under this pH value condition, resulting in an increase in the yield.

[0042] According to the present invention, for the sake of distinction and description, the metal salt used in the first-stage salting-out is called "metal salt I", and the metal salt used in the second-stage salting-out is called "metal salt II"; the first salting-out is called "first-stage salting-out", and the second salting-out is called "second-stage salting-out"; the first solid-liquid separation is called "first-stage solid-liquid separation", and the second solid-liquid separation is called "second-stage solid-liquid separation". The types of the metal salt I and the metal salt II may be the same or different, and are preferably independently selected from at least one of sodium sulfate, potassium sulfate, ammonium sulfate, sodium chloride and potassium chloride. The function of the first-stage salting-out is to precipitate impurities, and the function of the second-stage salting-out is to precipitate nattokinase. The salt saturation of the first-stage salting-out is preferably 5-50%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or any value therebetween. The salt saturation of the second-stage salting-out is preferably 30-80%, such as 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80% or any value therebetween. The time of the first-stage salting-out is preferably 1-25 h, such as 1 h, 2 h, 5 h, 8 h, 10 h, 12 h, 15 h, 18 h, 20 h or any value therebetween. The time of the second-stage salting-out is 1-48 h, such as 1 h, 2 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h or any value therebetween. The first-stage solid-liquid separation and the second-stage solid-liquid separation are both preferably centrifugal separation. The time of the two centrifugal separations is preferably independently 3-50 min, such as 3 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min or any value therebetween; the rotational speeds of the two centrifugal separations are preferably independently 4000-6000 r / min, such as 4000, 4200, 4500, 4800, 5000, 5200, 5500, 5800, 6000 r / min or any value therebetween.

[0043] According to the present invention, in step S4, the crude nattokinase product is purified by column chromatography and electrophoresis in sequence to obtain the nattokinase pure product. In a specific embodiment, the methods of column chromatography purification and electrophoresis purification are as follows: after activating the packing material, it is loaded into a chromatography column and balanced with a phosphate buffer solution. The crude nattokinase product is redissolved with water to prepare a sample loading solution and then loaded onto the column. Then, it is eluted with a phosphate buffer solution containing sodium chloride, and the eluate rich in nattokinase is collected and subjected to electrophoresis. The chromatography solution with a bright nattokinase band is vacuum dried to obtain the nattokinase pure product.

[0044] According to the present invention, the packing material used in the column chromatography purification can be various existing substances capable of separating nattokinase from impurities, and specific examples thereof include, but are not limited to, at least one of dextran gel, cation exchange resin, anion exchange resin, and macroporous adsorption resin. During the column chromatography purification process, the pH values of the phosphate buffer solution and the phosphate buffer solution containing sodium chloride are preferably independently 6-10, such as 6, 7, 7.4, 8, 9, or 10. The concentrations of the phosphate buffer solution and the phosphate buffer solution containing sodium chloride are preferably independently 0.1-10.0 mol / L, such as 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mol / L or any value therebetween. In addition, the concentration of sodium chloride in the phosphate buffer solution containing sodium chloride is preferably 0.05-5 mol / L, such as 0.05, 0.1, 0.11, 0.15, 0.5, 1, 2, 3, 4, 5 mol / L or any value therebetween. The concentration of nattokinase in the sample loading solution is preferably 5-100 mg / mL, such as 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 mg / mL or any value therebetween. The volume of the sample loading solution is preferably 1%-50% of the volume of the chromatography column, such as 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or any value therebetween. The elution flow rate is preferably 0.2-5 BV / h, such as 0.2, 0.5, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 BV / h or any value therebetween.

[0045] According to the present invention, the form of the electrophoresis purification only needs to be able to further separate nattokinase from impurities. Gel electrophoresis is preferably used, polyacrylamide gel electrophoresis (PAGE) is preferably used, and denaturing polyacrylamide gel electrophoresis (SDS-PAGE) is more preferably used.

[0046] The present invention will be described in detail below through examples.

[0047] In the following examples and comparative examples, trypsin was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., enzyme activity specification: 250 USPu / mg; papain was purchased from Shanghai Yuanye Bio-Technology Co., Ltd., enzyme activity specification: 800 u / mg; carboxypeptidase was purchased from Putai Bio-Technology Co., Ltd., enzyme activity specification: 100 u / mg; polyglutamic acid polypeptide enzyme was purchased from Shanghai Sigma Bio-Technology Co., Ltd., enzyme activity specification: 500 u / mg; β-lactamase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., enzyme activity specification: 300 u / mg.

[0048] In the following examples and comparative examples, the yield = the actual yield of nattokinase / the theoretical yield of nattokinase × 100%, the purity was measured by liquid chromatography, and the enzyme activity was measured by an ultraviolet spectrophotometer.

[0049] Preparation Example 1 Preparation of Nattokinase Fermentation Broth

[0050] (1) Strain activation: Under aseptic conditions, the Bacillus natto strain (CGMCC NO. 18081) was inoculated into a slant medium (formula: tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, glucose 5 g / L, pH value 7.0) by gradient dilution method, and cultured at 35 °C for 36 h for activation to obtain activated single colonies.

[0051] (2) Seed liquor preparation: Any single colony was picked from the cultured activated single colonies, and inoculated into a shake flask containing a medium (formula: tryptone 40 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 35 g / L, beef extract 10 g / L, pH = 7.0) at an inoculation amount of 3%, placed in a shaker and cultured at 35 °C and 180 r / min for 25 h, and then inoculated into a seed tank at an inoculation amount of 4%, and continued to culture for 3 generations under the same conditions for strain rejuvenation to obtain the seed liquor required for tank fermentation.

[0052] (3) Fermentation culture: The cultured seed liquor was inoculated into a fermenter at an inoculation amount of 4%, and fermentation culture was carried out under the conditions of a temperature of 35 °C and a pH of 7.0. During the whole fermentation process, the OD value and pH change of the fermentation broth were monitored, and the pH was adjusted in real time to keep it in the range of 7.0 - 7.5. When the culture continued to 25 h, the tank was discharged to obtain nattokinase fermentation broth with a viscosity of 320 mPa·s and an enzyme activity of 830 FU / mL.

[0053] Preparation Example 2 Preparation of Nattokinase Fermentation Broth

[0054] (1) Strain activation: Under aseptic conditions, the Bacillus natto strain (CGMCC NO. 18081) was inoculated into a slant medium (formula: tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, glucose 5 g / L, pH value 7.0) by gradient dilution method, and cultured at 35 °C for 24 h for activation to obtain activated single colonies.

[0055] (2) Seed liquid preparation: Pick any single colony from the well-cultured activated single colony, and inoculate it again into a shake flask containing a medium (formula: tryptone 40 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 35 g / L, beef extract 10 g / L, pH = 7.0) at an inoculation amount of 3%. After placing it in a shaker, culture it for 25 h under the conditions of 35 °C and 180 r / min. Then, inoculate it into a seed tank at an inoculation amount of 4% and continue to culture it for 2 generations under the same conditions for strain rejuvenation to obtain the seed liquid required for tank fermentation.

[0056] (3) Fermentation culture: Inoculate the well-cultured seed liquid into a fermenter at an inoculation amount of 4%, and carry out fermentation culture under the conditions of a temperature of 35 °C and a pH of 7.0. Monitor the changes in the OD value and pH of the fermentation broth throughout the fermentation process, and adjust the pH in real time to keep it within the range of 7.0 - 7.5. Continue to culture until 25 h and then discharge the tank to obtain nattokinase fermentation broth, whose viscosity is 280 mPa·s and enzyme activity is 680 FU / mL.

[0057] Preparation Example 3 Preparation of Nattokinase Fermentation Broth

[0058] (1) Strain activation: Under a sterile environment, inoculate the Bacillus natto strain (CGMCC NO.18081) into a slant medium (formula: tryptone 10 g / L, beef extract 5 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, glucose 5 g / L, pH value is 7.0) according to the gradient dilution method, and culture it at 35 °C for 30 h for activation to obtain activated single colonies.

[0059] (2) Seed liquid preparation: Pick any single colony from the well-cultured activated single colony, and inoculate it again into a shake flask containing a medium (formula: tryptone 40 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, glucose 35 g / L, beef extract 10 g / L, pH = 7.0) at an inoculation amount of 3%. After placing it in a shaker, culture it for 25 h under the conditions of 35 °C and 180 r / min. Then, inoculate it into a seed tank at an inoculation amount of 4% and continue to culture it for 3 generations under the same conditions for strain rejuvenation to obtain the seed liquid required for tank fermentation.

[0060] (3) Fermentation culture: Inoculate the well-cultured seed liquid into a fermenter at an inoculation amount of 4%, and carry out fermentation culture under the conditions of a temperature of 35 °C and a pH of 7.0. Monitor the changes in the OD value and pH of the fermentation broth throughout the fermentation process, and adjust the pH in real time to keep it within the range of 7.0 - 7.5. Continue to culture until 25 h and then discharge the tank to obtain nattokinase fermentation broth, whose viscosity is 300 mPa·s and enzyme activity is 709 FU / mL.

[0061] Example 1 Purification of Nattokinase

[0062] S1`, Enzymatic hydrolysis of fermentation broth: Add an equal volume of pure water to the nattokinase fermentation broth obtained in Preparation Example 1, stir evenly for preliminary dilution to obtain a nattokinase dilution with a viscosity of 250 mPa·s. Add papain accounting for 0.2% of the volume of the fermentation broth, stir evenly, heat to raise the internal temperature of the fermentation broth to 37 °C, and stir and control the temperature for 30 min. During stirring, continuously detect the viscosity of the fermentation broth. When the viscosity drops to 10 mPa·s, stop the enzymatic hydrolysis treatment to obtain an enzymatic hydrolysate.

[0063] S2`, Microfiltration: Microfilter the enzymatic hydrolysate using a 300 nm ceramic membrane, with a transmembrane pressure of 0.3 MPa. During microfiltration, control the material circulation temperature at 30 °C. After the microfiltration runs stably, sample and microscopically examine the permeate and retentate. Use the permeation amount of macromolecular proteins and bacteria to measure the microfiltration effect. When the volume recovery rate of the permeate reaches 85% of the enzymatic hydrolysis fermentation broth, stop microfiltration, and subject the permeate (nattokinase enzyme supernatant) to subsequent ultrafiltration and concentration.

[0064] S3`, Ultrafiltration: Ultrafilter and concentrate the nattokinase enzyme supernatant through an ultrafiltration membrane with a molecular weight cut-off of 15 kDa. The concentration conditions include a device frequency of 37 Hz and a membrane pressure of 0.06 MPa. During ultrafiltration, control the feed liquid circulation temperature at 30 °C. When the volume of the concentrated liquid reaches 1 / 7 of the volume of the fermentation broth, stop ultrafiltration and collect the retentate, which is the nattokinase concentrate.

[0065] S4`, Salting out: Accurately measure the nattokinase concentrate, add solid ammonium sulfate at a salt saturation of 20%, stir until all the salt is dissolved, and then let it stand at room temperature for 5 h for primary salting out. Then filter the salting-out solution through a 0.45 μm filter membrane, collect the filtrate, and add solid ammonium sulfate to the filtrate at a salt saturation of 60%. After the salt is completely dissolved, adjust the pH of the salting-out solution to 8.3 with 30% sodium hydroxide solution. Subsequently, let it stand at 6 °C for 15 h for secondary salting out. After standing, centrifuge at 5000 r / min for 10 min, and collect the lower brown precipitate, which is the crude nattokinase.

[0066] S5`, Purification: Fully swell Sephadex G-50 dextran gel and pack it into a column. Equilibrate the column with a phosphate buffer solution with a concentration of 0.2 mol / L and a pH value of 7.4. Prepare the crude nattokinase into a sample loading solution with a protein content of 25 mg / mL, and filter it through a 0.45 μm filter head. Load the sample at 15% of the column volume, and elute with the above phosphate buffer solution (containing 0.11 mol / L sodium chloride). The elution flow rate is 0.5 BV / h. Collect each part of the eluate for SDS-PAGE protein electrophoresis experiment. Combine the chromatographic solutions with bright nattokinase bands and vacuum freeze-dry them to obtain a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 90%, the purity is 99%, and the enzyme activity is 250760 FU / g.

[0067] Example 2 Purification of Nattokinase

[0068] S1`: Enzymatic hydrolysis of fermentation broth: Add an equal volume of pure water to the nattokinase fermentation broth obtained in Preparation Example 2, stir evenly for preliminary dilution to obtain a nattokinase dilution with a viscosity of 180 mPa·s. Add 5% of the volume of the fermentation broth of trypsin and stir evenly. Heat to raise the internal temperature of the fermentation broth to 30 °C and stir for temperature control for 120 min. During stirring, the viscosity of the fermentation broth is detected in real time. When the viscosity drops to 7 mPa·s, stop the enzymatic hydrolysis treatment to obtain an enzymatic hydrolysate.

[0069] S2`: Microfiltration: Microfilter the enzymatic hydrolysate with a 50 nm ceramic membrane. The transmembrane pressure is 0.1 MPa. During microfiltration, control the material circulation temperature at 30 °C. After the microfiltration runs stably, sample and microscopy the permeate and the retentate. Measure the microfiltration effect by the permeation amount of macromolecular proteins and bacteria. When the volume yield of the permeate reaches 90% of the enzymatic hydrolysis fermentation broth, stop microfiltration. The permeate (nattokinase enzyme clarified liquid) is subjected to subsequent ultrafiltration and concentration.

[0070] S3`: Ultrafiltration: Ultrafilter and concentrate the nattokinase enzyme clarified liquid through an ultrafiltration membrane with a molecular weight cut-off of 2 kDa. The concentration conditions include a device frequency of 37 Hz and a membrane pressure of 0.1 MPa. During ultrafiltration, control the material liquid circulation temperature at 30 °C. When the volume of the concentrated liquid reaches 1 / 3 of the volume of the fermentation broth, stop ultrafiltration and collect the retentate, which is the nattokinase concentrated liquid.

[0071] S4`: Salting out: Accurately measure the nattokinase concentrated liquid, add solid ammonium sulfate according to 5% salt saturation, stir until all the salt is dissolved, and then let it stand at room temperature for 25 h for primary salting out. Then filter the salting out solution through a 0.45 μm filter membrane, collect the filtrate, and add solid ammonium sulfate to the filtrate according to 30% salt saturation. After the salt is completely dissolved, adjust the pH of the salting out solution to 8.9 with 30% sodium hydroxide solution. Then let it stand at 10 °C for 48 h for secondary salting out. After standing, centrifuge at 5000 r / min for 10 min, and collect the brown precipitate at the bottom layer, which is the crude nattokinase.

[0072] S5`, Purification: After fully swelling the cation exchange resin XP-485, pack it into a column, balance the column with a phosphate buffer solution with a concentration of 0.2 mol / L and a pH value of 7.4, prepare the crude nattokinase into a sample loading solution with a protein content of 20 mg / mL, filter it with a 0.45 μm filter head, load the sample at 15% of the column volume, elute it with the above-mentioned phosphate buffer solution (containing 0.11 mol / L sodium chloride), the elution flow rate is 0.2 BV / h, collect each part of the eluate for SDS-PAGE protein electrophoresis experiment, and vacuum freeze-dry the chromatography solution with bright nattokinase bands combined to obtain the pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 57%, the purity is 76%, and the enzyme activity is 91000 FU / g.

[0073] Example 3 Purification of Nattokinase

[0074] S1`, Enzymatic hydrolysis of fermentation broth: Add an equal volume of pure water to the nattokinase fermentation broth obtained in Preparation Example 3, stir evenly for preliminary dilution to obtain a nattokinase dilution with a viscosity of 200 mPa·s, add 2% of the fermentation broth volume of carboxypeptidase and stir evenly, heat to raise the internal temperature of the fermentation broth to 50 °C and stir for temperature control for 15 min, and continuously detect the viscosity of the fermentation broth during stirring. When the viscosity drops to 8 mPa·s, stop the enzymatic hydrolysis treatment to obtain the enzymatic hydrolysate.

[0075] S2`, Microfiltration: Microfilter the enzymatic hydrolysate with a 500 nm ceramic membrane, the transmembrane pressure is 0.6 MPa, control the material circulation temperature at 30 °C during microfiltration, and take samples of the permeate and retentate for microscopic examination after the microfiltration runs stably. Measure the microfiltration effect by the permeation amount of macromolecular proteins and bacteria. When the volume recovery rate of the permeate reaches 88% of the enzymatic hydrolysis fermentation broth, stop microfiltration, and perform subsequent ultrafiltration concentration on the permeate (nattokinase enzyme clarified solution).

[0076] S3`, Ultrafiltration: Ultrafilter and concentrate the nattokinase enzyme clarified solution through an ultrafiltration membrane with a molecular weight cut-off of 25 kDa. The concentration conditions include a device frequency of 37 Hz and a membrane pressure of 0.5 MPa. Control the feed liquid circulation temperature at 30 °C during ultrafiltration. When the volume of the concentrated liquid reaches 1 / 20 of the fermentation broth volume, stop ultrafiltration and collect the retentate, which is the nattokinase concentrated liquid.

[0077] S4`, Salting out: Accurately measure the nattokinase concentrate, add solid ammonium sulfate at a salt saturation of 50%, stir until all the salt is dissolved, and then let it stand at room temperature for 2 h for primary salting out. Then, filter the salting-out solution through a 0.45-μm filter membrane, collect the filtrate, add solid ammonium sulfate to the filtrate at a salt saturation of 80%, adjust the pH of the salting-out solution to 8.5 with 30% sodium hydroxide solution after the salt is completely dissolved, and then let it stand at 4 °C for 10 h for secondary salting out. After standing, centrifuge at 5000 r / min for 10 min, and collect the brown precipitate at the bottom, which is the crude nattokinase product.

[0078] S5`, Purification: Fully swell the anion exchange resin D201 and load it into a column, perform column equilibration with a phosphate buffer solution with a concentration of 0.2 mol / L and a pH value of 7.4, prepare the crude nattokinase product into a sample loading solution with a protein content of 50 mg / mL, filter it with a 0.45-μm filter head, load the sample at 15% of the column volume, elute it with the above phosphate buffer solution (containing 0.11 mol / L sodium chloride), the elution flow rate is 5 BV / h, collect each part of the eluate for SDS-PAGE protein electrophoresis experiment, combine the chromatography solution with the bright nattokinase band and vacuum freeze-dry it to obtain the nattokinase pure product with a milky white appearance. After detection, the recovery rate of nattokinase is 75%, the purity is 80%, and the enzyme activity is 120380 FU / g.

[0079] Example 4 Purification of Nattokinase

[0080] S1`, Enzymatic hydrolysis of fermentation broth: Add an equal volume of pure water to the nattokinase fermentation broth obtained in Preparation Example 1, stir evenly for preliminary dilution to obtain a nattokinase dilution with a viscosity of 200 mPa·s, add polyglutamic acid polypeptide enzyme at 0.2% of the volume of the fermentation broth, stir evenly, heat to raise the internal temperature of the fermentation broth to 37 °C, and stir and control the temperature for 30 min. During stirring, the viscosity of the fermentation broth is detected in real time. When the viscosity drops to 8 mPa·s, stop the enzymatic hydrolysis treatment to obtain an enzymatic hydrolysate.

[0081] S2`, Microfiltration: Microfilter the enzymatic hydrolysate with a 300-nm ceramic membrane, the transmembrane pressure is 0.3 MPa, control the material circulation temperature at 30 °C during microfiltration, and take samples of the permeate and retentate for microscopic examination after the microfiltration runs stably. The microfiltration effect is measured by the permeation amount of macromolecular proteins and bacteria. When the volume recovery rate of the permeate reaches 87% of the enzymatic hydrolysis fermentation broth, stop microfiltration, and perform subsequent ultrafiltration concentration on the permeate (nattokinase enzyme clear solution).

[0082] S3`, Ultrafiltration: The nattokinase enzyme supernatant was ultrafiltered and concentrated through an ultrafiltration membrane with a molecular weight cut-off of 15 kDa. The concentration conditions included a device frequency of 37 Hz and a membrane pressure of 0.06 MPa. During ultrafiltration, the temperature of the feed liquid circulation was controlled at 30 °C. When the volume of the concentrated liquid reached 1 / 7 of the volume of the fermentation broth, ultrafiltration was stopped and the retentate was collected, which was the nattokinase concentrated liquid.

[0083] S4`, Salting out: Accurately measure the nattokinase concentrated liquid, add solid ammonium sulfate at a salt saturation of 20%, stir until all the salt is dissolved, and then let it stand at room temperature for 5 h for the first-stage salting out. Then, filter the salting-out solution through a 0.45 μm filter membrane, collect the filtrate, add solid ammonium sulfate to the filtrate at a salt saturation of 60%, adjust the pH of the salting-out solution to 8.5 with 30% sodium hydroxide solution after the salt is completely dissolved, and then let it stand at 9 °C for 15 h for the second-stage salting out. After standing, centrifuge at 5000 r / min for 10 min, and collect the brown precipitate at the bottom, which is the crude nattokinase.

[0084] S5`, Purification: The macroporous adsorption resin (purchased from Tianjin Yunkai Resin Technology Co., Ltd., with the brand number HPD400) was fully swollen and packed into a column, and the column was equilibrated with a phosphate buffer solution with a concentration of 0.2 mol / L and a pH value of 7.4. The crude nattokinase was prepared into a sample loading solution with a protein content of 25 mg / mL and filtered through a 0.45 μm filter head. The sample was loaded at 15% of the column volume, and the above phosphate buffer solution (containing 0.11 mol / L sodium chloride) was used for elution. The elution flow rate was 0.5 BV / h. The elution solutions of each part were collected for SDS-PAGE protein electrophoresis experiments, and the chromatography solutions with bright nattokinase bands were combined and vacuum freeze-dried to obtain the pure nattokinase with a milky white appearance. After detection, the recovery rate of nattokinase was 60%, the purity was 75%, and the enzyme activity was 85090 FU / g.

[0085] Purification of Nattokinase in Example 5

[0086] S1`, Enzymatic hydrolysis of fermentation broth: Add an equal volume of pure water to the nattokinase fermentation broth obtained in Preparation Example 1 and stir evenly for preliminary dilution to obtain a nattokinase dilution with a viscosity of 200 mPa·s. Add 0.2% of β-lactamase based on the volume of the fermentation broth and stir evenly. Heat to raise the internal temperature of the fermentation broth to 37 °C and stir for temperature control for 30 min. During stirring, the viscosity of the fermentation broth was detected in real time. When the viscosity decreased to 9 mPa·s, the enzymatic hydrolysis treatment was stopped to obtain the enzymatic hydrolysate.

[0087] S2`, Microfiltration: The enzymolysis solution is subjected to microfiltration using a 300 nm ceramic membrane. The transmembrane pressure is 0.3 MPa. During microfiltration, the material circulation temperature is controlled at 30 °C. After the microfiltration operation is stable, samples of the permeate and retentate are taken for microscopic examination. The microfiltration effect is measured by the permeation amount of macromolecular proteins and bacteria. When the volume yield of the permeate reaches 89% of the enzymolysis fermentation broth, microfiltration is stopped, and the permeate (nattokinase enzyme clear solution) is subjected to subsequent ultrafiltration and concentration.

[0088] S3`, Ultrafiltration: The nattokinase enzyme clear solution is ultrafiltered and concentrated through an ultrafiltration membrane with a molecular weight cut-off of 15 kDa. The concentration conditions include a device frequency of 37 Hz and a membrane pressure of 0.06 MPa. During ultrafiltration, the feed liquid circulation temperature is controlled at 30 °C. When the volume of the concentrated liquid reaches 1 / 7 of the volume of the fermentation broth, ultrafiltration is stopped and the retentate is collected, which is the nattokinase concentrated liquid.

[0089] S4`, Salting out: Accurately measure the nattokinase concentrated liquid, add solid ammonium sulfate at a salt saturation of 20%, stir until all the salt is dissolved, and then let it stand at room temperature for 5 h for primary salting out. Then, the salted-out solution is filtered through a 0.45 μm filter membrane, and the filtrate is collected. Then, solid ammonium sulfate is added to the filtrate at a salt saturation of 60%. After the salt is completely dissolved, the pH of the salted-out solution is adjusted to 8.5 with 30% sodium hydroxide solution. Subsequently, it is left to stand at 4 °C for 15 h for secondary salting out. After standing, it is centrifuged at 5000 r / min for 10 min, and the brown precipitate in the lower layer is collected, which is the crude nattokinase.

[0090] S5`, Purification: Sephadex G-100 dextran gel is fully swollen and packed into a column. The column is equilibrated with a phosphate buffer solution with a concentration of 0.2 mol / L and a pH value of 7.4. The crude nattokinase is prepared into a sample loading solution with a protein content of 25 mg / mL and filtered through a 0.45 μm filter head. The sample is loaded at 15% of the column volume, and elution is carried out with the above phosphate buffer solution (containing 0.11 mol / L sodium chloride). The elution flow rate is 0.5 BV / h. Each part of the eluate is collected for SDS-PAGE protein electrophoresis experiment. The chromatography solution with nattokinase bright bands is combined and vacuum freeze-dried to obtain a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 75%, the purity is 80%, and the enzyme activity is 140000 FU / g.

[0091] Purification of nattokinase in Example 6

[0092] Nattokinase was purified according to the method of Example 1, except that in step S4`, the pH value of secondary salting out was 7.5, and the other conditions were the same as those in Example 1, to obtain a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase was 60%, the purity was 80%, and the enzyme activity was 84500 FU / g.

[0093] Example 7 Purification of Nattokinase

[0094] Purify nattokinase according to the method of Example 1. The difference is that in step S2`, the pore size of the ceramic membrane used is 1000 nm, and the other conditions are the same as those in Example 1, obtaining a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 55%, the purity is 74%, and the enzyme activity is 76557 FU / g.

[0095] Example 8 Purification of Nattokinase

[0096] Purify nattokinase according to the method of Example 1. The difference is that in step S3`, the molecular weight cut-off of the ultrafiltration membrane used is 1 kDa, and the other conditions are the same as those in Example 1, obtaining a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 55%, the purity is 75%, and the enzyme activity is 79860 FU / g.

[0097] Example 9 Purification of Nattokinase

[0098] Purify nattokinase according to the method of Example 1. The difference is that in step S1`, the papain is replaced with trypsin of the same dosage, and the other conditions are the same as those in Example 1, obtaining a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 65%, the purity is 78%, and the enzyme activity is 68420 FU / g.

[0099] Example 10 Purification of Nattokinase

[0100] Purify nattokinase according to the method of Example 1. The difference is that in step S1`, the papain is replaced with carboxypeptidase of the same dosage, and the other conditions are the same as those in Example 1, obtaining a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 69%, the purity is 79%, and the enzyme activity is 79653 FU / g.

[0101] Example 11 Purification of Nattokinase

[0102] Purify nattokinase according to the method of Example 1. The difference is that in step S1`, the papain is replaced with β-lactamase of the same dosage, and the other conditions are the same as those in Example 1, obtaining a pure nattokinase product with a milky white appearance. After detection, the yield of nattokinase is 72%, the purity is 80%, and the enzyme activity is 85264 FU / g.

[0103] Example 12 Purification of Nattokinase

[0104] The nattokinase was purified according to the method of Example 4, except that in step S1`, the polyglutamic acid polypeptide enzyme was replaced with trypsin in the same dosage, and the other conditions were the same as those in Example 4, obtaining a nattokinase pure product with a milky white appearance. After detection, the yield of nattokinase was 52%, the purity was 73%, and the enzyme activity was 57860 FU / g.

[0105] Purification of Nattokinase in Example 13

[0106] The nattokinase was purified according to the method of Example 4, except that in step S1`, the polyglutamic acid polypeptide enzyme was replaced with carboxypeptidase in the same dosage, and the other conditions were the same as those in Example 4, obtaining a nattokinase pure product with a milky white appearance. After detection, the yield of nattokinase was 54%, the purity was 73%, and the enzyme activity was 60231 FU / g.

[0107] Purification of Nattokinase in Example 14

[0108] The nattokinase was purified according to the method of Example 4, except that in step S1`, the polyglutamic acid polypeptide enzyme was replaced with β-lactamase in the same dosage, and the other conditions were the same as those in Example 4, obtaining a nattokinase pure product with a milky white appearance. After detection, the yield of nattokinase was 53%, the purity was 74%, and the enzyme activity was 61259 FU / g.

[0109] Comparative Example 1

[0110] The nattokinase was purified according to the method of Example 1, except that in step S1`, the enzymolysis time was 10 min. At this time, the viscosity of the obtained enzymolysis solution was 150 mPa·s. Then, the enzymolysis solution was processed by the method of Example 1, obtaining a nattokinase pure product with a milky white appearance. After detection, the yield of nattokinase was 50%, the purity was 72%, and the enzyme activity was 55600 FU / g.

[0111] Comparative Example 2

[0112] The nattokinase was purified according to the method of Example 1, except that the papain was replaced with neutral protease in the same weight portion, and the other conditions were the same as those in Example 1, obtaining a nattokinase pure product with a milky white appearance. After detection, the yield of nattokinase was 45%, the purity was 60%, and the enzyme activity was 52133 FU / g.

[0113] Comparative Example 3

[0114] The nattokinase was purified according to the method of Example 1, except that the enzymatic hydrolysis step was not included. Instead, the nattokinase fermentation broth was directly diluted with water to obtain a nattokinase dilution with a viscosity of 250 mPa·s, and then microfiltered. The other conditions were the same as those in Example 1, and a nattokinase pure product with a milky white appearance was obtained. After detection, the yield of nattokinase was 40%, the purity was 60%, and the enzyme activity was 40000 FU / g.

[0115] Comparative Example 4

[0116] The nattokinase was purified according to the method of Example 1, except that the dosage of papain was 8% of the volume of the fermentation broth. The other conditions were the same as those in Example 1, and a nattokinase pure product with a milky white appearance was obtained. After detection, the yield of nattokinase was 34%, the purity was 53%, and the enzyme activity was 49635 FU / g.

[0117] Comparative Example 5

[0118] The nattokinase was purified according to the method of Example 1, except that the dosage of papain was 0.05% of the volume of the fermentation broth. The other conditions were the same as those in Example 1, and a nattokinase pure product with a milky white appearance was obtained. After detection, the yield of nattokinase was 31%, the purity was 49%, and the enzyme activity was 38695 FU / g.

[0119] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. A method for purifying nattokinase, characterized in that, The method includes enzymatically hydrolyzing nattokinase fermentation broth and then purifying the obtained hydrolysate; the method of enzymatic hydrolysis includes: diluting the nattokinase fermentation broth with water to a viscosity of 180 - 250 mPa·s, and then enzymatically hydrolyzing the obtained nattokinase dilution with an enzyme preparation into a hydrolysate. The enzyme preparation is selected from at least one of trypsin, papain, carboxypeptidase, polyglutamic acid polypeptide enzyme, and β-lactamase. The addition amount of the enzyme preparation is 0.1% - 5% of the volume of the nattokinase fermentation broth, and the conditions of enzymatic hydrolysis are such that the viscosity of the hydrolysate is reduced to 7 - 10 mPa·s.

2. The method for purifying nattokinase according to claim 1, characterized in that, The conditions of the enzymatic hydrolysis include a temperature of 30 - 50 °C and a time of 15 - 120 min.

3. The method for purifying nattokinase according to claim 1, characterized in that, The method of purification includes the following steps carried out in sequence: S1. Microfiltration: Microfiltering the hydrolysate with a ceramic membrane having a pore size of 50 - 500 nm, and the obtained permeate is nattokinase enzyme clarified liquid; S2. Ultrafiltration: Ultrafiltering the nattokinase enzyme clarified liquid with an ultrafiltration membrane having a molecular weight cut-off of 2 - 25 kDa, and the obtained retentate is nattokinase concentrate; S3. Salting out: Subjecting the crude nattokinase solution to salting out and then performing solid-liquid separation, and the obtained solid product is crude nattokinase; S4. Refinement: Subjecting the crude nattokinase to column chromatography purification and electrophoresis purification in sequence to obtain pure nattokinase.

4. The method for purifying nattokinase according to claim 3, characterized in that, In step S1, the conditions of the microfiltration include a temperature of the hydrolysate of 10 - 50 °C and a membrane pressure of 0.1 - 0.6 MPa. The time of the microfiltration is based on when the volume of the permeate reaches 85% - 90% of the volume of the hydrolysate.

5. The method for purifying nattokinase according to claim 3, characterized in that, In step S2, the conditions of the ultrafiltration include a temperature of the nattokinase enzyme clarified liquid of 8 - 40 °C and a membrane pressure of 0.01 - 0.5 MPa. The time of the ultrafiltration is based on when the volume ratio of the retentate to the nattokinase fermentation broth reaches 1:(3 - 20).

6. The method for purifying nattokinase according to claim 3, characterized in that, In step S3, the method of salting out is to add metal salt Ⅰ to the crude nattokinase solution, stir until metal salt Ⅰ is basically dissolved, and then let it stand to complete the first-stage salting out. Then, perform the first-stage solid-liquid separation on the obtained first-stage salting out liquid. Add metal salt Ⅱ to the obtained liquid product, stir until metal salt Ⅱ is basically dissolved, adjust the pH value of the solution to 7 - 10, and then let it stand at 4 - 10 °C to complete the second-stage salting out. Then, perform the second-stage solid-liquid separation on the obtained second-stage salting out liquid, and the obtained solid product is crude nattokinase.

7. The method for purifying nattokinase according to claim 6, characterized in that, The metal salt Ⅰ and the metal salt Ⅱ are each independently selected from at least one of sodium sulfate, potassium sulfate, ammonium sulfate, sodium chloride, and potassium chloride.

8. The method for purifying nattokinase according to claim 6, characterized in that, The salt saturation of the first-stage salting out is 5 - 50%, and the salt saturation of the second-stage salting out is 30 - 80%.

9. The method for purifying nattokinase according to claim 6, characterized in that, The time of the first-stage salting out is 1 - 25 h, and the time of the second-stage salting out is 1 - 48 h.

10. The method for purifying nattokinase according to claim 3, characterized in that, In step S4, the methods of column chromatography purification and electrophoresis purification are to activate the filler and load it into the chromatography column, balance it with phosphate buffer solution, dissolve the crude nattokinase with water to prepare a sample loading solution and then load the sample, and then elute it with phosphate buffer solution containing sodium chloride. Collect the eluate rich in nattokinase and perform electrophoresis. Vacuum dry the chromatography solution with a bright band of nattokinase to obtain pure nattokinase.

11. The method for purifying nattokinase according to claim 10, characterized in that, The pH values of the phosphate buffer solution and the phosphate buffer solution containing sodium chloride are independently 6 - 10, and the concentrations are independently 0.1 - 10.0 mol / L.

12. The method for purifying nattokinase according to claim 10, characterized in that, The concentration of sodium chloride in the phosphate buffer solution containing sodium chloride is 0.05 - 5 mol / L.

13. The method for purifying nattokinase according to claim 10, characterized in that, The concentration of nattokinase in the sample loading solution is 5 - 100 mg / mL.

14. The method for purifying nattokinase according to claim 10, characterized in that, The volume of the sample loading solution is 1% - 50% of the volume of the chromatography column.

15. The method for purifying nattokinase according to claim 10, characterized in that, The flow rate of the elution is 0.2 - 5 BV / h.

16. The method for purifying nattokinase according to claim 3, characterized in that, In step S4, the filler used for column chromatography purification is selected from at least one of dextran gel, cation exchange resin, anion exchange resin, and macroporous adsorption resin.