CN114292833A - Method for extracting and purifying protease from straw mushroom fruiting body - Google Patents
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Abstract
The invention discloses a method for extracting and purifying protease from straw mushroom fruiting bodies. The method comprises the steps of firstly treating straw mushroom fruiting bodies at 15 ℃ for 36-96h, homogenizing according to a certain material-liquid ratio, precipitating with ammonium sulfate, dialyzing to remove salt, then carrying out chromatography by using an ion column and a gel column to remove foreign proteins, analyzing the sizes of neutral proteases of the straw mushroom fruiting bodies by using SDS-polyacrylamide gel electrophoresis, and determining the optimal reaction conditions of the target neutral proteases. The method is simple and short in time consumption, the physicochemical property and the biological activity of the neutral protease of the straw mushroom fruiting body can be maintained to the greatest extent in the purification process, after the neutral protease of the straw mushroom fruiting body obtained by purification is subjected to enzymolysis on the soybean protein isolate, the proportion of hydrophobic amino acid in the enzymolysis liquid is higher than that in the trypsin enzymolysis liquid, the bitter taste in the hydrolysis liquid can be effectively reduced, and the method is suitable for industries such as food and the like.
Description
Technical Field
The invention belongs to the field of extraction and purification of proteins, and particularly relates to a method for extracting and purifying protease from straw mushroom fruiting bodies.
Background
Due to the differences of substrate specificity, structure and action mechanism of the protease, the protease has application in the production processes of detergents, medicines, foods, leather and the like, and the protease from microorganisms has wider application than plant protease. In contrast to animal, plant and bacterial proteases, fungal secreted proteases have a diversity including extracellular, membrane-bound and intracellular proteases, and are clearly superior to bacterial proteases in downstream industries. To obtain a wider variety of fungal proteases, mushrooms have also become a source of many proteases, such as serine proteases from Hypsizigus marmoreus (Hypsizigus marmoreus) and metallo-proteases from Raynariae (Lacccophalum mylitae), both of which are neutral proteases. Neutral protease has mild reaction conditions and high catalytic efficiency, and is widely applied to industries of food, medicine, feed and the like.
Volvariella volvacea (Bull. ex. Fr.) Sing, also called Hypsizygus marmoreus and Hypsizygus marmoreus, are tropical and subtropical high-temperature edible fungi, and their fruiting bodies are autolyzed at low temperature, and are preferably preserved at 15 deg.C. During the storage process of the straw mushroom fruiting body at 15 ℃, the activity of the neutral protease is gradually increased, and the content of the soluble protein is close to the lowest point at 36h, so that a large amount of protein is degraded into small molecular polypeptide and amino acid. Therefore, the neutral protease in the straw mushroom has stronger cracking capability and has application potential in the aspects of food, washing and the like. At present, the protease in the straw mushroom is purified by using a sporocarp as a protease source, and the straw mushroom protease is extracted from the sporocarp which is processed at a low temperature. Document 1 (Chenmingjie, Chen, Wannan, et al. isolation and purification of volvariella volvacea protease [ J ]. Proc. edible fungi, 1999,6(4):46-48.) reports a method for isolation and purification of volvariella volvacea protease, which purifies the protease in volvariella volvacea by Sephadex G-150 to finally obtain three protein peaks, but only one is active, the extraction efficiency is low, and the purity of the purified protease is not verified, indicating that the experimental design is incomplete. Document 2 (separation, purification and isoelectric Point of Pleurotus ostreatus protease in Li Shih, Chengming, and determination of isoelectric Point [ J ] fungus systems, 2003(02):161-164.) protease purified from Volvariella volvacea by ammonium sulfate precipitation and Dextran G-150 to a plurality of different isoelectric points, which is less pure.
Disclosure of Invention
The invention processes the straw mushroom fruiting body at 15 ℃ for 36-96h, separates and purifies the protease system produced by the straw mushroom fruiting body, studies the enzymology property of the obtained protease, and studies the application of the protease in enzymolysis of soybean protein isolate. The neutral protease extracted from the straw mushroom fruiting body has high enzyme activity and high purity, and the extraction and purification method has high efficiency.
The technical scheme for realizing the purpose of the invention is as follows:
the method for extracting and purifying the protease from the straw mushroom fruiting body comprises the following specific steps:
(1) treating straw mushroom fruiting bodies at 15 ℃ for 36-96h, adding distilled water into the straw mushroom fruiting bodies, homogenizing, standing at 4 ℃, centrifuging, retaining the supernatant to obtain a crude protease liquid, adding ammonium sulfate into the crude protease liquid to reach 80% of saturation, standing at 4 ℃, centrifuging, removing the supernatant, dissolving the precipitate with distilled water, dialyzing with a 10kDa dialysis bag, and freeze-drying and storing the dialyzed liquid;
(2) dissolving the freeze-dried solid obtained in the step (1) in a phosphate buffer solution with the concentration of 20mM and the pH value of 7.0 to obtain a neutral protease solution of the straw mushroom fruiting body, filtering the enzyme solution through a microporous membrane, loading the enzyme solution, balancing a DEAE FF ion column by using Tris-HCl buffer solution with the concentration of 20mM and the pH value of 7.0, performing gradient elution by using 1M NaCl solution at the flow rate of 1.0mL/min, adjusting the elution gradient after obtaining a separation gradient of a main absorption peak, sequentially performing elution by using 1M NaCl solutions with the concentration of 20%, 60% and 100%, finally obtaining the absorption peak, determining the protein content and the enzyme activity of the absorption peak, and determining the main absorption peak 1 by specific enzyme activity;
(3) purifying the main absorption peak 1 by using a gel column of Superdex 7510/300 GL, firstly balancing the gel column by using 20mM phosphate buffer solution with pH7.0, eluting by using 20mM phosphate buffer solution with pH7.0 containing 0.1M NaCl after loading, wherein the flow rate is 0.3mL/min, finally obtaining an absorption peak, and determining the protein content and the enzyme activity of the absorption peak to determine a main absorption peak 2;
(4) and (3) carrying out SDS-polyacrylamide gel electrophoresis on the concentrated solution of the main absorption peak 2 to obtain the straw mushroom fruiting body neutral protease with the molecular weight of 20 kDa.
Preferably, in the step (1), the ratio of the straw mushroom fruit body to the distilled water is 1: 10, standing for 2 hours, centrifuging at a speed of 5000-6000 r/min, and dialyzing for 36 hours.
Preferably, in step (1), the dialyzed sample is fractionated using an ultrafiltration concentration tube having a size of 30kDa to remove foreign proteins, thereby further improving the purification effect of gel column chromatography.
Preferably, in step (2), the pore size of the microporous membrane is 0.45 μm.
Preferably, in step (4), the concentrated solution of the main absorption peak 2 is obtained by concentrating with a 3kDa concentration tube.
The invention also provides the neutral protease of the straw mushroom fruiting body prepared by the extraction and purification method.
The optimal reaction pH of the neutral protease of the straw mushroom fruiting body is 7, the optimal reaction temperature is 40 ℃, and Fe2+Has effect in promoting casein decomposition by neutral protease of straw mushroom fruiting body, and metal ions (Ca) in balance2+、Ni2+、Co2+、Cu2+、Zn2+、Mg2+) The protease inhibitor and the surfactant have different degrees of inhibition on neutral protease of the straw mushroom fruiting body.
Further, the invention provides application of the straw mushroom fruiting body neutral protease in enzymolysis of soybean protein isolate.
Compared with the prior art, the invention has the following advantages:
(1) according to the invention, the straw mushroom fruiting body is treated at 15 ℃ for 36-96h, homogenate is carried out according to a certain material-liquid ratio, dialysis is carried out to remove salt after ammonium sulfate precipitation, and then chromatography is carried out by using an ion column and a gel column, so that impure protein is removed, the purification method is simple, the consumed time is short, the physicochemical property and the biological activity of neutral protease of the straw mushroom fruiting body can be maintained to the greatest extent in the purification process, and the enzyme activity and the purity of the neutral protease of the purified straw mushroom fruiting body are high;
(2) after the straw mushroom fruiting body neutral protease is used for enzymolysis of soybean protein isolate, the proportion of hydrophobic amino acid in an enzymolysis liquid is higher than that in a trypsin enzymolysis liquid, so that the bitterness in the hydrolysis liquid can be effectively reduced, and the straw mushroom fruiting body neutral protease has a wide application prospect in the food industry.
Drawings
FIG. 1 is a DEAE-FF ion column elution peak 1 in example 1;
FIG. 2 is a graph showing the elution peak 2 of Superdex 7510/300 GL gel column in example 1;
FIG. 3 is a DEAE-FF ion column elution peak 3 in comparative example 1;
FIG. 4 is a graph showing the elution peak 4 of Superdex 7510/300 GL gel column in comparative example 1;
FIG. 5 is an SDS-PAGE electrophoresis pattern in example 1;
FIG. 6 is a graph showing the change in enzyme activity at different pH values in example 2;
FIG. 7 is a graph showing the change in enzyme activity at different temperatures in example 2;
FIG. 8 is a graph showing the change in enzyme activity under different metal ions in example 2;
FIG. 9 is a graph showing the change in enzyme activity with different chemicals in example 2;
FIG. 10 shows the degree of hydrolysis of two proteases in example 3.
Detailed Description
The present invention will be described in further detail with reference to the following examples and accompanying drawings.
Example 1
(1) Weighing 100g of straw mushroom fruiting bodies, treating for 36h at 15 ℃, and treating according to the weight ratio of 10: 1, adding distilled water, homogenizing, standing at 4 deg.C for 2 hr, centrifuging for 10min (5000r/min), and collecting supernatant to obtain crude protease solution. Adding ammonium sulfate into the crude protease solution to reach 80% saturation, standing at 4 deg.C for 2 hr, centrifuging at 6000r/min, removing supernatant, dissolving the precipitate with distilled water, dialyzing the dissolved solution with 10kDa dialysis bag for 36 hr, lyophilizing the dialyzed solution, and storing.
(2) Dissolving the lyophilized solid with phosphate buffer solution (20mM pH7.0) to obtain neutral protease solution of straw mushroom fruiting body, passing the obtained enzyme solution through 0.45 μ M microporous membrane, loading, balancing DEAE FF (1cm × 5cm) ion column with Tris-HCl (20mM pH7.0) buffer solution, and performing gradient elution with 1M NaCl solution at flow rate of 1.0 mL/min; after obtaining the main absorption peak separation gradient, adjusting the elution gradient, and eluting with 20%, 60% and 100% 1M NaCl solution; and finally obtaining an absorption peak, determining the protein content of the absorption peak by using a BCA method protein content determination kit, and determining the enzyme activity of the absorption peak by using a Folin method in appendix B in national standard GBT23527-2009, thereby determining a main absorption peak 1 by the specific enzyme activity.
(3) The main absorption peak 1 obtained by the ion column purification was further purified by using a gel column of Superdex 7510/300 GL (1 cm. times.30 cm), which was equilibrated with a phosphate buffer (20mM pH7.0), and eluted with a phosphate buffer containing 0.1M NaCl (20mM pH7.0) at a flow rate of 0.3mL/min after the loading; and finally obtaining an absorption peak, determining the protein content of the absorption peak by using a BCA method protein content determination kit, and determining the enzyme activity of the absorption peak by using a Folin method in appendix B in national standard GBT23527-2009, thereby determining a main absorption peak 2 by the specific enzyme activity.
(4) The steps (2) and (3) are modified according to the Folin method in appendix B of national standard GBT23527-2009, standard curves are firstly determined by using L-tyrosine standard solutions with equal concentration gradients of 0 mug/mL, 10 mug/mL, 20 mug/mL, 30 mug/mL, 40 mug/mL and 50 mug/mL, adding 5.0mL of sodium carbonate solution (0.4mol/L) and 1.0mL of welan reagent solution into 1.0mL of L-tyrosine standard solution with each concentration, shaking uniformly, taking out after water bath for 20min at 40 deg.C, measuring absorbance at 680nm with spectrophotometer, a standard curve is drawn with the absorbance A as the ordinate and the tyrosine concentration c as the abscissa, and the amount of tyrosine (. mu.g) at an absorbance of 1, i.e., the absorption constant K value, which should be in the range of 95 to 100, is calculated by a regression equation. The enzyme activity in the sample is measured by the following method: firstly, preserving heat of a casein solution (10g/L) in a water bath at 40 ℃ for 5min, then adding 200 mu L of the casein solution (10g/L) into 200 mu L of an enzyme solution, uniformly mixing, putting the mixture into the water bath at 40 ℃ for incubation for 10min, taking out the mixture, adding 400 mu L of trichloroacetic acid (0.4mol/L), oscillating and uniformly mixing, centrifuging the mixture for 10min at the rotating speed of 8000r/min, taking 200 mu L of supernatant, then adding 1mL of a sodium carbonate solution (0.4mol/L) and 200 mu L of a formalin reagent use solution, preserving heat of the mixture in the water bath at 40 ℃ for 20min after oscillating and uniformly mixing, and measuring the absorbance at 680nm by using a spectrophotometer; comparison: the method comprises the steps of firstly preserving heat of a casein solution (10g/L) in a water bath at 40 ℃ for 5min, then adding 400 mu L of trichloroacetic acid (0.4mol/L) into 200 mu L of an enzyme solution, oscillating and mixing the solution uniformly, putting the solution into the water bath at 40 ℃ for incubation for 10min, taking out the solution, adding 200 mu L of the casein solution (10g/L), centrifuging the solution for 10min at the rotating speed of 8000r/min, taking 200 mu L of supernatant, then adding 1mL of a sodium carbonate solution (0.4mol/L) and 200 mu L of a formalin reagent using solution, preserving heat of the solution in the water bath at 40 ℃ for 20min after oscillating and mixing the solution uniformly, and measuring absorbance at 680nm by using a spectrophotometer. The tyrosine content is calculated by a regression equation, and the measured value is subtracted from the control value to obtain the enzyme activity of 200 mu L of enzyme solution, wherein the unit is expressed by U.
(5) And (3) concentrating the main absorption peak 2 by using a concentration tube with the size of 3kDa, and analyzing the main absorption peak 2 by adopting SDS-polyacrylamide gel electrophoresis to obtain the straw mushroom fruiting body neutral protease with the size of about 20 kDa.
The results of the protein content and the enzyme activity after purification in each step in example 1 are shown in Table 1.
TABLE 1
As can be seen from Table 1, the neutral protease of the straw mushroom fruiting body is subjected to ammonium sulfate extraction and precipitation, and then purified by a DEAE FF ion column and a Superdex 7510/300 GL gel column to obtain the electrophoresis grade pure neutral protease of the straw mushroom fruiting body, the protease is purified by 7.90 times, the specific enzyme activity reaches 216.17U/mg, the protease has high biological activity, and the protease can be widely used in the industries of food, detergents and the like.
Example 2
(1) The purified neutral protease from the fruiting body of straw mushroom was obtained in example 1.
(2) Preparing buffer solution systems with different pH values, wherein 0.05mol/L sodium acetate-acetic acid buffer solution is used at the pH value of 4-5, 0.05mol/L sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution is used at the pH value of 6-7, and 0.05mol/L Tris-HCl buffer solution is used at the pH value of 8-9. The neutral protease of the straw mushroom fruiting body is reacted with casein in buffer solution systems with different pH values, the activity of the neutral protease of the straw mushroom fruiting body under different pH values is measured by the method in the step (4) in the embodiment 1, the maximum enzyme activity is 100%, and other enzyme activities are expressed by relative enzyme activities, so that the optimum pH value of the enzyme activity reaction is determined.
(3) Catalyzing neutral protease of the straw mushroom fruiting body to react with casein at different reaction temperatures (20, 30, 40, 50, 60 and 70 ℃), measuring the enzyme activity of the neutral protease of the straw mushroom fruiting body by the method of the step (4) in the embodiment 1, and representing the maximum enzyme activity by 100% and other enzyme activities by relative enzyme activities, thereby determining the optimum reaction temperature.
(4) Mixing neutral protease of straw mushroom fruiting body with different metal ion mother liquor (CaCl)2、NiCl2、CoCl2、CuSO4、FeSO4、ZnSO4、MgSO4) Mixing, making final concentration of metal ions reach 5mM, respectively measuring enzyme activity by the method of step (4) in example 1 under the optimal reaction condition of neutral protease of straw mushroom fruiting body, determining enzyme activity of neutral protease of straw mushroom fruiting body not affected by metal ions as 100%, and measuring Ca2+、Cu2+、Fe2+、Mg2+、Zn2+、Ni2+、Co2+Influence on enzyme activity.
(5) Respectively mixing a protease inhibitor and a surfactant with neutral protease of the fruiting body of the straw mushroom to reach a certain concentration, respectively keeping the final concentrations of 1mmol/L, 2mmol/L, 4mmol/L, 10mmol/L, 5% and 5% of benzyl xanthyl chloride (PMSF), beta-mercaptoethanol (beta-mercaptoethanol), Ethylene Diamine Tetraacetic Acid (EDTA), Cetyl Trimethyl Ammonium Bromide (CTAB), tween-80 and triton after uniform mixing, keeping the temperature at the optimum temperature and pH for 10min, measuring the enzyme activity according to the method of the step (4) in the example 1, and respectively measuring the influence of the protease inhibitor and the surfactant on the neutral protease activity of the fruiting body of the straw mushroom with the untreated enzyme activity being 100%.
Example 2 middle stepThe results of steps (4) and (5) are shown in FIGS. 6 and 7. As can be seen from FIGS. 6, 7 and 8, the optimal conditions for the neutral protease reaction of the purified straw mushroom fruit body are as follows: the protease has the optimum pH value of 7.0 and the optimum reaction temperature of 40 ℃, is sensitive to PMSF, is serine protease, has obvious inhibition effect on beta-mercaptoethanol, but does not have obvious inhibition effect on EDTA; among the seven metal ions, only Fe2+Has effect in enhancing protease, Ca2+And Mg2+The influence on the enzyme activity is small, and the rest are inhibitory effects; the cationic detergents CTAB and triton both inhibit the activity of S4 protease, and the discrete detergent Tween-80 slightly enhances the neutral protease of straw mushroom.
Example 3
(1) The purified volvariella volvacea fruiting body neutral protease is obtained by the method of example 1; trypsin was purchased as a control enzyme.
(2) Preparing soybean protein isolate into 5% solution with certain pH, placing in 85 deg.C water bath for 15min, adding protease solution according to enzyme and substrate ratio of 100U/g, oscillating at 40 deg.C for reaction for a period of time, inactivating in boiling water bath for 15min after reaction, determining protein hydrolysis degree, centrifuging at 8000r/min for 10min, and collecting supernatant for storage.
(3) The method for measuring the hydrolysis degree comprises the steps of preparing a soybean protein isolate solution with certain pH, adding enzyme for enzymolysis for certain time, adjusting the pH to the pH of an initial substrate solution by using 0.5mol/L sodium hydroxide after the enzymolysis is finished, recording the volume of the consumed sodium hydroxide solution, and calculating according to the following formula:
DH(％)＝B*N/(α*Mp*hot)，
in the formula, B is the consumption of sodium hydroxide, N is the concentration of sodium hydroxide, α is the average degree of dissociation of the soy protein isolate (α ═ 0.44 at pH 7), Mp is the mass of the protein to be hydrolyzed, hot is the number of millimoles of peptide bonds per gram of protein substrate, and hot (soy protein isolate) is 7.8 mmol/g.
(4) Free amino acids in the neutral protease enzymolysis liquid and the trypsin enzymolysis liquid of the straw mushroom fruiting body are measured, and the results are shown in table 2.
TABLE 2
As can be seen from Table 2 and FIG. 10, the maximum degree of hydrolysis of neutral protease of the fruiting body of Volvariella volvacea is not much different from that of trypsin, and the ratio of hydrophobic amino acids (Val, Ile, Leu, Ala) and umami amino acids (Asp, Glu, Lys, Gly) in the protease enzymatic hydrolysis product is higher than that of trypsin enzymatic hydrolysis product by measuring the free amino acids of the two enzymatic hydrolysis products (Table 2), and respectively reaches 37.12% and 56.10%, which shows that the protease can reduce the bitter taste in the enzymatic hydrolysis solution and has great application potential in the food industry.
Comparative example 1
(1) The volvariella volvacea mycelia were extracted and dialyzed according to the method of example 1.
(2) The ion column elution was the same as in example 1, giving main absorption peak 3.
(3) The gel column elution method was the same as in example 1, yielding peak 4.
(4) Protease activity was measured in the same manner as in example 1.
From the comparative examples, it is understood from example 1 that the fruiting body of volvariella volvacea is more effectively purified than the mycelium, and much impure protein contained in the mycelium is difficult to remove. From comparative example 1, it can be seen that only one protein peak (peak 3) and activity are obtained from the linear elution result of the DEAE ion column, while three protein peaks are obtained from the DEAE ion column in example 1 by gradient elution, but only peak 1 has the strongest activity, and the other two peaks are both hetero-proteins; in the gel column purification peak diagram, the purification effect of the example 1 is better because the impurity peaks of the example 1 are fewer and the separation effect of the target peak and the impurity peaks is better.
Claims (10)
1. The method for extracting and purifying the protease from the straw mushroom fruiting body is characterized by comprising the following specific steps:
(1) treating straw mushroom fruiting bodies at 15 ℃ for 36-96h, adding distilled water into the straw mushroom fruiting bodies, homogenizing, centrifuging at 4 ℃, keeping supernatant to obtain crude protease liquid, adding ammonium sulfate into the crude protease liquid to reach 80% saturation, standing at 4 ℃, centrifuging, removing supernatant, dissolving precipitates with distilled water, dialyzing with a 10kDa dialysis bag, and freeze-drying and storing the dialyzed liquid;
(2) dissolving the freeze-dried solid obtained in the step (1) in a phosphate buffer solution with the concentration of 20mM and the pH value of 7.0 to obtain a neutral protease solution of the straw mushroom fruiting body, filtering the enzyme solution through a microporous membrane, loading the enzyme solution, balancing a DEAE FF ion column by using Tris-HCl buffer solution with the concentration of 20mM and the pH value of 7.0, performing gradient elution by using 1M NaCl solution at the flow rate of 1.0mL/min, adjusting the elution gradient after obtaining a separation gradient of a main absorption peak, sequentially performing elution by using 1M NaCl solutions with the concentration of 20%, 60% and 100%, finally obtaining the absorption peak, determining the protein content and the enzyme activity of the absorption peak, and determining the main absorption peak 1 by specific enzyme activity;
(3) purifying the main absorption peak 1 by using a gel column of Superdex 7510/300 GL, firstly balancing the gel column by using 20mM phosphate buffer solution with pH7.0, eluting by using 20mM phosphate buffer solution with pH7.0 containing 0.1M NaCl after loading, wherein the flow rate is 0.3mL/min, finally obtaining an absorption peak, and determining the protein content and the enzyme activity of the absorption peak to determine a main absorption peak 2;
(4) and (3) carrying out SDS-polyacrylamide gel electrophoresis on the concentrated solution of the main absorption peak 2 to obtain the straw mushroom fruiting body neutral protease with the molecular weight of 20 kDa.
2. The method according to claim 1, wherein in the step (1), the ratio of the straw mushroom fruiting body to the distilled water is 1: 10.
3. the method according to claim 1, wherein the standing time in step (1) is 2 hours.
4. The method according to claim 1, wherein in the step (1), the centrifugation rate is 5000 to 6000 r/min.
5. The method according to claim 1, wherein in step (1), the dialysis time is 36 h.
6. The method of claim 1, wherein in step (1), the dialyzed sample is fractionated using a 30kDa ultrafiltration concentrator tube to remove contaminating proteins.
7. The method according to claim 1, wherein in the step (2), the pore size of the microporous membrane is 0.45 μm.
8. The method of claim 1, wherein in step (4), the concentrated solution of the main absorption peak 2 is obtained by concentrating with a 3kDa concentration tube.
9. The neutral protease of the fruiting body of straw mushroom obtained by the method according to any one of claims 1 to 8.
10. The use of the neutral protease from straw mushroom fruiting body according to claim 9 in enzymolysis of soy protein isolate.
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