CN113999832A - Straw mushroom fruiting body neutral protease, extraction and purification method and application thereof - Google Patents
Straw mushroom fruiting body neutral protease, extraction and purification method and application thereof Download PDFInfo
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- 240000006794 Volvariella volvacea Species 0.000 title claims abstract description 72
- 108091005507 Neutral proteases Proteins 0.000 title claims abstract description 60
- 102000035092 Neutral proteases Human genes 0.000 title claims abstract description 60
- 108090000145 Bacillolysin Proteins 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 47
- 238000000746 purification Methods 0.000 title claims abstract description 36
- 238000000605 extraction Methods 0.000 title claims abstract description 22
- 239000007788 liquid Substances 0.000 claims abstract description 16
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 238000000502 dialysis Methods 0.000 claims abstract description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 7
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- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 7
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- 239000004365 Protease Substances 0.000 claims description 21
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- 230000000052 comparative effect Effects 0.000 description 9
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 8
- 235000004501 Volvariella volvacea Nutrition 0.000 description 8
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- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- -1 silk Substances 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 3
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- 238000006460 hydrolysis reaction Methods 0.000 description 3
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- 229910000029 sodium carbonate Inorganic materials 0.000 description 3
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- 239000004094 surface-active agent Substances 0.000 description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 3
- 208000035404 Autolysis Diseases 0.000 description 2
- 206010057248 Cell death Diseases 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
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- 238000005227 gel permeation chromatography Methods 0.000 description 2
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- 238000002955 isolation Methods 0.000 description 2
- 239000010985 leather Substances 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000028043 self proteolysis Effects 0.000 description 2
- 229940071440 soy protein isolate Drugs 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 235000019583 umami taste Nutrition 0.000 description 2
- WAPPRYMYMNVAFR-UHFFFAOYSA-N 2-sulfanylethanol Chemical compound OCCS.OCCS WAPPRYMYMNVAFR-UHFFFAOYSA-N 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 description 1
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 description 1
- 235000007685 Pleurotus columbinus Nutrition 0.000 description 1
- 240000001462 Pleurotus ostreatus Species 0.000 description 1
- 235000001603 Pleurotus ostreatus Nutrition 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002310 Welan gum Polymers 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
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- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
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- 238000013401 experimental design Methods 0.000 description 1
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- 238000004108 freeze drying Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
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- 238000006703 hydration reaction Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000011034 membrane dialysis Methods 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000000751 protein extraction Methods 0.000 description 1
- 230000007065 protein hydrolysis Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000011403 purification operation Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- LEAHFJQFYSDGGP-UHFFFAOYSA-K trisodium;dihydrogen phosphate;hydrogen phosphate Chemical compound [Na+].[Na+].[Na+].OP(O)([O-])=O.OP([O-])([O-])=O LEAHFJQFYSDGGP-UHFFFAOYSA-K 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- 238000005303 weighing Methods 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a neutral protease of straw mushroom fruiting body, an extraction and purification method and application thereof. The method comprises the steps of firstly treating straw mushroom fruiting bodies at 4 ℃, homogenizing according to a certain material-liquid ratio, carrying out dialysis desalting after precipitation by using ammonium sulfate, then carrying out chromatography by using an ion column and a gel column to remove impurity 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, the time consumption is short, the physicochemical property and the biological activity of the neutral protease of the straw mushroom fruiting body can be maintained to the maximum extent in the purification process, and the neutral protease of the straw mushroom fruiting body obtained by purification can be widely applied to the industries of medicines, foods, detergents and the like.
Description
Technical Field
The invention belongs to the field of protein extraction and purification, and particularly relates to neutral protease of straw mushroom fruiting bodies, an extraction and purification method and application thereof.
Background
The volvaria volvacea (Volvariella volvacea) is a typical high-temperature edible fungus variety, contains rich protein, has high growth and development speed and high respiratory intensity, is easy to open after being harvested, and loses edible value. As the straw mushroom is a high-temperature edible mushroom and is sensitive to temperature change, the straw mushroom usually undergoes hydration autolysis at 0-5 ℃, the rotting is accelerated, and the low-temperature autolysis phenomena such as softening, liquefaction and the like occur. In the process, macromolecular proteins in the straw mushroom are decomposed into micromolecular proteins, and the neutral protease activity of the straw mushroom fruiting body is higher under the storage condition of 4 ℃.
The protease is widely applied to the aspects of leather, fur, silk, medicine, food, brewing and the like. Neutral protease is one of protease preparations which are firstly applied to industrial production, and is widely applied to the fields of food, medicine, leather, feed, chemical industry, waste treatment and the like due to mild action conditions and high catalytic rate. The enzymatic washing powder is a new product in detergent, contains alkaline protease and can remove bloodstain and protein dirt on clothes.
At present, the extraction and purification method of protease is mainly ammonium sulfate precipitation method. The method has mild extraction conditions, and is not easy to destroy the active center of protease. In the purification process, because the components in the extracted crude protease are complex and a single purification operation cannot meet the purification requirement, a multi-unit purification mode can be adopted, such as a mode of combining an ion column and a gel column or a mode of combining membrane dialysis and the ion column. Document 1 (chenmingjie, chenne, wannan, et al. isolation and purification of volvariella volvacea protease [ J ]. journal of edible fungi, 1999,6(4):46-48.) reports a method for isolation and purification of volvariella volvacea protease, V23 volvariella volvacea fruit body is treated at 4 ℃ for 24 hours, after homogenization, the supernatant is collected, ammonium sulfate concentration is 100% for precipitation, after dialysis at 4 ℃, concentration is 1mL, purification is performed with sephadex g-150 gel column, finally three protein peaks are obtained, but only one is active, extraction efficiency is low, and the purity of protease obtained by purification is not verified, which indicates that experimental design thereof is incomplete. Document 2 (separation and purification of Pleurotus ostreatus protease and determination of isoelectric Point [ J ] bacterial substance System, 2003(02): 161-.
Disclosure of Invention
The invention aims to provide straw mushroom fruiting body neutral protease, an extraction and purification method thereof and application of the straw mushroom fruiting body neutral protease in enzymolysis of soybean protein isolate. The neutral protease of 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 neutral protease of the straw mushroom fruiting body comprises the following specific steps:
(1) treating straw mushroom fruiting bodies at 4 ℃ for 36-60h, 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 24 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 50 ℃, and Ca is used2+Has effect in promoting casein decomposition by neutral protease of straw mushroom fruiting body, and metal ions (Fe)2+、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 4 ℃ for 36h, homogenized according to a certain material-liquid ratio, precipitated by ammonium sulfate, dialyzed to remove salt, and then chromatographed 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 ratio of medicinal amino acid in an enzymolysis solution is higher than that in a trypsin enzymolysis solution, and the straw mushroom fruiting body neutral protease has wide application prospects in the medicine and food industries.
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 an SDS-PAGE electrophoresis pattern in example 1;
FIG. 4 is a DEAE-FF ion column elution peak 3 in comparative example 1;
FIG. 5 is a graph showing the elution peak 4 of Superdex 7510/300 GL gel column in comparative example 1;
FIG. 6 is a graph showing DEAE-FF ion column elution peak 5 in example 2;
FIG. 7 is a graph showing the elution peak 6 of Superdex 7510/300 GL gel column in example 2;
FIG. 8 is a DEAE-FF ion column elution peak 7 in comparative example 2;
FIG. 9 is a graph showing the elution peak 8 of Superdex 7510/300 GL gel column in comparative example 2;
FIG. 10 is a graph showing the change in enzyme activity at different pH values in example 3;
FIG. 11 is a graph showing the change in enzyme activity at different temperatures in example 3;
FIG. 12 is a graph showing the change in enzyme activity with respect to different metal ions in example 3;
FIG. 13 is a graph showing the change in enzyme activity with different chemicals in example 3;
FIG. 14 shows the degree of hydrolysis of two proteases in example 4.
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, standing at 4 ℃ for 36h, and mixing according to the weight ratio of 1: 10, adding distilled water for homogenizing, standing at 4 ℃ for 2 hours, centrifuging at 5000r/min, and reserving supernatant to obtain crude protease liquid. 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 24 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 purification of the neutral protease is 13.48 times, the specific enzyme activity reaches 286.82U/mg, the neutral protease has high biological activity, and the neutral protease can be widely used in the industries of medicines, foods, detergents and the like.
Example 2
(1) The extraction method and dialysis method were the same as in example 1.
(2) The dialyzed sample is fractionated by using an ultrafiltration concentration tube with the size of 30kDa, the protein content of an absorption peak is determined by using a BCA method protein content determination kit, and the enzyme activity of the absorption peak is determined by using the Folin method in appendix B in the national standard GBT23527-2009, so that the active fraction is determined by the specific enzyme activity.
(3) The ion column elution was the same as in example 1, giving main absorption peak 5.
(4) The gel column elution method was the same as in example 1, yielding peak 6.
(5) Protease activity was measured in the same manner as in example 1.
Example 3
(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%.
The results of steps (4) and (5) in example 3 are shown in FIGS. 12 and 13. From FIGS. 10, 11 and 12, it can be seen that the optimal conditions for the neutral protease reaction of the purified straw mushroom fruit body are as follows: pH7, temperature 50 ℃ and Ca only2+The ionic surfactant CTAB has a remarkable promoting effect on the neutral protease of the straw mushroom fruiting body, other metal ions have inhibiting effects of different degrees, beta-mercaptoethanol has a strong inhibiting effect on the protease of the straw mushroom fruiting body, so that the neutral protease contains a mercapto group, EDTA and PMSF have inhibiting effects on the neutral protease of the straw mushroom fruiting body, and compared with a non-ionic surfactant Tween-80 and Triton, the ionic surfactant CTAB has a weaker inhibiting effect on the neutral protease of the straw mushroom fruiting body.
Example 4
(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. 14, the maximum degree of hydrolysis of neutral protease of the fruiting body of Volvariella volvacea is much higher than that of trypsin, reaching 7.58%; the free amino acids of the enzymolysis products of the two are determined to find (table 2), the proportion of umami amino acids (Asp, Glu, Lys, Gly) in the neutral protease enzymolysis liquid of the straw mushroom fruit body and the trypsin enzymolysis liquid in the total amino acids is not very different, and the neutral protease enzymolysis liquid and the trypsin enzymolysis liquid both have more umami components, but the proportion of the medicinal amino acids (Asp, Leu, Met, Lys, Arg, Glu, Phe, Tyr, Gly) is the highest in the neutral protease enzymolysis liquid of the straw mushroom fruit body, reaches 73.53 percent and is 10.10 percent higher than the medicinal amino acids in the trypsin enzymolysis liquid, which indicates that the neutral protease of the straw mushroom fruit body has larger application potential in medicines and foods.
Comparative example 1
(1) The extraction method and dialysis method were the same as in example 1.
(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 linear elution with 1M NaCl solution at flow rate of 1.0 mL/min; 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 3 by the specific enzyme activity.
(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.
Comparative example 2
(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 7.
(3) The gel column elution method was the same as in example 1, yielding peak 8.
(4) Protease activity was measured in the same manner as in example 1.
Through the comparative examples, it is understood from examples 1-2 and comparative example 2 that the fruiting body of volvariella volvacea is better 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 through gradient elution, but only peak 1 has the strongest activity, and the other two peaks are both hetero-proteins, so that the purification effect of example 1 is better; from example 2, it can be seen that, a step of ultrafiltration concentration tube fraction is added on the basis of example 1, so that more macromolecular hybrid proteins are removed, and from the purification peak chart of gel column chromatography, the gel chromatography result of example 2 only has two protein peaks (fig. 7), the main activity peak is peak 6, and the separation effect is obviously better than that of the gel chromatography purification result of example 1 (fig. 2).
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6251917B1 (en) * | 1996-11-26 | 2001-06-26 | Basf Aktiengesellschaft | Benzamidoaldehydes and their use as cysteine protease inhibitors |
| CN101182504A (en) * | 2007-11-12 | 2008-05-21 | 惠静璇 | Purification method of white gourd prolease |
| CN101294151A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | A kind of extraction method of marine Brevibacterium oxidans alkaline protease |
| CN102212513A (en) * | 2010-04-09 | 2011-10-12 | 北京师范大学 | Proteolytic enzyme separated from cordyceps sinensis and purification method thereof |
| CN102268422A (en) * | 2011-07-06 | 2011-12-07 | 北京市农林科学院 | Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease |
| CN104531644A (en) * | 2014-12-16 | 2015-04-22 | 江南大学 | Method for separating and purifying protease capable of degrading soybean protein allergens |
| CN110551707A (en) * | 2019-10-17 | 2019-12-10 | 山西大学 | Method for purifying neutral or alkaline protease |
-
2021
- 2021-11-30 CN CN202111448386.9A patent/CN113999832A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6251917B1 (en) * | 1996-11-26 | 2001-06-26 | Basf Aktiengesellschaft | Benzamidoaldehydes and their use as cysteine protease inhibitors |
| CN101294151A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | A kind of extraction method of marine Brevibacterium oxidans alkaline protease |
| CN101182504A (en) * | 2007-11-12 | 2008-05-21 | 惠静璇 | Purification method of white gourd prolease |
| CN102212513A (en) * | 2010-04-09 | 2011-10-12 | 北京师范大学 | Proteolytic enzyme separated from cordyceps sinensis and purification method thereof |
| CN102268422A (en) * | 2011-07-06 | 2011-12-07 | 北京市农林科学院 | Cordyceps sobolifera protease capable of inhibiting human immunodeficiency virus-1 (HIV-1) reverse transcriptase and preparation method for Cordyceps sobolifera protease |
| CN104531644A (en) * | 2014-12-16 | 2015-04-22 | 江南大学 | Method for separating and purifying protease capable of degrading soybean protein allergens |
| CN110551707A (en) * | 2019-10-17 | 2019-12-10 | 山西大学 | Method for purifying neutral or alkaline protease |
Non-Patent Citations (10)
| Title |
|---|
| 代玉梅等: "沙蚕消化道细菌D2胞外蛋白酶的分离纯化及其性质", 《中国生物制品学杂志》 * |
| 宗红等: "一种米曲霉蛋白酶的分离纯化及酶解特性研究", 《食品工业科技》 * |
| 李世贵等: "草菇蛋白酶的分离、纯化及等电点的测定", 《菌物系统》 * |
| 李世贵等: "草菇蛋白酶的鉴定", 《食用菌学报》 * |
| 李玉环等: "枯草芽孢杆菌HS18碱性蛋白酶分离纯化及其性质研究", 《食品工业科技》 * |
| 王美艳等: "草菇低温诱导蛋白的分离与纯化", 《农业生物技术学报》 * |
| 蒙义文，陈素文，莫卫平，王佳兴，韩宇健: "中性蛋白酶的离子交换树脂纯化工艺研究", 《中国医药工业杂志》 * |
| 赵丛等: "枯草芽孢杆菌ZC-7中性蛋白酶的分离纯化及酶学性质研究", 《中国生物工程杂志》 * |
| 陈明杰等: "草菇蛋白酶的分离与纯化", 《食用菌学报》 * |
| 黄光荣等: "嗜热芽孢杆菌HS08耐热中性蛋白酶的分离纯化及部分特性研究", 《中国食品学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116715724A (en) * | 2023-05-24 | 2023-09-08 | 上海市农业科学院 | Antioxidant peptides derived from straw mushroom fruiting bodies and their applications |
| CN116715724B (en) * | 2023-05-24 | 2024-03-15 | 上海市农业科学院 | Antioxidant peptides derived from straw mushroom fruiting bodies and their applications |
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