CN113151217A - Method for extracting xylanase from fungus bag - Google Patents
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- CN113151217A CN113151217A CN202110667650.1A CN202110667650A CN113151217A CN 113151217 A CN113151217 A CN 113151217A CN 202110667650 A CN202110667650 A CN 202110667650A CN 113151217 A CN113151217 A CN 113151217A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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Abstract
The invention discloses a method for extracting xylanase from fungus bags, which comprises the steps of crushing and uniformly mixing fungus bag cultivation waste, adding a mixed solution, and preparing a supernatant enzyme solution; taking supernatant enzyme solution, precipitating with ammonium sulfate, and collecting precipitate; adding buffer solution until the precipitate is completely dissolved, and dialyzing on a magnetic stirrer; and preparing a supernatant to obtain a crude enzyme solution; eluting with buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with enzyme activity, freezing and concentrating to obtain xylanase; performing salt ion gradient elution, and collecting each elution peak; detecting the enzyme activity of each elution peak, and freeze-drying to obtain xylanase again; the method has the advantages of rich raw materials, low cost, effective utilization of waste materials, simple extraction method, high activity and purity of the extracted xylanase, higher extraction rate, realization of recycling of the edible fungus waste fungus bag, reduction of resource waste, extension of the source of the xylanase and higher economic value.
Description
Technical Field
The invention belongs to the technical field of xylanase production and extraction, and particularly relates to a method for extracting xylanase from a fungus bag.
Background
Xylanase is a generic term for a group of enzymes that can degrade xylan into oligosaccharides and xylose, and mainly includes beta-1, 4 xylanase and beta-xylosidase, which can degrade xylan in plants into monosaccharides. Xylanase is a novel enzyme preparation, is mainly applied to the feed and brewing industries at present, and becomes one of five industrial enzyme preparations with the largest production and sale quantity in the world.
The xylanase as a feed additive can effectively hydrolyze anti-nutritional factors such as non-starch polysaccharide and the like, break plant cell walls, promote the release of plant effective substances, reduce the viscosity of intestinal chyme, enhance the absorption of animals to nutrient substances and the production capacity of animals, improve the utilization rate of the feed, reduce the cost of the feed, enhance the disease resistance of livestock and poultry, reduce the morbidity and reduce the pollution caused by animal excreta. Therefore, the xylanase is an environment-friendly additive and has very important significance and value in the development of environment-friendly animal husbandry. The acidic xylanase is used in the beer industry, can degrade xylose in the cell walls of grains such as barley and the like, is beneficial to accelerating the action of the enzyme, improving the filtration speed of wort, preventing beer from being turbid and reducing the production cost.
The raw materials for producing xylanase are various, including corncob, bean cake powder, bran, corn steep liquor, whey powder and the like, but the enzymatic activity of the product is not high. The edible fungus bag can generate a large amount of cultivation material degrading enzymes in the cultivation process, wherein the xylanase has high activity and considerable extraction and application values. However, the method for extracting xylanase by using fungus bags in the prior art has the disadvantages of low extraction rate, great waste, complex process and low purity of the extracted xylanase.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for extracting xylanase from a fungus bag, which has the advantages of abundant raw materials, low cost, effective utilization of waste materials, simple extraction method, high activity and high purity of the extracted xylanase, higher extraction rate, realization of recycling of the edible fungus waste fungus bag, reduction of resource waste, extension of the source of the xylanase and higher economic value.
In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting xylanase from a fungal bag, comprising the steps of:
a, crushing and uniformly mixing the fungus bag cultivation waste, adding a mixed solution of water and a buffer solution according to a material-liquid ratio of 1:5, uniformly mixing the water and the buffer solution according to a ratio of 1:4, and standing the mixture for 1.5 hours at 20 ℃; filtering with gauze, centrifuging at 25 deg.C at 10000r/min for 10-15min, and collecting supernatant enzyme solution;
b, taking 10mL of supernatant enzyme solution, performing primary ammonium sulfate precipitation by using 30wt.% of ammonium sulfate, performing secondary ammonium sulfate precipitation by using 70wt.% of ammonium sulfate, salting out for 3-6h at 10 ℃, centrifuging for 10-15min at 10000r/min at 20 ℃, and collecting the precipitate;
c, putting buffer solution into the mixture until the precipitate is completely dissolved, taking 10ml of solution, and dialyzing on a magnetic stirrer at the temperature of 20 ℃; replacing the dialyzate every 1h until 0.1mol/LBaCl2 solution is added into the dialyzate, and detecting that no white precipitate is generated; collecting the solution, centrifuging at 25 deg.C at 10000r/min for 10-15min, freeze drying the supernatant to obtain soluble protein, placing into buffer solution until the soluble protein is completely dissolved, centrifuging at 25 deg.C at 10000r/min for 10-15min, and collecting the supernatant to obtain crude enzyme solution;
d, carrying out SephadexG-100 sephadex gel chromatography by buffer solution balance at the flow rate of 1mL/min until the value of a reference line on a recorder is constant, and stopping balance; taking the crude enzyme solution, eluting with a buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with the enzyme activity, freezing and concentrating to obtain xylanase;
e, balancing DEAEC-52 by using a trihydroxymethyl aminomethane hydrochloride buffer solution to obtain a balanced buffer solution; dissolving enzyme solution subjected to SephadexG-100 sephadex gel chromatography with an equilibrium buffer solution, loading to a column, taking 3ml, performing salt ion gradient elution by adopting a tris (hydroxymethyl) aminomethane hydrochloride equilibrium buffer solution containing sodium chloride at concentrations of 0M, 0.3M and 0.7M respectively, and collecting each elution peak; and detecting the enzyme activity of each elution peak, collecting, freezing and drying to obtain the xylanase.
Preferably, the buffer in steps a-d is 0.02m ph6.0 sodium phosphate dibasic-citric acid buffer; the tris hydrochloride buffer described in step e was 0.02m ph7.0 tris hydrochloride equilibration buffer.
Preferably, the protein purity of the xylanases obtained in step d and step e is characterized by staining with Coomassie Brilliant blue R-250 using 10% strength polyacrylamide, pH8.5 Tris-hydrochloride buffer, using the separation gel.
Compared with the prior art, the invention has the following beneficial effects:
the method has the advantages of abundant raw materials, low cost, effective utilization of waste materials, simple extraction method, time saving, high efficiency, high activity and purity of the extracted xylanase, high extraction rate, realization of recycling of the edible fungus waste fungus bags, reduction of resource waste, extension of the source of the xylanase and high economic value.
Detailed Description
The technical solutions of the present invention are described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
This example provides a method for extracting xylanase from a fungal bag, comprising the steps of:
a, crushing and uniformly mixing the fungus bag cultivation waste, adding a mixed solution of water and a buffer solution according to a material-liquid ratio of 1:5, uniformly mixing the water and the buffer solution according to a ratio of 1:4, and standing the mixture for 1.5 hours at 20 ℃; filtering with gauze, centrifuging at 25 deg.C at 10000r/min for 10min, and collecting supernatant enzyme solution;
b, taking 10mL of supernatant enzyme solution, performing primary ammonium sulfate precipitation by using 30wt.% of ammonium sulfate, performing secondary ammonium sulfate precipitation by using 70wt.% of ammonium sulfate, salting out for 3h at 10 ℃, centrifuging for 10min at 10000r/min at 20 ℃, and collecting the precipitate;
c, putting buffer solution into the mixture until the precipitate is completely dissolved, taking 10ml of solution, and dialyzing on a magnetic stirrer at the temperature of 20 ℃; replacing the dialyzate every 1h until 0.1mol/LBaCl2 solution is added into the dialyzate, and detecting that no white precipitate is generated; collecting the solution, centrifuging at 25 deg.C at 10000r/min for 10min, freeze drying the supernatant to obtain soluble protein, placing into buffer solution until the soluble protein is completely dissolved, centrifuging at 25 deg.C at 10000r/min for 10min, and collecting the supernatant to obtain crude enzyme solution;
d, carrying out SephadexG-100 sephadex gel chromatography by buffer solution balance at the flow rate of 1mL/min until the value of a reference line on a recorder is constant, and stopping balance; taking the crude enzyme solution, eluting with a buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with the enzyme activity, freezing and concentrating to obtain xylanase;
e, balancing DEAEC-52 by using a trihydroxymethyl aminomethane hydrochloride buffer solution to obtain a balanced buffer solution; dissolving enzyme solution subjected to SephadexG-100 sephadex gel chromatography with an equilibrium buffer solution, loading to a column, taking 3ml, performing salt ion gradient elution by adopting a tris (hydroxymethyl) aminomethane hydrochloride equilibrium buffer solution containing sodium chloride at concentrations of 0M, 0.3M and 0.7M respectively, and collecting each elution peak; and detecting the enzyme activity of each elution peak, collecting, freezing and drying to obtain the xylanase.
In this example, the buffer in steps a-d is 0.02M pH6.0 disodium phosphate-citric acid buffer; the tris hydrochloride buffer described in step e was 0.02m ph7.0 tris hydrochloride equilibration buffer.
Example 2
This example provides a method for extracting xylanase from a fungal bag, comprising the steps of:
a, crushing and uniformly mixing the fungus bag cultivation waste, adding a mixed solution of water and a buffer solution according to a material-liquid ratio of 1:5, uniformly mixing the water and the buffer solution according to a ratio of 1:4, and standing the mixture for 1.5 hours at 20 ℃; filtering with gauze, centrifuging at 25 deg.C at 10000r/min for 15min, and collecting supernatant enzyme solution;
b, taking 10mL of supernatant enzyme solution, performing primary ammonium sulfate precipitation by using 30wt.% of ammonium sulfate, performing secondary ammonium sulfate precipitation by using 70wt.% of ammonium sulfate, salting out for 6h at 10 ℃, centrifuging for 15min at 20 ℃ at 10000r/min, and collecting the precipitate;
c, putting buffer solution into the mixture until the precipitate is completely dissolved, taking 10ml of solution, and dialyzing on a magnetic stirrer at the temperature of 20 ℃; replacing the dialyzate every 1h until 0.1mol/LBaCl2 solution is added into the dialyzate, and detecting that no white precipitate is generated; collecting the solution, centrifuging at 25 deg.C at 10000r/min for 15min, freeze drying the supernatant to obtain soluble protein, placing into buffer solution until the soluble protein is completely dissolved, centrifuging at 25 deg.C at 10000r/min for 15min, and collecting the supernatant to obtain crude enzyme solution;
d, carrying out SephadexG-100 sephadex gel chromatography by buffer solution balance at the flow rate of 1mL/min until the value of a reference line on a recorder is constant, and stopping balance; taking the crude enzyme solution, eluting with a buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with the enzyme activity, freezing and concentrating to obtain xylanase;
e, balancing DEAEC-52 by using a trihydroxymethyl aminomethane hydrochloride buffer solution to obtain a balanced buffer solution; dissolving enzyme solution subjected to SephadexG-100 sephadex gel chromatography with an equilibrium buffer solution, loading to a column, taking 3ml, performing salt ion gradient elution by adopting a tris (hydroxymethyl) aminomethane hydrochloride equilibrium buffer solution containing sodium chloride at concentrations of 0M, 0.3M and 0.7M respectively, and collecting each elution peak; and detecting the enzyme activity of each elution peak, collecting, freezing and drying to obtain the xylanase.
In this example, the buffer in steps a-d is 0.02M pH6.0 disodium phosphate-citric acid buffer; the tris hydrochloride buffer described in step e was 0.02m ph7.0 tris hydrochloride equilibration buffer.
Example 3
This example provides a method for extracting xylanase from a fungal bag, comprising the steps of:
a, crushing and uniformly mixing the fungus bag cultivation waste, adding a mixed solution of water and a buffer solution according to a material-liquid ratio of 1:5, uniformly mixing the water and the buffer solution according to a ratio of 1:4, and standing the mixture for 1.5 hours at 20 ℃; filtering with gauze, centrifuging at 25 deg.C at 10000r/min for 12min, and collecting supernatant enzyme solution;
b, taking 10mL of supernatant enzyme solution, performing primary ammonium sulfate precipitation by using 30wt.% of ammonium sulfate, performing secondary ammonium sulfate precipitation by using 70wt.% of ammonium sulfate, salting out for 5h at 10 ℃, centrifuging for 12min at 20 ℃ at 10000r/min, and collecting the precipitate;
c, putting buffer solution into the mixture until the precipitate is completely dissolved, taking 10ml of solution, and dialyzing on a magnetic stirrer at the temperature of 20 ℃; replacing the dialyzate every 1h until 0.1mol/LBaCl2 solution is added into the dialyzate, and detecting that no white precipitate is generated; collecting the solution, centrifuging at 25 deg.C at 10000r/min for 12min, freeze drying the supernatant to obtain soluble protein, placing into buffer solution until the soluble protein is completely dissolved, centrifuging at 25 deg.C at 10000r/min for 12min, and collecting the supernatant to obtain crude enzyme solution;
d, carrying out SephadexG-100 sephadex gel chromatography by buffer solution balance at the flow rate of 1mL/min until the value of a reference line on a recorder is constant, and stopping balance; taking the crude enzyme solution, eluting with a buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with the enzyme activity, freezing and concentrating to obtain xylanase;
e, balancing DEAEC-52 by using a trihydroxymethyl aminomethane hydrochloride buffer solution to obtain a balanced buffer solution; dissolving enzyme solution subjected to SephadexG-100 sephadex gel chromatography with an equilibrium buffer solution, loading to a column, taking 3ml, performing salt ion gradient elution by adopting a tris (hydroxymethyl) aminomethane hydrochloride equilibrium buffer solution containing sodium chloride at concentrations of 0M, 0.3M and 0.7M respectively, and collecting each elution peak; and detecting the enzyme activity of each elution peak, collecting, freezing and drying to obtain the xylanase.
In this example, the buffer in steps a-d is 0.02M pH6.0 disodium phosphate-citric acid buffer; the tris hydrochloride buffer described in step e was 0.02m ph7.0 tris hydrochloride equilibration buffer.
The method has the advantages of abundant raw materials, low cost, effective utilization of waste materials, simple extraction method, time saving, high efficiency, high activity and high purity of the extracted xylanase, and the extraction rate is up to more than 92 percent, so that the method not only can realize the recycling of the edible fungus waste fungus bags and reduce the resource waste, but also expands the sources of the xylanase, and has high economic value.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (3)
1. A method for extracting xylanase from a fungus bag, which is characterized by comprising the following steps:
a, crushing and uniformly mixing the fungus bag cultivation waste, adding a mixed solution of water and a buffer solution according to a material-liquid ratio of 1:5, uniformly mixing the water and the buffer solution according to a ratio of 1:4, and standing the mixture for 1.5 hours at 20 ℃; filtering with gauze, centrifuging at 25 deg.C at 10000r/min for 10-15min, and collecting supernatant enzyme solution;
b, taking 10mL of supernatant enzyme solution, performing primary ammonium sulfate precipitation by using 30wt.% of ammonium sulfate, performing secondary ammonium sulfate precipitation by using 70wt.% of ammonium sulfate, salting out for 3-6h at 10 ℃, centrifuging for 10-15min at 10000r/min at 20 ℃, and collecting the precipitate;
c, putting buffer solution into the mixture until the precipitate is completely dissolved, taking 10ml of solution, and dialyzing on a magnetic stirrer at the temperature of 20 ℃; replacing the dialyzate every 1h until 0.1mol/LBaCl2 solution is added into the dialyzate, and detecting that no white precipitate is generated; collecting the solution, centrifuging at 25 deg.C at 10000r/min for 10-15min, freeze drying the supernatant to obtain soluble protein, placing into buffer solution until the soluble protein is completely dissolved, centrifuging at 25 deg.C at 10000r/min for 10-15min, and collecting the supernatant to obtain crude enzyme solution;
d, carrying out SephadexG-100 sephadex gel chromatography by buffer solution balance at the flow rate of 1mL/min until the value of a reference line on a recorder is constant, and stopping balance; taking the crude enzyme solution, eluting with a buffer solution, collecting each elution peak, detecting the enzyme activity of each tube, combining the tubes with the enzyme activity, freezing and concentrating to obtain xylanase;
e, balancing DEAEC-52 by using a trihydroxymethyl aminomethane hydrochloride buffer solution to obtain a balanced buffer solution; dissolving enzyme solution subjected to SephadexG-100 sephadex gel chromatography with an equilibrium buffer solution, loading to a column, taking 3ml, performing salt ion gradient elution by adopting a tris (hydroxymethyl) aminomethane hydrochloride equilibrium buffer solution containing sodium chloride at concentrations of 0M, 0.3M and 0.7M respectively, and collecting each elution peak; and detecting the enzyme activity of each elution peak, collecting, freezing and drying to obtain the xylanase.
2. The method of claim 1, wherein the buffer in steps a-d is 0.02m ph6.0 disodium phosphate-citrate buffer; the tris hydrochloride buffer described in step e was 0.02m ph7.0 tris hydrochloride equilibration buffer.
3. The method for extracting xylanase from a bale according to claim 1, wherein the protein purity of the xylanase obtained in steps d and e is characterized by using separation gel at a concentration of 10% polyacrylamide, pH8.5 tris hydrochloride buffer, and staining with Coomassie Brilliant blue R-250.
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| CN202110667650.1A CN113151217A (en) | 2021-06-16 | 2021-06-16 | Method for extracting xylanase from fungus bag |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030008379A1 (en) * | 2001-03-19 | 2003-01-09 | Bhosle Narayan Baburao | Novel 'pseudomonas stutzeri' strain and process for preparation of xylanase |
| CN101812431A (en) * | 2009-02-23 | 2010-08-25 | 湖南省畜牧兽医研究所 | Method utilizing bacterisugar to prepare feed enzyme |
| CN104312999A (en) * | 2014-10-09 | 2015-01-28 | 福建农林大学 | Method for separating and purifying xylanase from waste flammulina velutiper cultivation material |
| CN105154412A (en) * | 2015-09-29 | 2015-12-16 | 福建农林大学 | Method for extracting xylanase from waste tremella fungus bags |
-
2021
- 2021-06-16 CN CN202110667650.1A patent/CN113151217A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030008379A1 (en) * | 2001-03-19 | 2003-01-09 | Bhosle Narayan Baburao | Novel 'pseudomonas stutzeri' strain and process for preparation of xylanase |
| CN101812431A (en) * | 2009-02-23 | 2010-08-25 | 湖南省畜牧兽医研究所 | Method utilizing bacterisugar to prepare feed enzyme |
| CN104312999A (en) * | 2014-10-09 | 2015-01-28 | 福建农林大学 | Method for separating and purifying xylanase from waste flammulina velutiper cultivation material |
| CN105154412A (en) * | 2015-09-29 | 2015-12-16 | 福建农林大学 | Method for extracting xylanase from waste tremella fungus bags |
Non-Patent Citations (2)
| Title |
|---|
| 杨云龙等: "金针菇菌糠中木聚糖酶的分离纯化及其酶学性质研究", 《江西农业大学学报》 * |
| 马怀良等: "黑木耳菌糠粗纤维素酶和木聚糖酶浸提条件研究", 《北方园艺》 * |
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