CN112522240A - Method for purifying xylanase - Google Patents
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- CN112522240A CN112522240A CN201910878770.9A CN201910878770A CN112522240A CN 112522240 A CN112522240 A CN 112522240A CN 201910878770 A CN201910878770 A CN 201910878770A CN 112522240 A CN112522240 A CN 112522240A
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- C—CHEMISTRY; METALLURGY
- 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
- C12N9/2482—Endo-1,4-beta-xylanase (3.2.1.8)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
Abstract
The invention belongs to the technical field of biological enzymes, and particularly relates to a method for purifying xylanase. The method takes commercially available crude xylanase powder as a raw material, and the purified xylanase is produced by the steps of pH adjustment, hollow fiber membrane filtration, ultrafiltration, freeze drying and the like. The method adopts ultrafiltration grading treatment, can effectively improve the purification multiple and efficiency, reduces the production cost, adopts liquid nitrogen to realize the quick freezing storage of enzyme liquid, reduces the structural change of enzyme crystals and the loss of enzyme activity caused by slow pre-freezing, and improves the product performance. The method has the characteristics of simple production process, convenient production, high product yield, low equipment investment and operation cost, high comprehensive utilization rate of resources and the like. The xylanase product purified by the method has high activity, can meet the requirement of the pharmaceutical industry on high-purity enzyme preparations, and can be widely applied to the industries of medicine, food, chemical industry and the like.
Description
Technical Field
The invention belongs to the technical field of biological enzymes, and particularly relates to a method for purifying xylanase.
Background
Xylanases belong to the class of hydrolases and are an enzyme system that degrades xylan into xylooligosaccharides, and this enzyme system mainly includes beta-1, 4-endoxylanase (EC 3.2.1.8), beta-D-xylosidase (EC 3.2.1.37), alpha-D-glucuronidase (EC 3.2.1.139), alpha-L-arabinofuranosidase (EC 3.2.1.55), acetyl xylanase (EC 3.3.1.72), and the like. Among them, beta-1, 4-endoxylanase is the most critical enzyme for complete hydrolysis of xylan, and hydrolyzes xylan into xylooligosaccharides such as small oligosaccharides and xylobiose, and small amounts of xylose and arabinose by hydrolyzing the beta-1, 4-glycosidic bond of xylan molecule. The xylanase is widely applied to the fields of medicines, foods, energy sources, paper making, feeds and the like, is one of the fastest-developing industrial enzyme preparations in recent years, and has higher commercial value and market potential. However, the xylanase preparation sold in the market has low purity, cannot meet the requirement of enzyme for preparing high value-added products (such as the field of medicine), and limits the application of xylanase.
At present, methods for purifying xylanase include non-specific methods and specific methods. The nonspecific purification method mainly comprises the steps of ammonium sulfate fractional precipitation or centrifugation, and further purification by ion exchange chromatography, aqueous two-phase extraction, gel filtration chromatography, hydrophobic chromatography and the like. Specific purification methods are affinity chromatography methods developed primarily based on specific binding to xylanases. However, large-scale production has not been achieved for some time because of the high purification cost. Therefore, the development of a xylanase purification method which is low in cost and suitable for industrialization has important application value.
The existing method for purifying xylanase, such as 'purification, identification and enzymology property research of xylanase' in 'feed research' 2017, 15 th, discloses a method comprising the following steps: the xylanase enzyme solution is obtained by using commercially available solid xylanase as a raw material and purifying the solid xylanase by steps of dissolving in deionized water, cooling and precipitating, centrifuging, performing sephadex filtration chromatography and the like. The main disadvantages of this process are: firstly, the sephadex filtration chromatography has high operation cost and limited production capacity, and is not convenient for industrial mass production; secondly, the xylanase concentration in the xylanase enzyme liquid product obtained by elution is low, and the subsequent industrial application is inconvenient. Also, as in the article "xylanase ultrafiltration purification method research" of volume 11 of 2010, volume 38, Anhui agricultural science, the disclosed method is: the xylanase stock solution obtained by culturing the trichoderma reesei is used as a raw material, and is subjected to ultrafiltration by ultrafiltration columns with different molecular weights after being diluted properly, or the xylanase stock solution is directly subjected to ultrafiltration without being diluted, and a citric acid buffer is added in the ultrafiltration process for dilution. The main disadvantages of this process are: firstly, xylanase stock solution raw materials are not pretreated, the resource utilization rate is low, the purity of the produced product is low, and the requirement of preparing products with high added values cannot be met; secondly, the obtained product is liquid and is inconvenient for long-term storage and transportation.
Disclosure of Invention
The invention aims to overcome the defects of the conventional xylanase purification method, and provides the xylanase purification method which has the characteristics of simple production process, convenience in production, high product yield, low equipment investment and operation cost, high comprehensive resource utilization rate and the like. The product prepared by the method has the characteristics of high activity, good stability and the like.
The mechanism of the invention is as follows: xylanase is an enzyme system consisting of multiple hydrolases, and requires the synergistic action of the multiple hydrolases in the process of complete degradation of xylan. However, the role played by β -1, 4-endoxylanases is most critical. Therefore, the invention takes the improvement of the purity of the beta-1, 4-endo-xylanase in the xylanase as a main target, and establishes a corresponding separation and purification process based on the difference of isoelectric points and molecular weights caused by different structures of hydrolytic enzymes in the xylanase. The isoelectric point of the beta-1, 4-endoxylanase is mostly from acidity to neutrality, so that the pH of a crude enzyme system is adjusted to alkalinity to reduce the solubility of part of protein, and then a hollow fiber membrane filter is adopted for separation to realize the primary purification of the xylanase. The molecular weight of the beta-1, 4-endo-xylanase is mostly concentrated in 15-50 kDa, macromolecular proteins above 50kDa can be removed by an ultrafiltration membrane with the molecular weight cutoff of 40-50 kDa, then substances below 15kDa, such as proteins, polysaccharides, amino acids and the like can be removed by an ultrafiltration membrane with the molecular weight cutoff of 8-12 kDa, and the xylanase is further purified by two-stage ultrafiltration separation. For xylanase concentrated solution, the activity of enzyme protein molecules at ultralow temperature can be maintained by prefreezing the xylanase concentrated solution by liquid nitrogen, and the enzyme can be rapidly frozen and stored. Meanwhile, when liquid nitrogen is pre-frozen, a small amount of glycerol is added into the xylanase concentrated solution, so that a large amount of ice crystals generated during rapid pre-freezing can be prevented from damaging the structure of the enzyme, and the effect of protecting the enzyme is further achieved.
The purpose of the invention is realized as follows: a method for purifying xylanase uses the commercial crude xylanase powder as raw material and comprises the steps of pH regulation, hollow fiber membrane filtration, ultrafiltration, freeze drying and the like. The method comprises the following specific steps:
(1) preparation of crude xylanase liquid
Dispersing commercially available crude xylanase powder into purified water according to the mass (g) of the crude xylanase powder to volume (mL) ratio of the purified water of 1: 100-200, adjusting the pH value to 8.0-10.0 by using 0.1-0.4 mol/L sodium hydroxide solution, and uniformly stirring to obtain a mixed solution; and then pumping the mixed solution into a hollow fiber membrane filter for filtration, controlling the aperture of the filter membrane to be 0.2 mu m, and pumping at a speed of 500-1000 mL/min, and respectively collecting membrane filtrate and membrane retentate. The collected membrane trapped fluid mainly contains protein, and can be used as a feed additive after being dried and crushed; the collected membrane filtrate, i.e., the crude xylanase solution, is used for the next step of treatment.
(2) Preparation of xylanase ultrafiltrate
And (3) after the step (1) is finished, pumping the crude xylanase liquid collected in the step (1) into an ultrafiltration membrane separation device with the molecular weight cutoff of 40-50 kDa, carrying out first ultrafiltration separation under 0.2-0.4 MPa until the volume of the first ultrafiltration trapped fluid is reduced to 3-5% of the original volume, and respectively collecting first ultrafiltration permeate liquid and first ultrafiltration trapped fluid. The collected first ultrafiltration trapped fluid mainly contains macromolecular protein, and can be combined with the membrane trapped fluid collected in the step (1) to prepare feed additives; and collecting the first ultrafiltration permeate, namely xylanase ultrafiltrate, and using the collected first ultrafiltration permeate as next treatment.
(3) Preparation of xylanase concentrate
And (3) after the step (2) is finished, pumping the xylanase ultrafiltrate collected in the step (2) into an ultrafiltration membrane separation device with the molecular weight cutoff of 8-12 kDa, carrying out second ultrafiltration separation under 0.1-0.3 MPa until the volume of the second ultrafiltration trapped fluid is reduced to 5-10% of the original volume, and respectively collecting the second ultrafiltration permeate and the second ultrafiltration trapped fluid. The collected second ultrafiltration permeating liquid is mainly purified water, contains a small amount of micromolecular protein, polysaccharide, amino acid and the like, and can be continuously used for dispersing the crude xylanase powder in the step (1); and collecting the second ultrafiltration retentate, namely xylanase concentrated solution, and using the second ultrafiltration retentate as next treatment.
(4) Preparation of purified xylanase
After the step (3) is finished, adding glycerol into the xylanase concentrated solution according to the volume ratio of the glycerol to the xylanase concentrated solution collected in the step (3) of 1: 200-300, and uniformly stirring to obtain the xylanase concentrated solution added with the glycerol; and pumping the xylanase concentrated solution added with the glycerol into liquid nitrogen according to the volume ratio of the xylanase concentrated solution added with the glycerol to the liquid nitrogen of 1: 30-100, controlling the pumping speed to be 10-50 mL/min, then quickly filtering, and respectively collecting filter residues and filtrate. The collected filtrate is mainly liquid nitrogen and can be continuously used; and placing the collected filter residues in a freeze drying device, and carrying out freeze drying for 18-24 h at the temperature of 5-10 Pa and-55-65 ℃, so as to prepare the purified xylanase, wherein the purification multiple is 13.6-20.1, the enzyme activity is 70-103U/mug, and the yield is 47.2-59.8%.
After the technical scheme is adopted, the invention mainly has the following effects:
1. the method takes the xylanase powder sold in the market as the raw material, has the characteristics of simple purification process and equipment and easy control, is suitable for large-scale production, and can also be popularized and applied to the purification production of other enzyme systems.
2. In the production process, ultrafiltration grading treatment is adopted, so that the purification multiple and efficiency can be effectively improved, and the production cost is reduced.
3. In the production process, the liquid nitrogen is adopted to realize the quick freezing storage of the enzyme liquid, reduce the structural change of the enzyme crystal and the loss of the enzyme activity caused by slow pre-freezing and improve the product performance.
4. The product produced by the method of the invention can be stored for half a year at 4 ℃ without enzyme activity loss.
5. The method has the advantages of no discharge of three wastes, high resource utilization rate and high product activity, and can meet the requirements of the pharmaceutical industry on high-purity enzyme preparations.
The product prepared by the method can be widely applied to the industries of medicine, food, chemical industry and the like.
Fourth, detailed description of the invention
The present invention will be further described with reference to the following specific embodiments.
Example 1
A method for purifying xylanase comprises the following specific steps:
(1) preparation of crude xylanase liquid
Taking commercially available crude xylanase powder as a raw material, dispersing the crude xylanase powder in purified water according to the ratio of the mass (g) of the crude xylanase powder to the volume (mL) of the purified water of 1: 100, adjusting the pH value to 8.0 by using 0.1mol/L sodium hydroxide solution, and uniformly stirring to obtain a mixed solution; and then pumping the mixed solution into a hollow fiber membrane filter for filtration, controlling the aperture of the filter membrane to be 0.2 mu m, and pumping at the speed of 500mL/min, and respectively collecting membrane filtrate and membrane retentate. The collected membrane trapped fluid mainly contains protein, and can be used as a feed additive after being dried and crushed; the collected membrane filtrate, i.e., the crude xylanase solution, is used for the next step of treatment.
(2) Preparation of xylanase ultrafiltrate
And (3) after the step (1) is finished, pumping the crude xylanase liquid collected in the step (1) into an ultrafiltration membrane separation device with the molecular weight cutoff of 40kDa, carrying out first ultrafiltration separation under 0.2MPa until the volume of the first ultrafiltration trapped fluid is reduced to 3% of the original volume, and respectively collecting first ultrafiltration permeate and first ultrafiltration trapped fluid. The collected first ultrafiltration trapped fluid mainly contains macromolecular protein, and can be combined with the membrane trapped fluid collected in the step (1) to prepare feed additives; and collecting the first ultrafiltration permeate, namely xylanase ultrafiltrate, and using the collected first ultrafiltration permeate as next treatment.
(3) Preparation of xylanase concentrate
And (3) after the step (2) is finished, pumping the xylanase ultrafiltrate collected in the step (2) into an ultrafiltration membrane separation device with the molecular weight cutoff of 8kDa, carrying out second ultrafiltration separation under 0.1MPa until the volume of the second ultrafiltration retentate is reduced to 5% of the original volume, and respectively collecting the second ultrafiltration permeate and the second ultrafiltration retentate. The collected second ultrafiltration permeating liquid is mainly purified water, contains a small amount of micromolecular protein, polysaccharide, amino acid and the like, and can be continuously used for dispersing the crude xylanase powder in the step (1); and collecting the second ultrafiltration retentate, namely xylanase concentrated solution, and using the second ultrafiltration retentate as next treatment.
(4) Preparation of purified xylanase
After the step (3) is finished, adding glycerol into the xylanase concentrated solution according to the volume ratio of the glycerol to the xylanase concentrated solution collected in the step (3) of 1: 200, and uniformly stirring to obtain the xylanase concentrated solution added with the glycerol; and pumping the xylanase concentrated solution added with the glycerol into liquid nitrogen according to the volume ratio of the xylanase concentrated solution added with the glycerol to the liquid nitrogen of 1: 30, controlling the pumping speed to be 10mL/min, then quickly filtering, and respectively collecting filter residues and filtrate. The collected filtrate is mainly liquid nitrogen and can be continuously used; and placing the collected filter residues in a freeze drying device, and carrying out freeze drying for 18h at 5Pa and-55 ℃, so as to prepare the purified xylanase, wherein the purification multiple is 13.6, the enzyme activity is 70U/microgram, and the yield is 47.2%.
Example 2
A method for purifying xylanase comprises the following specific steps:
(1) preparation of crude xylanase liquid
Taking commercially available crude xylanase powder as a raw material, dispersing the crude xylanase powder in purified water according to the ratio of the mass (g) of the crude xylanase powder to the volume (mL) of the purified water of 1: 150, adjusting the pH value to 9.0 by using 0.2mol/L sodium hydroxide solution, and uniformly stirring to obtain a mixed solution; and then pumping the mixed solution into a hollow fiber membrane filter for filtration, controlling the aperture of the filter membrane to be 0.2 mu m, and pumping at the speed of 800mL/min, and respectively collecting membrane filtrate and membrane retentate. The collected membrane trapped fluid mainly contains protein, and can be used as a feed additive after being dried and crushed; the collected membrane filtrate, i.e., the crude xylanase solution, is used for the next step of treatment.
(2) Preparation of xylanase ultrafiltrate
And (3) after the step (1) is finished, pumping the crude xylanase liquid collected in the step (1) into an ultrafiltration membrane separation device with the molecular weight cutoff of 45kDa, carrying out first ultrafiltration separation under 0.3MPa until the volume of the first ultrafiltration trapped fluid is reduced to 4% of the original volume, and respectively collecting first ultrafiltration permeate and first ultrafiltration trapped fluid. The collected first ultrafiltration trapped fluid mainly contains macromolecular protein, and can be combined with the membrane trapped fluid collected in the step (1) to prepare feed additives; and collecting the first ultrafiltration permeate, namely xylanase ultrafiltrate, and using the collected first ultrafiltration permeate as next treatment.
(3) Preparation of xylanase concentrate
And (3) after the step (2) is finished, pumping the xylanase ultrafiltrate collected in the step (2) into an ultrafiltration membrane separation device with the molecular weight cutoff of 10kDa, carrying out second ultrafiltration separation under 0.2MPa until the volume of the second ultrafiltration retentate is reduced to 8% of the original volume, and respectively collecting the second ultrafiltration permeate and the second ultrafiltration retentate. The collected second ultrafiltration permeating liquid is mainly purified water, contains a small amount of micromolecular protein, polysaccharide, amino acid and the like, and can be continuously used for dispersing the crude xylanase powder in the step (1); and collecting the second ultrafiltration retentate, namely xylanase concentrated solution, and using the second ultrafiltration retentate as next treatment.
(4) Preparation of purified xylanase
After the step (3), adding glycerol into the xylanase concentrated solution according to the volume ratio of glycerol to the xylanase concentrated solution collected in the step (3) of 1: 250, and uniformly stirring to obtain the xylanase concentrated solution added with the glycerol; and pumping the xylanase concentrated solution added with the glycerol into liquid nitrogen according to the volume ratio of the xylanase concentrated solution added with the glycerol to the liquid nitrogen of 1: 60, controlling the pumping speed to be 30mL/min, then quickly filtering, and respectively collecting filter residues and filtrate. The collected filtrate is mainly liquid nitrogen and can be continuously used; and (3) placing the collected filter residues in a freeze drying device, and carrying out freeze drying for 20 hours at the temperature of 8Pa and-60 ℃ to prepare the purified xylanase, wherein the purification multiple is 18.3, the enzyme activity is 94U/microgram, and the yield is 53.4%.
Example 3
A method for purifying xylanase comprises the following specific steps:
(1) preparation of crude xylanase liquid
Taking commercially available crude xylanase powder as a raw material, dispersing the crude xylanase powder in purified water according to the ratio of the mass (g) of the crude xylanase powder to the volume (mL) of the purified water of 1: 200, adjusting the pH value to 10.0 by using 0.4mol/L sodium hydroxide solution, and uniformly stirring to obtain a mixed solution; and then pumping the mixed solution into a hollow fiber membrane filter for filtration, controlling the aperture of the filter membrane to be 0.2 mu m, and pumping at the speed of 1000mL/min, and respectively collecting membrane filtrate and membrane retentate. The collected membrane trapped fluid mainly contains protein, and can be used as a feed additive after being dried and crushed; the collected membrane filtrate, i.e., the crude xylanase solution, is used for the next step of treatment.
(2) Preparation of xylanase ultrafiltrate
And (3) after the step (1) is finished, pumping the crude xylanase liquid collected in the step (1) into an ultrafiltration membrane separation device with the molecular weight cutoff of 50kDa, carrying out first ultrafiltration separation under 0.4MPa until the volume of the first ultrafiltration trapped fluid is reduced to 5% of the original volume, and respectively collecting first ultrafiltration permeate and first ultrafiltration trapped fluid. The collected first ultrafiltration trapped fluid mainly contains macromolecular protein, and can be combined with the membrane trapped fluid collected in the step (1) to prepare feed additives; and collecting the first ultrafiltration permeate, namely xylanase ultrafiltrate, and using the collected first ultrafiltration permeate as next treatment.
(3) Preparation of xylanase concentrate
And (3) after the step (2) is finished, pumping the xylanase ultrafiltrate collected in the step (2) into an ultrafiltration membrane separation device with the molecular weight cutoff of 12kDa, carrying out second ultrafiltration separation under 0.3MPa until the volume of the second ultrafiltration retentate is reduced to 10% of the original volume, and respectively collecting the second ultrafiltration permeate and the second ultrafiltration retentate. The collected second ultrafiltration permeating liquid is mainly purified water, contains a small amount of micromolecular protein, polysaccharide, amino acid and the like, and can be continuously used for dispersing the crude xylanase powder in the step (1); and collecting the second ultrafiltration retentate, namely xylanase concentrated solution, and using the second ultrafiltration retentate as next treatment.
(4) Preparation of purified xylanase
After the step (3) is finished, adding glycerol into the xylanase concentrated solution according to the volume ratio of the glycerol to the xylanase concentrated solution collected in the step (3) of 1: 300, and uniformly stirring to obtain the xylanase concentrated solution added with the glycerol; and pumping the xylanase concentrated solution added with the glycerol into liquid nitrogen according to the volume ratio of the xylanase concentrated solution added with the glycerol to the liquid nitrogen of 1: 100, controlling the pumping speed to be 50mL/min, then quickly filtering, and respectively collecting filter residues and filtrate. The collected filtrate is mainly liquid nitrogen and can be continuously used; and (3) placing the collected filter residues in a freeze drying device, and carrying out freeze drying at 10Pa and-65 ℃ for 24h to prepare the purified xylanase, wherein the purification multiple is 20.1, the enzyme activity is 103U/microgram, and the yield is 59.8%.
Claims (1)
1. A method for purifying xylanase is characterized by comprising the following specific steps:
(1) preparation of crude xylanase liquid
Dispersing commercially available crude xylanase powder into purified water according to the mass ratio of the crude xylanase powder to the volume ratio of the purified water of 1 g: 100-200 mL, adjusting the pH value to 8.0-10.0 by using 0.1-0.4 mol/L sodium hydroxide solution, and uniformly stirring to obtain a mixed solution; then pumping the mixed solution into a hollow fiber membrane filter for filtration, controlling the aperture of the filter membrane to be 0.2 mu m, and the pumping speed to be 500-1000 mL/min, and respectively collecting membrane filtrate and membrane retentate; the collected membrane filtrate, namely the crude xylanase liquid, is used for the next treatment;
(2) preparation of xylanase ultrafiltrate
After the step (1) is finished, pumping the crude xylanase liquid collected in the step (1) into an ultrafiltration membrane separation device with the molecular weight cutoff of 40-50 kDa, carrying out first ultrafiltration separation under 0.2-0.4 MPa until the volume of the first ultrafiltration trapped fluid is reduced to 3-5% of the original volume, and respectively collecting first ultrafiltration permeate and first ultrafiltration trapped fluid; collecting the first ultrafiltration permeate, namely xylanase ultrafiltrate, and using the collected first ultrafiltration permeate as next treatment;
(3) preparation of xylanase concentrate
After the step (2) is finished, pumping the xylanase ultrafiltrate collected in the step (2) into an ultrafiltration membrane separation device with the molecular weight cutoff of 8-12 kDa, carrying out second ultrafiltration separation under 0.1-0.3 MPa until the volume of the second ultrafiltration trapped fluid is reduced to 5-10% of the original volume, and respectively collecting second ultrafiltration permeate and second ultrafiltration trapped fluid; collecting the second ultrafiltration retentate, namely xylanase concentrated solution, and using the second ultrafiltration retentate as next treatment;
(4) preparation of purified xylanase
After the step (3) is finished, adding glycerol into the xylanase concentrated solution according to the volume ratio of the glycerol to the xylanase concentrated solution collected in the step (3) of 1: 200-300, and uniformly stirring to obtain the xylanase concentrated solution added with the glycerol; pumping the xylanase concentrated solution added with the glycerol into liquid nitrogen according to the volume ratio of the xylanase concentrated solution added with the glycerol to the liquid nitrogen being 1: 30-100, controlling the pumping speed to be 10-50 mL/min, then quickly filtering, and respectively collecting filter residues and filtrate; and placing the collected filter residues in a freeze drying device, and carrying out freeze drying for 18-24 h at the temperature of 5-10 Pa and-55-65 ℃, so as to prepare the purified xylanase, wherein the purification multiple is 13.6-20.1, the enzyme activity is 70-103U/mug, and the yield is 47.2-59.8%.
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