CN113373134B - Extraction method of N-acetylglucosamine deacetylase - Google Patents
Extraction method of N-acetylglucosamine deacetylase Download PDFInfo
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- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- 241001149952 Amylomyces rouxii Species 0.000 description 1
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- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
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Classifications
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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)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01033—N-Acetylglucosamine deacetylase (3.5.1.33)
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses an extraction method of N-acetylglucosamine deacetylase, belonging to the technical field of bioengineering. The invention takes fermentation liquor containing N-acetylglucosamine deacetylase as raw material, collects microorganism suspension through flocculation centrifugation or ceramic microfiltration membrane, and simultaneously removes residual nutrient components in the fermentation process; then the cell wall is broken by high pressure homogenization to release the cell content and the target enzyme, and finally the solution rich in N-acetylglucosamine deacetylase is permeated out by a ceramic microfiltration membrane or an ultrafiltration membrane, and meanwhile, the cell debris components are trapped and removed. The invention improves the extraction recovery rate of the enzyme, has less material consumption and less wastewater production in the extraction process, and realizes good economic benefit and environmental protection and safety effects.
Description
Technical Field
The invention relates to an extraction method of N-acetylglucosamine deacetylase, belonging to the technical field of bioengineering.
Background
The D-glucosamine has important physiological significance in human body and is applied to the fields of medicine, food, cosmetics and the like. Chitin is widely distributed in biological raw materials such as shrimp shells, crab shells and the like, but strong acid and strong alkali are needed in the production process, so that serious environmental pollution is easily caused. The recombinant microorganism for producing the N-acetylglucosamine can be constructed by utilizing a genetic engineering technology, and the large-scale production of the N-acetylglucosamine can be realized, but the recombinant microorganism still has a step away from D-glucosamine, and can be obtained after the N-acetylglucosamine deacetylase is catalyzed and hydrolyzed.
N-acetylglucosamine deacetylase (EC 3.5.1.33) is an important deacetylase with a molecular weight of 30-60 kDa and some enzyme complexes between 160-200 kDa. The catalyzed biological reaction formula is: N-acetyl-D-glucosamine +H 2 O=d-glucosamine + acetic acid. Roseman in 1957 first discovered that this enzyme catalyzes and participates in the metabolic process of D-glucosamine. Similar functional deacetylases in the same family also include chitose deacetylases (EC 3.5.1.41), diacetylebiose deacetylases (EC 3.5.1.105) and N-acetylglucosamine-6-phosphate deacetylases (EC 3.5.1.25). Microorganisms which have been found to exist in the N-acetylglucosamine deacetylase gene are mainly Escherichia coli, lactococcus lactis, thermococcus kodaka, horikoshi et al (J.biol. Chem.2004,279, 30021-30027). The high-efficiency expression and production can be realized by the aerobic fermentation of the recombinant escherichia coli, the recombinant bacillus subtilis and other microorganisms. Extraction of N-acetylglucosamineDeacetylase is one of the key steps in the production of D-glucosamine.
In 1993, U.S. patent (US 5219749) discloses a method for purifying chitosan deacetylase, which comprises enriching chitosan deacetylase in Mucor rouxii fermentation broth by ammonium sulfate precipitation, sequentially subjecting to three units of hydrophobic chromatography, strong anion exchange chromatography and strong cation exchange chromatography to obtain the final purified chitosan deacetylase.
Chitin deacetylase is extracted from Saccharomyces cerevisiae by Bouriotis et al, university of Greek, 2002, and the extraction steps mainly comprise: enriching crude enzyme by ammonium sulfate precipitation, adding into phenyl-Sepharose HiLoad chromatographic column, gradient eluting, loading onto Mono S cation exchange chromatographic column, and separating to obtain pure chitosan deacetylase with recovery rate of 24.5%.
The chitin deacetylase is obtained by aerobic fermentation of Scopulariella brachycarpa in university Cai Jun of Wuhan in 2006, the crude enzyme is enriched by an ammonium sulfate precipitation method, and the chitin deacetylase is finally obtained by sequentially passing through two chromatographic units of Sephadex G-25 and Sephadex G-100.
U.S. Pat. No. 5,172,72,1 and Chinese patent No. 4,241,42A disclose the genetic modification, fermentation process and enzymatic reaction conditions of N-acetylglucosamine-6-phosphate deacetylase (EC 3.5.1.25), N-acetylglucosamine deacetylase (EC 3.5.1.33), chitin deacetylase and acylase.
The enzyme extraction efficiency depends on factors such as the composition of impurities in raw materials, physical and chemical property differences of target substances and impurities, the relative concentration of target products and the like. The prior art has a relatively small number of publications about the extraction method of N-acetylglucosamine deacetylase, and has relatively more chitosan deacetylase with similar properties and functions, but the prior art is mainly realized on a laboratory scale, the basic steps comprise salt precipitation enrichment and multistage chromatographic purification, the separation steps are more, the extraction of related materials is expensive, the reagents used in the separation process are usually organic solvents, the green and safe production is not favored, and the recovery rate of the final product is low.
Disclosure of Invention
Aiming at the defects of high material consumption, low efficiency and the like in the prior art, the invention provides a novel extraction method of N-acetylglucosamine deacetylase, which takes recombinant microorganism fermentation liquor rich in N-acetylglucosamine deacetylase as a raw material, realizes solid-liquid separation through ceramic membrane microfiltration or flocculation precipitation, collects and obtains microorganism thalli and simultaneously removes extracellular impurities such as nutrients and the like remained in a culture medium; disrupting the cell wall by high pressure homogenization to release the cell contents comprising N-acetylglucosamine deacetylase; finally, a ceramic microfiltration membrane or a ceramic ultrafiltration membrane is adopted to intercept cell fragments and the like, so that N-acetylglucosamine deacetylase is dialyzed out, and a purer enzyme solution is obtained.
The first object of the present invention is to provide a method for separating and purifying N-acetylglucosamine deacetylase, which comprises the following steps:
(1) Taking microbial fermentation liquor containing N-acetylglucosamine deacetylase as a raw material, and adopting a ceramic microfiltration membrane concentration method or a flocculation centrifugation method to realize solid-liquid separation to obtain microbial concentrated suspension containing the N-acetylglucosamine deacetylase;
(2) Homogenizing the microorganism concentrated suspension at high pressure to break the cell wall of the microorganism and release N-acetylglucosamine deacetylase from the cell to obtain wall-broken suspension containing the N-acetylglucosamine deacetylase;
(3) Separating the wall-broken suspension by a ceramic microfiltration membrane or a ceramic ultrafiltration membrane, and obtaining the N-acetylglucosamine deacetylase extract with higher purity from the permeate.
In one embodiment, the microbial fermentation broth of step (1) is a fermentation broth of a microorganism after fermentation in a medium comprising a carbon source, a nitrogen source, and an inorganic salt for a period of time, comprising N-acetylglucosamine deacetylase, a cellular metabolite, and an incompletely consumed medium component; the microbial fermentation liquid can be recombinant escherichia coli fermentation liquid, recombinant bacillus subtilis fermentation liquid, lactococcus fermentation liquid and recombinant yeast fermentation liquid.
In one embodiment, the microfiltration membrane in the step (1) is a membrane component made of ceramic, the average pore diameter of the microfiltration membrane is 1-5 μm, the operating pressure is 0.3-0.8 MPa, and the temperature in the operating process is controlled to be 4-30 ℃.
In one embodiment, the flocculant used in step (1) is a food-safe flocculant, including, but not limited to, an organic flocculant or an inorganic flocculant;
in one embodiment, the flocculant is one or a combination of more than two of polyacrylamide, dimethylamine-epichlorohydrin copolymer, polymeric ferric chloride, ferric sulfate, ferrous sulfate;
in one embodiment, the flocculant is added in the form of (a) or (b):
(a) The flocculant of a single component was added at the following doses: the addition amount of the polyacrylamide is 0.01-0.2% of the dry weight of biomass in the raw materials; the addition amount of the dimethylamine-epichlorohydrin copolymer is 0.01 to 0.1 percent of the biomass dry weight; the addition amount of the polymeric ferric chloride is 0.1-2.0% of the biomass dry weight; the addition amount of ferric chloride is 0.1-1.0% of the biomass dry weight; the addition amount of the ferric sulfate is 0.1 to 1.5 percent of the dry weight of the biomass; the addition amount of the ferrous sulfate is 0.2-2.0% of the dry weight of the biomass;
(b) When two or more flocculants are added, each flocculant is added in an amount of 20 to 50% of the amount of the single component as described in (a).
In one embodiment, the centrifuge apparatus of step (1) may be a continuously operated centrifuge or a batch operated centrifuge; the continuous centrifugal equipment can be a disc type centrifugal machine, and the centrifugal factor is 8000-15000 g; the batch centrifuge may be an automatic discharge centrifuge with a centrifuge factor of 4000 to 8000g.
In one embodiment, the operation condition of the high-pressure homogenizing wall breaking device in the step (2) is that the high-pressure homogenizing pressure is 60-120MPa, the homogenizing times are 1-3 times, and the temperature is controlled to be 4-30 ℃.
In one embodiment, the ceramic membrane separation device in the step (3) may be a microfiltration membrane or an ultrafiltration membrane, wherein the aperture of the microfiltration membrane is 0.1-1.0 μm, the molecular weight cut-off of the ultrafiltration membrane is 300-1000kDa, the operating pressure is 0.3-0.8 MPa, and the temperature in the operating process is controlled at 4-30 ℃.
In one embodiment, the amount of water added during the ceramic membrane separation in step (3) is 1 to 5 times the volume of the raw material liquid. The N-acetylglucosamine deacetylase with higher purity is obtained in the permeate.
In one embodiment, after the step (3) is completed, the used production equipment and pipelines are sterilized or disinfected by using steam, so as to prevent microbial contamination in the production process, wherein the pressure of the steam is 0.2-0.6 MPa, and the sterilization time is 5-40 min.
The beneficial effects are that:
compared with the prior art, the invention has the following advantages:
(1) The invention realizes the high-efficiency purification of the N-acetylglucosamine deacetylase on the industrial production scale. The production process has the advantages of high recovery rate, low raw material consumption, environmental protection and safety, the highest recovery rate of the product can reach 88 percent, and the highest specific activity of the produced N-acetylglucosamine deacetylase can reach 22U/g protein;
(2) The method has the advantages that the adopted enzyme extraction condition is mild, organic solvents and high-concentration salting out are not used, cell wall breaking is realized through membrane separation and high-pressure homogenization, so that the enzyme is efficiently extracted, the amount of discharged pollutants is small, and the process is environment-friendly and safe;
(3) The invention sterilizes and disinfects separating equipment and pipelines by steam, adopts a pure physical method to realize and maintain the cleanness and sanitation of the production process, and effectively solves the problems of easy bacterial contamination and sanitation and safety in the enzyme extraction process.
(4) The process is suitable for the production of N-acetylglucosamine deacetylase under different scales, and has wide industrial application value.
Drawings
FIG. 1, N-extraction scheme for acetylglucosamine deacetylase.
Detailed Description
Technical terms:
microbial concentrate suspension: a flocculated sediment containing microbial cells in a fluid state.
Intercepting liquid: a solution containing a protein having a molecular weight greater than the molecular weight cut-off of the ultrafiltration membrane, which is cut-off by the ultrafiltration membrane.
Biomass (on a dry basis): refers to the weight of microbial cell dry matter per unit volume. Placing a certain volume of fermentation liquor into a centrifuge tube, centrifuging at 8000r/min for 10min to remove supernatant, washing precipitate with clear water for 2-3 times, and drying the obtained precipitate at 105 ℃ until the weight of dry matters of microorganisms is measured. The unit is g/L.
Recovery rate of enzyme activity: the recovery rate of the enzyme activity was calculated as follows,
the deacetylase activity unit (U) is defined as: the enzyme amount required for obtaining 1mmol of glucosamine by reacting at 30 ℃ in the presence of 50mmol/L sodium phosphate buffer (pH 7.5) with N-acetylglucosamine as a substrate is 1U enzyme activity unit per 1 min.
The purity of the deacetylase is expressed in units of enzyme activity contained in each gram of protein and is expressed as U/g protein.
The analysis method comprises the following steps:
the determination of deacetylase activity is described in J.Microbiol. Biotechnol.2018,28 (11), 1850-1858; the method for determining the protein is a colorimetric method of the bicinchoninic acid (Bicinchoninic Acid).
Example 1
According to the process route shown in the way A in figure 1, taking recombinant escherichia coli fermentation broth containing N-acetylglucosamine deacetylase as a raw material,
the recombinant escherichia coli fermentation broth containing N-acetylglucosamine deacetylase is a fermentation broth obtained by aerobic fermentation of recombinant escherichia coli in a culture medium containing glucose, peptone, yeast powder, ammonium sulfate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, calcium carbonate and other components, wherein the fermentation broth contains a culture medium component which is not completely metabolized and contains recombinant microorganism cells, a plurality of cell residual fragments, metabolic byproducts, such as acetic acid, propionic acid, amino acid, pigment and the like.
The operation steps are as follows:
(1) Taking 50m 3 Recombinant escherichia coli fermentation broth (the enzyme activity of the fermentation broth is 101U/L) containing N-acetylglucosamine deacetylase, the biomass dry weight of each L of fermentation broth is 60g, 20L of ferric sulfate solution with the concentration of 100g/L is pumped into a fermentation broth storage tank, 25L of food-grade polyacrylamide solution with the concentration of 10g/L is pumped, and flocculation is carried out for 20min after stirring and mixing are completed;
(2) Continuously pumping the flocculated solution containing microbial cells obtained in the step (1) into a disk centrifuge with the rotation speed of 12000rpm, and collecting 12m 3 The microorganism concentrated suspension enters a temporary storage tank; the clear liquid flows into the clear liquid tank and is discharged to the sewage treatment unit after being detected.
(3) Homogenizing the concentrated microorganism suspension collected in the step (2) under high pressure to break the cell wall of the microorganism and release N-acetylglucosamine deacetylase from the cell to obtain a wall-broken suspension containing the N-acetylglucosamine deacetylase; the high-pressure homogenizing pressure is 110MPa, and the feeding flow rate is 3m 3 And (3) controlling the temperature at 10 ℃, repeating the homogenizing operation for 1 time after the homogenization is completed, and carrying out 2 times of homogenization.
(4) 12m obtained in the step (3) 3 Separating the wall-broken suspension by a ceramic ultrafiltration membrane. The molecular weight cut-off of the ultrafiltration membrane is 500kDa, the operating pressure is 0.5MPa, and the temperature in the operating process is controlled between 4 and 30 ℃. When the volume of the trapped liquid is about 10m 3 At the time, deionized water was intermittently added for a total of 36m 3 times 3 Washing the trapped liquid, collecting the trapped liquid at 38m 3 And (3) penetrating the liquid to obtain an N-acetamido glucose deacetylase extracting solution with the purity of 18U/g protein, and discharging the trapped liquid to a sewage treatment unit.
(5) And sterilizing or disinfecting the used production equipment and pipelines by utilizing steam, wherein the pressure of the steam is 0.3MPa, and the sterilization time is 20min, so that microbial pollution is prevented in the production process. The extraction effect is shown in Table 1.
Example 2
According to the process route shown in the route B in FIG. 1, the recombinant escherichia coli fermentation broth containing N-acetylglucosamine deacetylase is taken as a raw material, and the operation steps are as follows:
(1) Taking 50m 3 The recombinant escherichia coli fermentation broth containing N-acetylglucosamine deacetylase (the enzyme activity of the fermentation broth is 111U/L) is pumped into a ceramic microfiltration membrane component for solid-liquid separation, a microorganism concentrated suspension is collected, the average pore diameter of the microfiltration membrane is 2 mu m, the operating pressure is 0.4MPa, and the temperature in the operating process is controlled at 4-30 ℃. When the volume of the trapped liquid is about 20m 3 At the time, deionized water was intermittently added for a total of 50m 3 times 3 Concentrating to 20m 3 And discharging the washed microorganism concentrated suspension and the permeate to a sewage treatment unit.
(2) Homogenizing the concentrated microorganism suspension collected in the step (1) under high pressure to break the cell wall of the microorganism and release N-acetylglucosamine deacetylase from the cell to obtain a wall-broken suspension containing the N-acetylglucosamine deacetylase; the high-pressure homogenizing pressure is 100MPa, and the feeding flow rate is 3m 3 And (3) controlling the temperature at 15 ℃, repeating the homogenizing operation for 1 time after the homogenization is completed, and carrying out 2 times of homogenization.
(3) 20m obtained in the step (2) 3 Separating the wall-broken suspension by a ceramic ultrafiltration membrane. The molecular weight cut-off of the ultrafiltration membrane is 500kDa, the operating pressure is 0.4MPa, and the temperature in the operating process is controlled between 4 and 30 ℃. When the volume of the trapped liquid is about 10m 3 At the time, deionized water was intermittently added for 3 times for a total of 30m 3 Washing the trapped liquid, collecting 40m 3 And (3) penetrating the liquid to obtain an N-acetylglucosamine deacetylase extracting solution with higher purity, and discharging the trapped liquid to a sewage treatment unit.
(4) And sterilizing or disinfecting the used production equipment and pipelines by utilizing steam, wherein the pressure of the steam is 0.3MPa, and the sterilization time is 20min, so that microbial pollution is prevented in the production process. The extraction effect is shown in Table 1.
Example 3
The embodiment is different from example 1 in that for 12m obtained in the step (3) 3 Separating the wall-broken suspension by a ceramic microfiltration membrane. The aperture of the microfiltration membrane is 0.2 μm,the operating pressure is 0.4MPa, and the temperature in the operating process is controlled between 4 and 10 ℃. When the volume of the trapped liquid is about 8m 3 At the time, deionized water was intermittently added for a total of 24m 3 times 3 Washing the trapped liquid, collecting the trapped liquid at 24m 3 And (3) penetrating the liquid to obtain an N-acetylglucosamine deacetylase extracting solution with higher purity, and discharging the trapped liquid to a sewage treatment unit. The extraction effect is shown in Table 1.
Example 4
The embodiment is different from example 2 in that for 20m obtained in step (2) 3 Separating the wall-broken suspension by a ceramic microfiltration membrane. The aperture of the microfiltration membrane is 0.1 mu m, the operating pressure is 0.5MPa, and the temperature in the operating process is controlled between 4 and 30 ℃. When the volume of the trapped liquid is about 10m 3 At the time, deionized water was intermittently added for 3 times for 25m 3 Washing the trapped liquid, collecting 25m 3 And (3) penetrating the liquid to obtain an N-acetylglucosamine deacetylase extracting solution with higher purity, and discharging the trapped liquid to a sewage treatment unit. The extraction effect is shown in Table 1.
TABLE 1 extraction effects of N-acetylglucosamine deacetylase Using different technologies
Example 5
Specific embodiments are the same as example 1 except that the flocculant ferric sulfate in example 1 is replaced with one or a combination of two or more of polyacrylamide, dimethylamine-epichlorohydrin copolymer, polymeric ferric chloride, and ferrous sulfate;
wherein the flocculant is added in the following modes (a) or (b):
(a) The flocculant of a single component was added at the following doses: the addition amount of the polyacrylamide is 0.01-0.2% of the dry weight of biomass in the raw materials; the addition amount of the dimethylamine-epichlorohydrin copolymer is 0.01 to 0.1 percent of the biomass dry weight; the addition amount of the polymeric ferric chloride is 0.1-2.0% of the biomass dry weight; the addition amount of ferric chloride is 0.1-1.0% of the biomass dry weight; the addition amount of the ferric sulfate is 0.1 to 1.5 percent of the dry weight of the biomass; the addition amount of the ferrous sulfate is 0.2-2.0% of the dry weight of the biomass;
(b) When two or more flocculants are added, each flocculant is added in an amount of 20 to 50% of the amount of the single component as described in (a).
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (2)
1. The method for separating and purifying the N-acetylglucosamine deacetylase is characterized by comprising the following steps of:
(1) Taking microbial fermentation liquor containing N-acetylglucosamine deacetylase as a raw material, and adopting a flocculation centrifugation method to realize solid-liquid separation to obtain microbial concentrated suspension containing the N-acetylglucosamine deacetylase;
(2) Homogenizing the microorganism concentrated suspension collected in the step (1) under high pressure, and collecting wall-broken suspension containing N-acetylglucosamine deacetylase;
(3) Separating the wall-broken suspension obtained in the step (2) through a ceramic microfiltration membrane or a ceramic ultrafiltration membrane, and collecting N-acetylglucosamine deacetylase extracting solution in the permeate; the aperture of the ceramic microfiltration membrane is 0.1-1.0 mu m, the molecular weight cut-off of the ceramic ultrafiltration membrane is 300-1000kDa, the operating pressure is 0.3-0.8 MPa, and the operating process control temperature is 4-30 ℃;
the microbial fermentation broth in the step (1) is recombinant escherichia coli fermentation broth;
the flocculating agent of the step (1) is polyacrylamide and ferric sulfate;
the centrifugation in the step (1) adopts a continuously operated centrifuge or a intermittently operated centrifuge; the continuous centrifugal equipment is a disc type centrifugal machine, and the centrifugal factor is 8000-15000 g; the intermittently operated centrifugal machine is an automatic discharging centrifugal machine, and the centrifugal factor is 4000-8000 g;
the high-pressure homogenization of step (2) is operated under the following conditions: the pressure is 60-120MPa, the temperature is 4-30 ℃, and the homogenization times are 1-3.
2. The method according to claim 1, wherein after the step (3) is completed, the used production equipment and pipelines are sterilized or disinfected by steam, the pressure of the steam is 0.2-0.6 mpa, and the sterilization time is 5-40 min.
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Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04349884A (en) * | 1991-05-27 | 1992-12-04 | Agency Of Ind Science & Technol | Production of deacetylase |
| WO1993007262A2 (en) * | 1991-10-09 | 1993-04-15 | Biotechnology Trading Company, Inc. | Purified chitin deacetylase |
| JPH07231787A (en) * | 1994-02-24 | 1995-09-05 | Toyobo Co Ltd | Purified chitin deacetylase |
| CN110484525A (en) * | 2019-09-24 | 2019-11-22 | 华东理工大学 | A kind of heat-resisting N-acetylglucosamine deacetylase and its encoding gene and application |
| CN110511917A (en) * | 2019-09-24 | 2019-11-29 | 华东理工大学 | A kind of deacetylase and its encoding gene and application |
| CN110951708A (en) * | 2018-09-27 | 2020-04-03 | 华东理工大学 | N-acetylglucosamine deacetylase, and coding gene and application thereof |
| CN111411132A (en) * | 2020-05-14 | 2020-07-14 | 山东惠仕莱生物科技有限公司 | Conversion and extraction method for producing levodopa by enzyme method |
| CN111518857A (en) * | 2020-06-11 | 2020-08-11 | 江苏海飞生物科技有限公司 | Enzyme method for producing glucosamine salt and purification method thereof |
| CN111647027A (en) * | 2020-06-11 | 2020-09-11 | 江苏海飞生物科技有限公司 | Method for separating and purifying N-acetylglucosamine |
-
2021
- 2021-07-30 CN CN202110873349.6A patent/CN113373134B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH04349884A (en) * | 1991-05-27 | 1992-12-04 | Agency Of Ind Science & Technol | Production of deacetylase |
| WO1993007262A2 (en) * | 1991-10-09 | 1993-04-15 | Biotechnology Trading Company, Inc. | Purified chitin deacetylase |
| JPH07231787A (en) * | 1994-02-24 | 1995-09-05 | Toyobo Co Ltd | Purified chitin deacetylase |
| CN110951708A (en) * | 2018-09-27 | 2020-04-03 | 华东理工大学 | N-acetylglucosamine deacetylase, and coding gene and application thereof |
| CN110484525A (en) * | 2019-09-24 | 2019-11-22 | 华东理工大学 | A kind of heat-resisting N-acetylglucosamine deacetylase and its encoding gene and application |
| CN110511917A (en) * | 2019-09-24 | 2019-11-29 | 华东理工大学 | A kind of deacetylase and its encoding gene and application |
| CN111411132A (en) * | 2020-05-14 | 2020-07-14 | 山东惠仕莱生物科技有限公司 | Conversion and extraction method for producing levodopa by enzyme method |
| CN111518857A (en) * | 2020-06-11 | 2020-08-11 | 江苏海飞生物科技有限公司 | Enzyme method for producing glucosamine salt and purification method thereof |
| CN111647027A (en) * | 2020-06-11 | 2020-09-11 | 江苏海飞生物科技有限公司 | Method for separating and purifying N-acetylglucosamine |
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