WO2021248697A1 - 酶法生产氨基葡萄糖盐及其提纯方法 - Google Patents

酶法生产氨基葡萄糖盐及其提纯方法 Download PDF

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WO2021248697A1
WO2021248697A1 PCT/CN2020/111623 CN2020111623W WO2021248697A1 WO 2021248697 A1 WO2021248697 A1 WO 2021248697A1 CN 2020111623 W CN2020111623 W CN 2020111623W WO 2021248697 A1 WO2021248697 A1 WO 2021248697A1
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glucosamine
membrane
column
solution
concentration
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French (fr)
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丁春华
章文劼
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江苏海飞生物科技有限公司
拜启私人有限公司
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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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • C07H1/08—Separation; Purification from natural products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
    • C07H5/06—Aminosugars
    • 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
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/001—Amines; Imines
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/10—Process efficiency

Definitions

  • the invention relates to an enzymatic production of glucosamine salt and a purification method thereof, and belongs to the technical field of bioengineering.
  • Glucosamine is an important aminohexose. It is formed by the substitution of a hydroxyl group of glucose by an amino group. It is easily soluble in water and hydrophilic solvents. Exist widely in nature, the chemical name is: 2-amino-2-deoxy-D-glucose, usually in the form of N-acetyl derivatives (such as chitin) or N-sulfate and N-acetyl-3-O-lactic acid
  • the ether (teichoic acid) form exists in microbial, animal-derived polysaccharides and conjugated polysaccharides.
  • Glucosamine hydrochloride molecular weight 215.5Da, white crystals, odorless, slightly sweet, easily soluble in water, slightly soluble in methanol, insoluble in organic solvents such as ethanol.
  • Glucosamine molecules are not very stable and are prone to oxidation or degradation. It can be prepared as glucosamine salts, such as glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, and glucosamine pyruvate, and its stability can be significantly improved.
  • Glucosamine has important physiological functions to the human body, participates in liver and kidney detoxification, plays an anti-inflammatory, liver and kidney function, has a good effect on the treatment of rheumatoid arthritis and gastric ulcer, is the main raw material for the synthesis of antibiotics and anticancer drugs, and can also Used in food, cosmetics and feed additives.
  • GlcNAc production methods there are three main types of GlcNAc production methods: chemical method, enzymatic method and microbial method.
  • Natural raw materials such as shrimp and crab shells and fungal cell walls are rich in chitin, and glucosamine monomer can be obtained only after acid hydrolysis or enzymatic hydrolysis.
  • the enzymatic method mainly hydrolyzes chitin specifically by chitinase, and the enzymes involved mainly include endochitinase, exochitinase, ⁇ -N-acetylhexosaminidase and deacetylase. After enzymatic hydrolysis of chitin, glucosamine monomer can be obtained.
  • the use of recombinant microorganisms can directly biosynthesize GlcNAc with glucose as a substrate, and the product concentration can even exceed 100g/L, which has laid a good foundation for the large-scale production of GlcNAc.
  • Base. The use of microbial fermentation to produce glucosamine has the advantages of high conversion rate, high product concentration and short production cycle.
  • the main raw materials for extracting glucosamine are shrimp and crab shells and microbial fermentation broth. Whether it is fermentation broth or enzymatic hydrolysate, the main product obtained after the reaction is accompanied by the formation of various by-products and unreacted residues. . Therefore, it is necessary to develop corresponding glucosamine extraction processes for different raw materials.
  • the current extraction methods in the actual production process often have disadvantages such as complex process routes, low separation efficiency, high energy consumption, and large environmental pollution.
  • the main methods of deacetylation are acid hydrolysis and enzymatic hydrolysis.
  • Acid hydrolysis consumes a large amount of inorganic acid, and a large amount of lye needs to be added in the subsequent extraction process to neutralize the previously added inorganic acid solution, so that a large amount of salt will be produced during the extraction process.
  • the extraction process consumes a large amount of acid and alkali, and produces a large amount of difficult-to-treat high-salt wastewater.
  • the enzymatic deacetylation method does not need to use a large amount of acid and alkali solutions, so it is more and more favored.
  • ZL2016112278411 discloses a method for separating D-glucosamine hydrochloride by deacetylation coupling adsorption.
  • the reaction temperature involved exceeds 90°C, pigments are easily produced during the treatment process, and the ion exchange resin is easily broken and lost.
  • ZL2013106719979 discloses a purification method of glucosamine hydrochloride mother liquor.
  • Glucosamine hydrochloride mother liquor is used as a raw material, and glucosamine is adsorbed on a positive column through an acidic cation exchange column. After eluting the positive column with hydrochloric acid solution, the obtained analytical solution passes through the anion exchange column to remove acetic acid, chloride ion and other anions, thereby obtaining glucosamine instead of glucosamine hydrochloride. Since glucosamine cannot be stored stably for a long time, this method has limited industrial application value.
  • the present invention provides a new method for the production, separation and purification of glucosamine salt, which uses a fermentation broth rich in acetylglucosamine or an enzymatic hydrolysate of chitin as raw materials , On the basis of obtaining glucosamine salt by cation exchange, using anion exchange to obtain acetate, and recovering unreacted acetyl glucosamine. While increasing the yield of the target product, the by-products can be used as resources while reducing The consumption of raw and auxiliary materials, waste water, and solid waste discharge have reached the goals of energy saving, consumption reduction, and environmental protection and safety.
  • the method can also obtain the corresponding glucosamine salt by simply adjusting the type of acidic eluent of the cation exchange resin, and extract high-purity crystals of various glucosamine salts on an industrial production scale.
  • the first object of the present invention is to provide a method for the production, separation and purification of glucosamine salt, the method comprising the following steps:
  • a clear solution containing glucosamine as a raw material, optionally, when the solution containing glucosamine is a turbid solution, filter with an ultrafiltration membrane, and use the filtered clear solution containing glucosamine as a raw material;
  • the molecular weight cut-off of the ultrafiltration membrane is 5-200kDa;
  • step (1) Adsorb the glucosamine-containing solution of step (1) with a cation exchange resin, so that the cationic resin adsorbs glucosamine;
  • Adsorption with anion exchange resin step (2) Pass the column liquid under the positive column of the cation exchange resin to make the anion resin adsorb acetate ions; return the column liquid under the negative column containing acetyl glucosamine that has passed through the anion exchange resin to prepare glucosamine ;
  • the anion adsorption resin is eluted with an alkaline eluent, and the resulting analytical solution is rich in sodium acetate, which can be used in the nitrogen and phosphorus removal process of sewage treatment plants, and can also be used as a raw material for chemical reactions and other suitable purposes.
  • the glucosamine-containing solution described in step (1) is the reaction product of N-acetyl glucosamine after biological or chemical deacetylation, or it may be a solution containing glucosamine from other sources.
  • the ultrafiltration membrane described in step (1) may be a membrane module made of ceramic material, or a membrane module made of organic material.
  • the glucosamine-containing solution described in step (1) is prepared from a solution containing N-acetyl glucosamine as a raw material, and a deacetylase extract or a deacetylase preparation as a catalyst through a catalytic reaction. have to.
  • the reuse in step (3) for preparing glucosamine is used as an enzymatic hydrolysis raw material for preparing glucosamine for the deacetylation reaction catalyzed by deacetylase.
  • the second object of the present invention is to provide a preparation method of glucosamine salt, which method firstly enzymatically removes acetyl groups from a solution containing acetyl glucosamine, and then separates and purifies it according to the separation and purification method.
  • the acetylglucosamine-containing solution can be obtained by microbial fermentation, can also be obtained by enzymatic hydrolysis of biological raw materials containing chitin, or can be obtained by chemical hydrolysis of chitin-containing raw materials .
  • the enzymatic hydrolysis is based on an acetylglucosamine solution with a concentration of 40-150g/L as a raw material, and deacetylase is added at a ratio of 10-40U/g acetylglucosamine; the pH range of the enzymatic hydrolysis reaction The reaction temperature is 4-8, the reaction temperature is 25-55°C, and the reaction is stirred for 10-40 minutes; the acetyl glucosamine solution is a raw material liquid containing acetyl glucosamine obtained by microbial fermentation or chitin hydrolysis.
  • the deacetylase may be derived from microorganisms and obtained through microbial fermentation, or extracted from other organisms; the microorganisms may be microorganisms screened in nature, or they may be obtained through genetic Engineered recombinant microorganisms.
  • the enzymatic hydrolysis is performed by a deacetylase enzyme reaction to specifically remove the acetyl group in the N-acetylglucosamine molecule, and the reaction obtains an enzymatic hydrolysate whose main components are glucosamine and acetic acid.
  • the method includes the following steps:
  • step (2) The enzymatic hydrolysate after the reaction in step (1) can directly enter step (3), or it can be filtered by ultrafiltration membrane to obtain the ultrafiltration membrane dialysate and membrane concentrate containing glucosamine respectively, and the membrane concentrate
  • the enzyme solution is reused in step (1) to participate in the next batch of enzyme reaction process; the molecular weight cut-off of the ultrafiltration membrane is 5-200kDa;
  • step (3) Adsorb the glucosamine membrane dialysate obtained in step (2) with a cation exchange resin, and the cation exchange resin is continuously eluted with an acidic eluent to obtain an analytical solution containing glucosamine salt;
  • the equipment used in step (3) for cation exchange chromatography can be a fixed bed, a continuous ion exchange bed, or an ion exchange simulated moving bed;
  • the acidic eluent can be Hydrochloric acid can also be sulfuric acid, phosphoric acid, pyruvic acid, or citric acid;
  • the corresponding glucosamine salts obtained by elution are glucosamine hydrochloride, glucosamine sulfate, and glucosamine phosphate.
  • Glucosamine pyruvate and Glucosamine citrate the concentration of the acidic eluent is 0.30-3.0 mol/L.
  • the adsorption and elution temperature of step (3) cation exchange is 20-70°C; the adsorption temperature and elution temperature of step (4) anion exchange is 20-65°C; the above-mentioned two-stage ion exchange layer
  • the feed flow rate of the analysis is 2.0-10.0BV/h; the flow rate of the eluent is 1.0-8.0BV/h.
  • the equipment used in step (4) for anion exchange chromatography can be a fixed bed, a continuous bed of ion exchange, or a simulated moving bed of ion exchange;
  • the alkaline eluent can be It can be a NaOH solution or a KOH solution.
  • the alkaline eluent has a concentration of 0.30-3.0mol/L;
  • the analytical solution after eluting the anion exchange column can be recovered into sodium acetate or potassium acetate, sodium acetate or Potassium acetate can be collected through pipelines and transported to the sewage treatment plant to be used as a supplementary carbon source for nitrogen and phosphorus removal in the sewage treatment process, and it can also be used as a raw material for other chemical reaction processes.
  • the nanofiltration membrane in step (5) is a ceramic membrane, the pore size of the nanofiltration membrane is between 0.5-2nm, and the operating pressure is 2-5atm;
  • the reverse osmosis membrane is an organic roll type Membrane or ceramic membrane, the molecular weight cut-off of the reverse osmosis membrane is 50-100Da, and the operating pressure is 4-10atm.
  • step (5) concentration, crystallization, and drying are sequentially performed.
  • the concentration is evaporative concentration; the evaporative concentration may be single-effect evaporation, double-effect evaporation, or multiple-effect evaporation.
  • the crystallization temperature is 5-40°C.
  • the crystallization mother liquor is also decolorized; the decolorization method is activated carbon adsorption decolorization; the decolorized crystallization mother liquor is recycled for the concentration process; in the decolorization method, the amount of activated carbon is the amount of the raw material liquid 0.01-2.0% (w/v).
  • the drying is vacuum drying or flash drying; the inlet air temperature of the flash drying is 110-290°C, and the outlet air temperature is 70-90°C; the temperature of the vacuum low-temperature drying is 40-80°C, the vacuum degree is 70-95kPa.
  • the multiple-effect evaporation concentration is three-effect evaporation, and the temperature is 80°C, 70°C, and 60°C, respectively.
  • the vacuum degree of the final effect evaporator is 80-98kPa.
  • the present invention has the following advantages:
  • the present invention realizes the enzymatic production and high-efficiency purification of a variety of different glucosamine salts on an industrial production scale.
  • the production process has the advantages of high conversion rate, high recovery rate, low raw material consumption, and environmental protection and safety.
  • the product recovery rate exceeds 95%, and the purity of the produced glucosamine salt can exceed 99.5%;
  • the enzyme reaction conditions used in the present invention are mild, the reaction rate is efficient, and the recycling process of deacetylase is designed. On the one hand, the enzyme and the product can be effectively separated through ultrafiltration membrane filtration, and on the other hand, the enzyme can be recycled. , Which reduces the cost of using enzymes;
  • the present invention forms a cyclic process through the combination of two-stage ion exchange and reverse osmosis membrane/nanofiltration membrane.
  • the by-product acetate and the incompletely reacted substrate acetylglucosamine can be effectively reused and effectively improved
  • the conversion rate of the substrate and the extraction recovery rate of the product are effectively reduced, and the consumption of resin and eluent in the ion exchange process is effectively reduced, and the triple benefits of low consumption, energy saving and environmental protection are achieved.
  • the present invention uses continuous moving bed and simulated moving bed to carry out the ion exchange process, which can improve the continuity and automation level of process operation while improving the separation efficiency of ion exchange;
  • the ion exchange reaction conditions of the present invention are mild. Compared with the commonly used fixed bed, the resin loss can be reduced by about 60%, and the acid and lye consumption required for resin regeneration can be reduced by about 50%, and at the same time, it can be greatly reduced The amount of waste water produced.
  • the process of the present invention is suitable for the production of various glucosamine salts.
  • the acidic eluent of the cation exchange resin can be changed to obtain the glucosamine salt of the corresponding acid, which has a wide range of industrial application value.
  • Glucosamine salt refers to the glucosamine salt product obtained after eluting the cation exchange column with different acids, including but not limited to glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate and glucosamine Any kind of citrate.
  • Membrane dialysate the liquid obtained through the membrane material in the membrane filtration process.
  • Membrane concentrate The liquid that cannot penetrate the membrane material and is retained during the membrane filtration process.
  • Analysis solution The liquid obtained after the eluent (acid or lye) flows through saturated ion exchange resin during the ion exchange process.
  • Bottom column liquid During the ion exchange process, the raw material liquid is not absorbed by the ion exchange resin but flows out directly and is washed out by deionized water.
  • glucosamine salt Purification and recovery rate of glucosamine salt: As the enzymatic production of glucosamine salt and its purification process involves units such as enzyme reaction and ion exchange, the molecular structure of the reactant will change. The recovery rates are all calculated on the basis of the quality of glucosamine.
  • the quantification of glucosamine salt and acetyl glucosamine was performed by HPLC analysis method.
  • the liquid chromatograph is Agilent 1260 series, the chromatographic column is Thermo ODS-2 Hypersil C18 column (250mm ⁇ 4.0mm), the glucosamine salt sample to be tested is filtered through 0.22 ⁇ m microfiltration membrane and then injected with 10 ⁇ l injection volume In the chromatographic column.
  • the analysis of acetylglucosamine and acetic acid used the column HPX-87H column (Bio-Rad, USA).
  • the detector adopts a differential refractometer detector.
  • the mobile phase is 5mM H 2 SO 4 , the flow rate is 0.6ml/min, and the detection temperature is 40°C.
  • the deacetylase preparation can be a commercial enzyme that can hydrolyze and remove the acetyl group in N-acetylglucosamine; this step specifically removes N-acetyl
  • the acetyl group in the glucosamine molecule reacts to obtain an enzymatic hydrolysate whose main components are glucosamine and acetic acid, and the pH of the enzyme reaction is preferably 7.0-8.0;
  • the molecular weight cut-off of the ultrafiltration membrane is 5-200kDa, preferably 5-30kDa; the membrane concentrate is dialyzed with water, and the glucosamine-containing products are collected separately Ultrafiltration membrane dialysate and membrane concentrate containing deacetylase, the recovery rate of deacetylase after membrane dialysis is 80-90%;
  • step (3) Return the enzyme solution after membrane concentration in step (2) to the deacetylase solution storage tank to participate in the next batch of enzyme reaction process;
  • the ultrafiltration membrane dialysate containing glucosamine obtained in step (2) is continuously pumped into a simulated moving bed filled with acidic resin (for example, cation exchange resin with sulfonic acid group), and the feed flow rate is 2.0- 10.0BV/h, so that the glucosamine in the dialysate is adsorbed on the positive column;
  • acidic resin for example, cation exchange resin with sulfonic acid group
  • acidic eluents such as hydrochloric acid, sulfuric acid, phosphoric acid, pyruvic acid, and citric acid with a concentration of 0.30-3.0mol/L to continuously elute the positive column.
  • the elution temperature is 20-70°C, preferably 25°C, and the corresponding amino group can be obtained.
  • Analysis solution of glucose salt is 20-70°C, preferably 25°C, and the corresponding amino group can be obtained.
  • the deacetylase preparation can be a commercial enzyme that can hydrolyze and remove the acetyl group in N-acetylglucosamine; this step specifically removes N-acetyl
  • the acetyl group in the glucosamine molecule reacts to obtain an enzymatic hydrolysate whose main components are glucosamine and acetic acid, and the pH of the enzyme reaction is preferably 7.0-8.0;
  • the molecular weight cut-off of the ultrafiltration membrane is 5-200kDa, preferably 5-30kDa; the membrane concentrate is dialyzed with water, and the glucosamine-containing products are collected separately Ultrafiltration membrane dialysate and membrane concentrate containing deacetylase, the recovery rate of deacetylase after membrane dialysis is 80-90%;
  • step (3) Return the enzyme solution after membrane concentration in step (2) to the deacetylase solution storage tank to participate in the next batch of enzyme reaction process;
  • the ultrafiltration membrane dialysate containing glucosamine obtained in step (2) is continuously pumped into a simulated moving bed filled with acidic resin (for example, cation exchange resin with sulfonic acid group), and the feed flow rate is 2.0- 10.0BV/h, so that the glucosamine in the dialysate is adsorbed on the positive column;
  • acidic resin for example, cation exchange resin with sulfonic acid group
  • acidic eluents such as hydrochloric acid, sulfuric acid, phosphoric acid, pyruvic acid, and citric acid with a concentration of 0.30-3.0mol/L to continuously elute the positive column.
  • the elution temperature is 20-70°C, preferably 25°C, and the corresponding amino group can be obtained.
  • the analytical solution of glucose salt is used in the subsequent concentration, crystallization and drying process;
  • step (3) The column liquid under the positive column of step (3) is further transported to the negative column filled with basic anion exchange resin (for example, anion exchange resin with quaternary ammonium salt), and the feed flow rate is 2.0-10.0BV/ h; Control the adsorption temperature and elution temperature to 20-65°C, preferably 25°C; make acetic acid adsorb on the negative column;
  • basic anion exchange resin for example, anion exchange resin with quaternary ammonium salt
  • the analyte rich in acetate can be separated, and the analyte can be transported to sewage
  • the treatment workshop is used as a supplementary carbon source for the denitrification and phosphorus removal process, and can also be used for chemical raw materials;
  • the nanofiltration membrane is a ceramic membrane, the pore size of the nanofiltration membrane is 0.5-2nm, and the operating pressure is 2-5atm;
  • the reverse osmosis membrane is an organic roll membrane or a ceramic membrane, the molecular weight cut-off of the reverse osmosis membrane is 50-100 Da, and the operating pressure is 4-10 atm;
  • the final concentration of N-acetylglucosamine after membrane concentration can reach 10-15% (w/v).
  • the membrane concentrate is recycled to the N-acetylglucosamine storage tank for use in the next batch of step (1). Enzyme reaction process;
  • step (3) Pump the analytical solution of step (3) positive column to the multi-effect evaporator for evaporation and concentration.
  • the multi-effect evaporation and concentration is three-effect evaporation, and the temperature is controlled by gradient.
  • the temperature of the three-effect evaporator is set to 80°C, 70°C and 60°C respectively, and the vacuum degree of the three-effect evaporator is 80-98kPa;
  • step (6) multi-effect evaporator into the crystallizer, control the crystallization temperature at 5-40°C by controlling the jacket temperature of the crystallizer, and send the crystal suspension produced by the crystallizer into Solid-liquid separation equipment separates to obtain crystallization mother liquor and glucosamine salt crystal sludge;
  • the separation equipment can be a centrifuge with a continuous centrifugal function or a horizontal scraper discharge centrifuge;
  • step (8) Use activated carbon to decolorize the crystallization mother liquor obtained in step (7), and return it to the multi-effect evaporation equipment of step (6) after decolorization; transport the glucosamine salt crystalline sludge to the drying equipment to obtain dried Glucosamine salt crystals.
  • the recovery rate of the concentration, crystallization and drying unit can reach 98%.
  • step (1) Pump the enzyme hydrolysate after the reaction in step (1) into the ultrafiltration membrane equipment.
  • the molecular weight cut-off of the ultrafiltration membrane is 5000 Da.
  • step (3) Continuously pump the glucosamine dialysate obtained in step (2) into a simulated moving bed filled with 001 ⁇ 7 strong acidic styrene resin.
  • the feed flow rate is 4.0 BV/h, and the feed and wash
  • the removal temperature is 25°C; the glucosamine in the dialysate is adsorbed on the positive column, and the positive column is washed with deionized water to obtain the column liquid under the positive column containing neutral sugar and acetic acid;
  • a 2mol/L hydrochloric acid solution was used to continuously elute the positive column, and the elution flow rate was 3.0BV/h, so as to obtain 91.7m 3 of analytical solution with a concentration of 252kg/m 3 glucosamine hydrochloride;
  • step (3) The lower column liquid of step (3) the positive column is further transported to the negative column filled with anion exchange resin in the simulated moving bed at a flow rate of 4.0 BV/h, and the negative column is washed with deionized water to obtain N-acetamido
  • the first batch of the above reaction and purification process can obtain 23.1 tons of glucosamine hydrochloride, and the recovery rate reaches 66.2%.
  • the eluates of glucosamine hydrochloride obtained in step (3) of the first batch and the second batch are combined and then concentrated, crystallized and dried, as follows:
  • the crystallization mother liquor is sent to the activated carbon decolorization column at a flow rate of 0.5m 3 /h for decolorization, and then returned to the storage tank in front of the three-effect evaporation device after decolorization; the glucosamine hydrochloride crystal sludge separated by the decanter centrifuge passes through
  • the screw conveyor is sent to the flash dryer, the inlet air temperature of the flash drying is 150°C, and the outlet air temperature is 80°C to obtain glucosamine hydrochloride crystals.
  • the total recovery rate of the concentration, crystallization and drying units in steps (6)-(8) can reach 98%.
  • the difference is that on the basis of Example 3, the nanofiltration membrane concentration equipment is replaced by a reverse osmosis ceramic membrane equipment, that is, the column liquid under the cathode obtained in step (4) is collected into the reverse osmosis membrane equipment.
  • Permeable ceramic membrane equipment the reverse osmosis membrane has a pore size of 1 nm and an operating pressure of 0.5-1.0 MPa.
  • the concentrated liquid of the reverse osmosis ceramic membrane equipment is returned to the enzyme reaction tank to participate in the next batch of enzyme reaction process.
  • step (3) uses sulfuric acid solution instead of hydrochloric acid solution, that is, in step (3), 1mol/L sulfuric acid solution is used to adsorb on the positive column
  • step (3) 1mol/L sulfuric acid solution is used to adsorb on the positive column
  • the glucosamine on the surface is eluted, and the product obtained is glucosamine sulfate.
  • step (3) uses phosphoric acid solution instead of hydrochloric acid solution, that is, step (3) uses 1mol/L phosphoric acid solution to adsorb on the positive column
  • step (3) uses 1mol/L phosphoric acid solution to adsorb on the positive column
  • the glucosamine on the elution is eluted, and the product obtained is glucosamine phosphate.
  • step (3) uses citric acid solution instead of hydrochloric acid solution, that is, step (3) uses 1mol/L citric acid solution to adsorb on The glucosamine on the positive column is eluted, and the product obtained is glucosamine citrate.
  • step (3) uses pyruvate solution instead of hydrochloric acid solution, that is, step (3) uses 2mol/L pyruvic acid solution to adsorb on The glucosamine on the positive column is eluted, and the product obtained is glucosamine pyruvate.
  • the difference is that on the basis of Example 3, the simulated moving bed of step (3) is replaced with a continuous moving bed equipment, in which the filler of the male column and the female column are unchanged, and the filler of the male column is 001 ⁇ 7 strongly acidic styrene-based cationic resin, and 201 ⁇ 7 strongly basic styrene-based anionic resin as the filler.
  • the filler of the male column is 001 ⁇ 7 strongly acidic styrene-based cationic resin, and 201 ⁇ 7 strongly basic styrene-based anionic resin as the filler.
  • 16kg of cation exchange resin and 14kg of anion exchange resin are consumed, 600kg of concentrated hydrochloric acid and 650L 30% sodium hydroxide solution are consumed, and 15 tons of waste water are produced.
  • the difference is that on the basis of Example 3, the simulated moving bed of step (3) is replaced with a fixed bed ion exchange device, and the positive column packing is 201 ⁇ 7 strong basic styrene-based anion exchange
  • the resin and the negative column packing are 001 ⁇ 7 strongly acidic styrene cation exchange resin.
  • 30kg of cation exchange resin and 28kg of anion exchange resin are consumed, 1300kg of concentrated hydrochloric acid and 1400L 30% sodium hydroxide solution are consumed, resulting in 50 tons of wastewater.
  • the difference is that on the basis of Example 5, the three-effect evaporation concentration and crystallization are omitted, and the analytical solution rich in glucosamine sulfate eluted from the simulated moving bed positive column is directly transported to the spray Drying equipment, the feed flow rate is 5m 3 /h, and the inlet air temperature of spray drying is 150°C. After drying, 41.5 tons of glucosamine sulfate powder is obtained, and the purity of the product reaches 99%.
  • the difference is that on the basis of Example 3, the enzyme reaction solution obtained in step (1) is continuously pumped into the positive column described in step (3), and the feed flow rate is 3.0 BV/ h, the feed and elution temperature is 30°C, the subsequent steps are the same as in Example 2, and the product obtained is glucosamine hydrochloride.
  • glucosamine hydrochloride Collected 42 m 3 analysis solution, the concentration of glucosamine hydrochloride was 103 kg/m 3 , and the yield in this step was 88.6%. 0.2% activated carbon was added to the analytical solution for decolorization, and then pumped into the three-effect evaporation concentrator, and then crystallized, centrifuged and dried to obtain 4410 kg of glucosamine hydrochloride, with a total recovery rate of 80.2%. Because the reaction temperature of acetylglucosamine and cationic resin is relatively high, the reaction time is long, pigment is easy to produce during the reaction, and the loss of ion exchange resin increases. The production of glucosamine hydrochloride consumes 1100kg of concentrated hydrochloric acid and 120kg of cationic resin. 30m 3 wastewater with high mineral acid content and high COD, acetic acid cannot be recovered in the production process.
  • the inventor also tried to adjust the enzymatic hydrolysis, separation and purification process parameters.
  • the product recovery rate of the ion exchange unit can reach 99%, and the filler loss is controlled within the range of 20 kg/ton.
  • the amount of acid solution is controlled within the range of 600kg/ton of product, and the waste water production is less than 10m 3 /ton of product.
  • the difference is that the raw material is replaced by glucosamine hydrochloride mother liquor with an enzyme reaction solution containing glucosamine, and the operation step of transporting the analytical solution of the cation exchange column to the anion exchange column is omitted. Put the collected deacetylase reaction solution and the membrane dialysate after ultrafiltration membrane dialysis totaling 50m 3 (containing glucosamine and acetic acid) in a storage tank.
  • the solution with a concentration of glucosamine of 89kg/m 3 is pumped into the In the strong acid cation resin exchange column, keep the temperature at 32°C to allow glucosamine to be adsorbed on the cation resin, pour in pure water to clean the cation resin, and collect the lower column liquid containing acetic acid totaling 60m 3 , and then pour in 0.3 mol after the cleaning is complete /L hydrochloric acid solution to elute the cation exchange column, the flow rate of the elution process is 1.5BV/h. 31m 3 of glucosamine hydrochloride solution containing 126kg/m 3 was collected, and the yield was 87.8%.
  • Parsing by heating the liquid was warmed to 60 °C, 1% powdered active carbon, and filtered to give a filtrate containing 3 30.5m 125kg / m 3 glucosamine hydrochloride solution, and then pumped into a three-effect evaporator concentrator, After crystallization, centrifugation and drying, 3585kg of glucosamine hydrochloride crystals were obtained, the crystals were slightly yellow, and the total yield was 80.6%.
  • the method first decolorizes and then concentrates. The activated carbon consumption is large.
  • the production of glucosamine hydrochloride consumes 900kg of concentrated hydrochloric acid and 20kg of cationic resin per ton of glucosamine hydrochloride, and produces 30m 3 of wastewater with high COD content.

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Abstract

一种酶法生产氨基葡萄糖盐及其提纯方法，以N-乙酰氨基葡萄糖为原料，经过脱乙酰酶水解得到氨基葡萄糖和乙酸，再通过酸溶液洗脱阳离子交换柱分离得到氨基葡萄糖盐，同时通过阴离子交换回收得到副产品乙酸钠。所得氨基葡萄糖盐经过浓缩、结晶、脱色、干燥得到高纯度的氨基葡萄糖盐晶体。结合了酶的循环利用工艺、残余底物循环回收工艺和乙酸回收工艺，在提升N-乙酰氨基葡萄糖的转化率和氨基葡萄糖盐产品总得率的同时，使酶得到循环利用，使残余底物循环得到充分转化，乙酸得到资源化利用，并通过常温下的操作条件，使树脂损耗率低，盐酸废液产生量极少，实现了节能降耗的经济效益和环保安全的效果。

Description

酶法生产氨基葡萄糖盐及其提纯方法 技术领域
本发明涉及酶法生产氨基葡萄糖盐及其提纯方法，属于生物工程技术领域。
背景技术
氨基葡萄糖(GlcNAc)是一种重要的氨基己糖，由葡萄糖的一个羟基被氨基取代形成，易溶于水及亲水性溶剂。广泛存在于自然界，化学名称为：2-氨基-2-脱氧-D-葡萄糖，通常以N-乙酰基衍生物(如甲壳素)或以N-硫酸酯和N-乙酰-3-O-乳酸醚(胞壁酸)形式存在于微生物、动物来源的多糖和结合多糖中。氨基葡萄糖盐酸盐，分子量215.5Da，白色结晶，无气味，略有甜味，易溶于水，微溶于甲醇，不溶于乙醇等有机溶剂。氨基葡萄糖分子不是很稳定，容易发生氧化或降解。可以制备为氨基葡萄糖盐，如氨基葡萄糖盐酸盐、氨基葡萄糖硫酸盐、氨基葡萄糖磷酸盐和氨基葡萄糖丙酮酸盐等，其稳定性可得到显著提升。氨基葡萄糖对人体具有重要的生理功能，参与肝肾解毒，发挥抗炎护肝补肾作用，对治疗风湿性关节炎症和胃溃疡有良好的疗效，是合成抗生素和抗癌药物的主要原料，还可应用于食品，化妆品和饲料添加剂中。
目前GlcNAc的生产方法主要有三大类：化学法、酶法和微生物法。天然原料如虾蟹壳和真菌细胞壁含有较丰富的几丁质，通过酸水解或酶水解后才能得到氨基葡萄糖单体。酶法主要是通过几丁质酶专一性水解几丁质，涉及的酶主要包括内切几丁质酶、外切几丁质酶、β-N-乙酰己糖胺酶和脱乙酰酶。几丁质经酶水解后可以得到氨基葡萄糖单体。
随着基因工程、代谢工程和合成生物学的快速发展，利用重组微生物可以直接以葡萄糖为底物生物合成得到GlcNAc，产物浓度甚至可以超过100g/L，这为GlcNAc的大规模生产奠定了良好的基础。利用微生物发酵法生产氨基葡萄糖具有转化率高、产物浓度高和生产周期短等优势。然而，目前提取氨基葡萄糖的原料主要为虾蟹壳和微生物发酵液，无论是发酵液还是酶水解产物，反应后得到主产物的同时均伴随有各种副产物的生成和未反应完全的残余物。因此，针对不同的原料，需要开发相应的氨基葡萄糖提取工艺。但目前实际生产过程中的提取方法往往存在工艺路线复杂、分离效率低、能耗高、环境污染大等缺点。
以富含乙酰氨基葡萄糖的液体或几丁质水解液为原料时，脱去分子中的乙酰基是提取工作的第一步，脱乙酰的方法主要有酸水解法和酶水解法。酸水解需消耗大量的无机酸，在后续的提取过程中又需要加入大量的碱液以中和之前加入的无机酸液，使提取过程中会产生大量的盐。提取过程的酸、碱消耗量大，产生大量难处理的高盐废水。而酶法脱乙酰的方法则无需使用大量酸、碱溶液，因此受到越来越多的青睐。
ZL2016112278411(公开号CN 106831894 B)公开了一种脱乙酰基耦合吸附分离D-氨基葡萄糖盐酸盐的方法，该方法以乙酰氨基葡萄糖发酵液为出发原料，通过陶瓷膜分离去除微生物菌体，再通过活性炭脱色以及离子交换树脂去除培养基中的残余盐分，得到乙酰氨基葡萄糖，再通过酸性阳离子交换柱在91℃条件下实现脱乙酰反应和吸附，反应时间约为120min，通过盐酸洗脱后获得氨基葡萄糖盐酸盐。由于涉及的反应温度超过90℃，处理过程中易产生色素物质，离子交换树脂易破碎损耗。
ZL2013106719979(公开号CN 103626809 B)公开了一种氨基葡萄糖盐酸盐母液的纯化方法，以氨基葡萄糖盐酸盐母液为原料，通过酸性阳离子交换柱使氨基葡萄糖吸附于阳柱上。用盐酸溶液洗脱阳柱后，所得解析液再通过阴离子交换柱，将会去除乙酸、氯离子等阴离子，从而获得氨基葡萄糖，而不是氨基葡萄糖盐酸盐。由于氨基葡萄糖不能长期稳定储存，该方法在工业上的应用价值有限。
发明内容
针对现有技术存在的高能耗、高污染等缺陷，本发明提供了一种新的氨基葡萄糖盐生产和分离提纯方法，以富含乙酰氨基葡萄糖的发酵液或者几丁质的酶水解液为原料，在通过阳离子交换获得氨基葡萄糖盐的基础上，利用阴离子交换获得乙酸盐，并回收未反应完全的乙酰氨基葡萄糖，在提高目标产品得率的同时，使副产物得到资源化利用，同时降低原辅材料消耗、废水、固废的排放量，达到节能降耗和环保安全的目标。该方法还能够通过简单调整阳离子交换树脂的酸性洗脱液种类获得相应的氨基葡萄糖盐，在工业生产规模下提取获得多种氨基葡萄糖盐的高纯度晶体。
本发明的第一个目的是提供一种氨基葡萄糖盐的生产和分离纯化方法，所述方法包括如下步骤：
(1)以含有氨基葡萄糖的澄清溶液为原料，可选地，当含有氨基葡萄糖的溶液为浑浊溶液时，用超滤膜过滤，以过滤后的澄清的含氨基葡萄糖的溶液为原料；所述超滤膜的截留分子量为5-200kDa；
(2)用阳离子交换树脂对步骤(1)的含有氨基葡萄糖的溶液进行吸附，使阳离子树脂吸附氨基葡萄糖；
(3)用酸性洗脱液洗脱步骤(2)阳离子交换树脂，获得含有氨基葡萄糖盐的解析液；
用阴离子交换树脂吸附步骤(2)经过阳离子交换树脂的阳柱下柱液，使阴离子树脂吸附乙酸根离子；将经过阴离子交换树脂的含有乙酰氨基葡萄糖的阴柱下柱液回用于制备氨基葡萄糖；
(4)用碱性洗脱液洗脱阴离子吸附树脂，所得的解析液中富含乙酸钠可用于污水处理厂 的脱氮除磷工艺，也可以用作化学反应的原料等合适用途。
在一种实施方式中，步骤(1)所述的含氨基葡萄糖的溶液是N-乙酰氨基葡萄糖经过生物法或化学法脱乙酰后的反应产物，也可以是其它来源的含氨基葡萄糖的溶液。
在一种实施方式中，步骤(1)所述的超滤膜可以是陶瓷材质的膜组件，也可以是有机材质的膜组件。
在一种实施方式中，步骤(1)所述的含有氨基葡萄糖的溶液是以含N-乙酰氨基葡萄糖的溶液作为原料，以脱乙酰酶提取液或脱乙酰酶制剂为催化剂经催化反应后制得。
在一种实施方式中，步骤(3)所述的回用于制备氨基葡萄糖是作为制备氨基葡萄糖的酶解原料，用于脱乙酰酶催化的脱乙酰反应。
本发明的第二个目的是提供氨基葡萄糖盐的制备方法，所述方法先对含乙酰氨基葡萄糖的溶液进行酶解脱除乙酰基，再按照所述分离纯化方法进行分离、纯化。
在一种实施方式中，所述含乙酰氨基葡萄糖的溶液可以是通过微生物发酵所得，也可以是通过酶水解含有几丁质的生物原料所得，也可以是通过化学水解含有几丁质的原料所得。
在一种实施方式中，所述酶解是以浓度为40-150g/L的乙酰氨基葡萄糖溶液为原料，按照10-40U/g乙酰氨基葡萄糖的比例加入脱乙酰酶；酶解反应的pH范围为4-8，反应温度为25-55℃，搅拌反应10-40min；所述乙酰氨基葡萄糖溶液是利用微生物发酵或者几丁质水解得到的含有乙酰氨基葡萄糖的原料液。
在一种实施方式中，所述脱乙酰酶可以来源于微生物，并通过微生物发酵所得，也可以由其它生物体中提取所得；所述微生物可以是自然界中筛选得到的微生物，也可以是通过基因工程改造的重组微生物。
在一种实施方式中，所述酶解通过脱乙酰酶进行酶反应，专一性地脱除N-乙酰氨基葡萄糖分子中的乙酰基，反应得到主要成分为氨基葡萄糖和乙酸的酶水解产物。
在一种实施方式中，所述方法包括如下步骤：
(1)以浓度为80-150g/L的乙酰氨基葡萄糖溶液为原料，按照10-40U/g乙酰氨基葡萄糖的比例加入脱乙酰酶，酶反应的pH范围为4-8，反应温度为25-55℃，搅拌反应10-90min；
(2)将步骤(1)反应后的酶水解产物可以直接进入步骤(3)，也可以进行超滤膜过滤，分别得含氨基葡萄糖的超滤膜透析液和膜浓缩液，将膜浓缩液的酶液回用于步骤(1)，参与下一批次的酶反应过程；所述超滤膜的截留分子量为5-200kDa；
(3)用阳离子交换树脂对步骤(2)所得的氨基葡萄糖膜透析液进行吸附，阳离子交换树脂采用酸性洗脱液进行连续洗脱，获得含有氨基葡萄糖盐的解析液；
(4)用阴离子交换树脂吸附步骤(3)经过阳离子交换树脂的阳柱下柱液，阴离子吸附 树脂采用碱性洗脱液洗脱阴离子吸附树脂，分离出的富含乙酸钠的解析液可用于污水处理厂的脱氮除磷工艺；
(5)将步骤(4)经过阴柱的阴柱下柱液用纳滤膜或反渗透膜过滤；将纳滤膜或反渗透膜的浓缩液循环用于步骤(1)进行下一批次的酶反应过程。
在一种实施方式中，步骤(3)用于阳离子交换层析的设备可以是固定床，也可以是离子交换连续床，也可以是离子交换模拟移动床；所述的酸性洗脱剂可以是盐酸，也可以是硫酸，也可以是磷酸，也可以是丙酮酸，也可以是柠檬酸；洗脱得到的相应的氨基葡萄糖盐分别为氨基葡萄糖盐酸盐、氨基葡萄糖硫酸盐、氨基葡萄糖磷酸盐、氨基葡萄糖丙酮酸盐和氨基葡萄糖柠檬酸盐；所述酸性洗脱液浓度为0.30-3.0mol/L。
在一种实施方式中，步骤(3)阳离子交换的吸附和洗脱温度为20-70℃；步骤(4)阴离子交换的吸附温度和洗脱温度为20-65℃；上述两级离子交换层析的进料流速为2.0–10.0BV/h；洗脱液的流速为1.0-8.0BV/h。
在一种实施方式中，步骤(4)用于阴离子交换层析的设备可以是固定床，也可以是离子交换连续床，也可以是离子交换模拟移动床；所述的碱性洗脱剂可以是NaOH溶液，也可以是KOH溶液，所述碱性洗脱液浓度为0.30-3.0mol/L；通过洗脱阴离子交换柱后的解析液中可以分别回收到乙酸钠或乙酸钾，乙酸钠或乙酸钾可以通过管道收集并输送至污水处理车间，用于污水处理过程中脱氮除磷的补充碳源，也可以用于其它化学反应过程的原料。
在一种实施方式中，所述步骤(5)的纳滤膜为陶瓷膜，纳滤膜的孔径在0.5-2nm之间，操作压力为2-5atm；所述的反渗透膜为有机卷式膜或者陶瓷膜，反渗透膜的截留分子量为50-100Da，操作压力为4-10atm。
在一种实施方式中，所述步骤(5)之后还依次进行浓缩、结晶和干燥。
在一种实施方式中，所述浓缩为蒸发浓缩；所述蒸发浓缩可以是单效蒸发，也可以是双效蒸发，也可以是多效蒸发。
在一种实施方式中，所述结晶的温度为5-40℃。
在一种实施方式中，还对结晶母液进行脱色；所述的脱色方法为活性炭吸附脱色；经脱色后的结晶母液循环回用于浓缩过程；所述的脱色方法中，活性炭用量为原料液的0.01-2.0％(w/v)。
在一种实施方式中，所述干燥为真空干燥或者闪蒸干燥；所述闪蒸干燥的进风温度为110-290℃，出风温度为70-90℃；所述真空低温干燥的温度为40-80℃，真空度为70-95kPa。
在一种实施方式中，所述的多效蒸发浓缩为三效蒸发，温度分别为80℃、70℃和60℃。末效蒸发器的真空度为80-98kPa。
有益效果：
本发明与现有技术相比，具有如下优点：
(1)本发明在工业生产规模下实现了多种不同氨基葡萄糖盐的酶法生产和高效提纯。生产工艺具有高转化率、高回收率、低原料消耗和环保安全的优势，产物回收率超过95％，生产的氨基葡萄糖盐纯度可超过99.5％；
(2)本发明所采用的酶反应条件温和，反应速率高效，并设计了脱乙酰酶的循环利用工艺，通过超滤膜过滤一方面使酶和产物有效分离，另一方面使酶得到回收利用，降低了酶的使用成本；
(3)本发明通过两级离子交换和反渗透膜/纳滤膜结合，形成了循环工艺，一方面使副产物乙酸盐和反应未完全的底物乙酰氨基葡萄糖得到有效回用，有效提高了底物的转化率和产物的提取回收率，又有效降低了离子交换过程中的树脂和洗脱剂的耗用量，起到了低耗、节能和环保的三重效益。
(4)本发明通过连续移动床和模拟移动床进行离子交换过程，可以在提升离子交换的分离效率的同时，提高工艺操作的连续化和自动化水平；
(5)本发明的离子交换反应条件温和，与常用的固定床相比，可使树脂耗损量减少约60％，树脂再生所需的酸液和碱液消耗减少约50％，同时可以大幅减少废水的产生量。
(6)本发明的工艺适用于多种氨基葡萄糖盐的生产，可通过改变阳离子交换树脂的酸性洗脱剂种类，获得对应酸的氨基葡萄糖盐，具有广泛的工业化应用价值。
附图说明
图1氨基葡萄糖盐的提取工艺路线。
具体实施方式
技术术语：
氨基葡萄糖盐：是指采用不同酸洗脱阳离子交换柱后得到的氨基葡萄糖盐产物，包括但不限于氨基葡萄糖盐酸盐、氨基葡萄糖硫酸盐、氨基葡萄糖磷酸盐、氨基葡萄糖丙酮酸盐和氨基葡萄糖柠檬酸盐的任何一种。
膜透析液：膜过滤过程中透过膜材料而得到的液体。
膜浓缩液：膜过滤过程中不能透过膜材料而被截留下来的液体。
解析液：离子交换过程中，洗脱液(酸液或碱液)流过已饱和的离子交换树脂后所得到的液体。
下柱液：离子交换过程中，原料液中未被离子交换树脂吸附而直接流出和被去离子水清洗出来的液体。
脱乙酰酶活性单位定义为：1U＝1mmol/min，即1min反应得到1mmol氨基葡萄糖为1U酶活单位。
氨基葡萄糖盐的提纯回收率：由于酶法生产氨基葡萄糖盐及其提纯过程中涉及到酶反应和离子交换等单元，反应物的分子结构会发生变化。所述回收率均以氨基葡萄糖的质量为基准进行计算。
氨基葡萄糖盐和乙酰氨基葡萄糖的定量采用HPLC分析法。液相色谱仪为安捷伦1260 series，色谱柱为Thermo ODS-2 Hypersil C18 column(250mm×4.0mm)，待测的氨基葡萄糖盐样品先通过0.22μm的微滤膜过滤后再以10μl进样量注入色谱柱中。乙酰氨基葡萄糖和乙酸的分析采用色谱柱HPX-87H column(Bio-Rad,USA)。检测器采用示差折光仪检测器。流动相为5mM H 2SO 4，流速为0.6ml/min，检测温度为40℃。
实施例1
按图1所示工艺路线，操作步骤如下：
(1)以浓度为40-150g/L的乙酰氨基葡萄糖溶液为原料，按照10-30U/g乙酰氨基葡萄糖的比例加入脱乙酰酶溶液或脱乙酰酶制剂，酶反应的pH范围为4.0-8.0，反应温度为25-45℃，搅拌反应10-40min；脱乙酰酶制剂可选用商业化的能够水解脱除N-乙酰氨基葡萄糖中乙酰基的酶；该步骤专一性地脱除N-乙酰氨基葡萄糖分子中的乙酰基，反应得到主要成分为氨基葡萄糖和乙酸的酶水解产物，酶反应的pH优选为7.0-8.0；
(2)将步骤(1)反应后的酶水解产物输送至超滤膜设备中，超滤膜的截留分子量为5-200kDa，优选5-30kDa；用水透析膜浓缩液，分别收集含氨基葡萄糖的超滤膜透析液和含脱乙酰酶的膜浓缩液，膜透析完成后脱乙酰酶的回收率为80-90％；
(3)将步骤(2)膜浓缩后的酶液返回至脱乙酰酶液储罐中，参与下一批次的酶反应过程；
将步骤(2)所得的含氨基葡萄糖的超滤膜透析液连续泵入到模拟移动床填充了酸性树脂(例如带有磺酸基的阳离子交换树脂)的阳柱中，进料流速为2.0–10.0BV/h，使透析液中的氨基葡萄糖吸附在阳柱上；
采用去离子水清洗阳柱，获得含有N-乙酰氨基葡萄糖和乙酸的阳柱下柱液；
采用浓度为0.30-3.0mol/L的盐酸、硫酸、磷酸、丙酮酸、柠檬酸等酸性洗脱液连续洗脱阳柱，洗脱温度为20-70℃，优选25℃，可得含有相应氨基葡萄糖盐的解析液。
实施例2
按图1所示工艺路线，操作步骤如下：
(1)以浓度为40-150g/L的乙酰氨基葡萄糖溶液为原料，按照10-30U/g乙酰氨基葡萄 糖的比例加入脱乙酰酶溶液或脱乙酰酶制剂，酶反应的pH范围为4.0-8.0，反应温度为25-45℃，搅拌反应10-40min；脱乙酰酶制剂可选用商业化的能够水解脱除N-乙酰氨基葡萄糖中乙酰基的酶；该步骤专一性地脱除N-乙酰氨基葡萄糖分子中的乙酰基，反应得到主要成分为氨基葡萄糖和乙酸的酶水解产物，酶反应的pH优选为7.0-8.0；
(2)将步骤(1)反应后的酶水解产物输送至超滤膜设备中，超滤膜的截留分子量为5-200kDa，优选5-30kDa；用水透析膜浓缩液，分别收集含氨基葡萄糖的超滤膜透析液和含脱乙酰酶的膜浓缩液，膜透析完成后脱乙酰酶的回收率为80-90％；
(3)将步骤(2)膜浓缩后的酶液返回至脱乙酰酶液储罐中，参与下一批次的酶反应过程；
将步骤(2)所得的含氨基葡萄糖的超滤膜透析液连续泵入到模拟移动床填充了酸性树脂(例如带有磺酸基的阳离子交换树脂)的阳柱中，进料流速为2.0–10.0BV/h，使透析液中的氨基葡萄糖吸附在阳柱上；
采用去离子水清洗阳柱，获得含有N-乙酰氨基葡萄糖和乙酸的阳柱下柱液；
采用浓度为0.30-3.0mol/L的盐酸、硫酸、磷酸、丙酮酸、柠檬酸等酸性洗脱液连续洗脱阳柱，洗脱温度为20-70℃，优选25℃，可得含有相应氨基葡萄糖盐的解析液，用于后续的浓缩、结晶、干燥过程；
(4)将步骤(3)的阳柱下柱液进一步输送到填充了碱性阴离子交换树脂(例如带有季铵盐的阴离子交换树脂)的阴柱中，进料流速为2.0–10.0BV/h；控制吸附温度和洗脱温度为20-65℃，优选25℃；使乙酸吸附在阴柱上；
采用去离子水清洗阴柱，可得含N-乙酰氨基葡萄糖的阴柱下柱液；
采用0.30-3.0mol/L的NaOH或KOH等碱性洗脱液以1.0-8.0BV/h的速度连续洗脱阴柱，可分离出富含乙酸盐的解析液，解析液可输送至污水处理车间作为脱氮除磷过程的补充碳源，也可以用于化学原料；
(5)将步骤(4)经过阴柱的阴柱下柱液输送至纳滤膜或者反渗透膜浓缩设备中；
所述纳滤膜为陶瓷膜，纳滤膜的孔径为0.5-2nm，操作压力为2-5atm；
所述的反渗透膜为有机卷式膜或者陶瓷膜，反渗透膜的截留分子量为50-100Da，操作压力为4-10atm；
膜浓缩后的N-乙酰氨基葡萄糖终浓度可达到10-15％(w/v)，将膜浓缩液循环回收至N-乙酰氨基葡萄糖贮罐中，用于下一批次步骤(1)的酶反应过程；
(6)将步骤(3)阳柱的解析液泵送至多效蒸发器中进行蒸发浓缩，所述的多效蒸发浓缩为三效蒸发，梯度控制温度，例如，将一效、二效和三效蒸发器的温度分别设置为80℃、 70℃和60℃，三效蒸发器的真空度为80-98kPa；
(7)将步骤(6)多效蒸发器浓缩后的出料流入至结晶器，通过控制结晶器的夹套温度使结晶温度控制在5-40℃，将结晶器产生的晶体悬液送入固液分离设备分离，分别获得结晶母液和氨基葡萄糖盐晶泥；所述分离设备可选用具有连续离心功能的离心机或者卧式刮刀卸料离心机；
(8)采用活性炭对步骤(7)分离获得的结晶母液进行脱色，脱色后回送至步骤(6)的多效蒸发设备中；将所述氨基葡萄糖盐晶泥输送至干燥设备中获得干燥后的氨基葡萄糖盐晶体。浓缩、结晶和干燥单元的回收率可达到98％。
实施例3
按图1所示工艺路线，操作步骤如下：
(1)收集350m 3浓度为102kg/m 3的乙酰氨基葡萄糖溶液于储罐中，将乙酰氨基葡萄糖溶液泵入酶反应罐中，按照10-25U/g乙酰氨基葡萄糖的比例加入脱乙酰酶液，酶反应的pH为7.0-8.0，反应温度为37℃，搅拌反应30min；
(2)将步骤(1)反应后的酶水解产物泵入到超滤膜设备中，超滤膜的截留分子量为5000Da，添加60m 3纯水用于透析膜浓缩液，分别收集超滤膜透析液和膜浓缩液；共收集获得380m 3含量为56.3kg/m 3的氨基葡萄糖透析液；将膜浓缩液的30m 3酶液返回至脱乙酰酶液储罐中，参与下一批次的酶反应过程；
(3)将步骤(2)所得的氨基葡萄糖透析液连续泵入到模拟移动床填充了001×7强酸性苯乙烯系树脂的阳柱中，进料流速为4.0BV/h，进料和洗脱温度为25℃；透析液中的氨基葡萄糖吸附在阳柱上，用去离子水冲洗阳柱，获得含有中性糖和乙酸的阳柱下柱液；
采用2mol/L的盐酸溶液连续洗脱阳柱，洗脱流速为3.0BV/h，从而得到91.7m 3浓度为252kg/m 3氨基葡萄糖盐酸盐的解析液；
(4)将步骤(3)阳柱的下柱液以4.0BV/h流速进一步输送到模拟移动床填充了阴离子交换树脂的阴柱中，采用去离子水冲洗阴柱，可得N-乙酰氨基葡萄糖浓度约为3％的阴柱下柱液；
采用1.5mol/L的NaOH溶液在连续洗脱阴柱，分离出80.6m 3富含乙酸钠(107.5kg/m 3)的解析液；其中，阴柱填料为201×7强碱性苯乙烯系树脂；
(5)将步骤(4)经过阴柱的514.6m 3下柱液输送至纳滤膜浓缩设备中，纳滤膜的孔径为1nm，操作压力为0.5-1.0MPa，共浓缩得到89.3m 3的N-乙酰氨基葡萄糖浓度为125g/L的纳滤膜浓缩液；将纳滤膜浓缩液循环回收至乙酰氨基葡萄糖储罐中，用于下一批次的酶反应过程；
第一批次上述反应和提纯过程可获得23.1吨氨基葡萄糖盐酸盐，其回收率达到66.2％。
将纳滤膜浓缩得到的89.3m 3浓缩液循环至乙酰氨基葡萄糖溶液储罐中，并将步骤(2)膜浓缩液的酶液返回至酶反应罐中进行的在第二轮循环反应后可回收到45m 3含量为238kg/m 3氨基葡萄糖盐酸盐溶液，氨基葡萄糖盐酸盐的总回收率达到97％。
延续前述工艺，将第一批次和第二批次步骤(3)获得的氨基葡萄糖盐酸盐的洗脱液合并后进行浓缩、结晶和干燥，具体如下：
(6)将合并后的含氨基葡萄糖盐酸盐的溶液泵送至三效蒸发器中，控制进料流量为6m 3/h，末效冷凝器的真空度为90kPa；冷却水进水温度为8-15℃，出料产品浓度为720g/L；
(7)将三效蒸发器的出料流入至结晶器，通过控制结晶器的夹套温度使结晶温度控制在40℃。将结晶器产生的晶体悬液送入卧螺离心机分离，分离获得结晶母液和氨基葡萄糖盐酸盐晶泥；
(8)将结晶母液以0.5m 3/h的流速送入活性炭脱色柱脱色，脱色后回送至三效蒸发设备前的储罐中；卧螺离心机分离所得的氨基葡萄糖盐酸盐晶泥通过螺旋输送器送入闪蒸干燥器，闪蒸干燥的进风温度为150℃，出风温度为80℃，即得氨基葡萄糖盐酸盐晶体。
步骤(6)-(8)的浓缩、结晶和干燥单元的总回收率可达到98％。
在整个生产过程中，生产1吨氨基葡萄糖盐酸盐需消耗浓盐酸溶液500kg，30％NaOH溶液550L，纯水10吨和产生9.7吨废水，损耗阳离子交换树脂和阴离子交换树脂各约10kg。
实施例4
按图1所示工艺路线，区别在于，在实施例3的基础上，以反渗透陶瓷膜设备替换纳滤膜浓缩设备，也即，将步骤(4)获得的阴柱下柱液收集至反渗透陶瓷膜设备，所述的反渗透膜的孔径为1nm，操作压力为0.5-1.0MPa，反渗透陶瓷膜设备浓缩后的液体返回至酶反应罐参与下一批次的酶反应过程。
该生产过程中，生产1吨氨基葡萄糖盐酸盐需消耗纯水11吨，产生10.5吨废水。
实施例5
按图1所示工艺路线，区别在于，在实施例3的基础上，步骤(3)采用硫酸溶液代替盐酸溶液，也即，步骤(3)中采用1mol/L的硫酸溶液对吸附在阳柱上的氨基葡萄糖进行洗脱，得到的产品为氨基葡萄糖硫酸盐。
实施例6
按图1所示工艺路线，区别在于，在实施例3的基础上，步骤(3)采用磷酸溶液代替盐酸溶液，也即，步骤(3)中采用1mol/L的磷酸溶液对吸附在阳柱上的氨基葡萄糖进行洗脱，得到的产品为氨基葡萄糖磷酸盐。
实施例7
按图1所示工艺路线，区别在于，在实施例3的基础上，步骤(3)采用柠檬酸溶液代替盐酸溶液，也即，步骤(3)中采用1mol/L的柠檬酸溶液对吸附在阳柱上的氨基葡萄糖进行洗脱，得到的产品为氨基葡萄糖柠檬酸盐。
实施例8
按图1所示工艺路线，区别在于，在实施例3的基础上，步骤(3)采用丙酮酸溶液代替盐酸溶液，也即，步骤(3)中采用2mol/L的丙酮酸溶液对吸附在阳柱上的氨基葡萄糖进行洗脱，得到的产品为氨基葡萄糖丙酮酸盐。
实施例9
按图1所示工艺路线，区别在于，在实施例3的基础上，将步骤(3)的模拟移动床替换为连续移动床设备，其中阳柱和阴柱的填料不变，阳柱填料为001×7强酸性苯乙烯系阳离子树脂，阴柱填料为201×7强碱性苯乙烯系阴离子树脂。每生产1吨产品需消耗16kg阳离子交换树脂和14kg阴离子交换树脂，需消耗600kg浓盐酸和650L 30％的氢氧化钠溶液，产生15吨废水。
实施例10
按图1所示工艺路线，区别在于，在实施例3的基础上，步骤(3)的模拟移动床替换为固定床离子交换设备，阳柱填料为201×7强碱性苯乙烯系阴离子交换树脂，阴柱填料为001×7强酸性苯乙烯系阳离子交换树脂。每生产1吨产品需消耗30kg阳离子交换树脂和28kg阴离子交换树脂，需消耗1300kg浓盐酸和1400L 30％的氢氧化钠溶液，产生50吨废水。
实施例11
按图1所示工艺路线，区别在于，在实施例5的基础上，省略三效蒸发浓缩和结晶，将模拟移动床阳柱洗脱出来的富含氨基葡萄糖硫酸盐的解析液直接输送至喷雾干燥设备，进料流速为5m 3/h，喷雾干燥的进风温度为150℃。干燥后即得41.5吨氨基葡萄糖硫酸盐粉末，产品纯度达到99％。
实施例12
按图1所示工艺路线，区别在于，在实施例3的基础上，将步骤(1)所得的酶反应液连续泵入步骤(3)所述的阳柱中，进料流速为3.0BV/h，进料和洗脱温度为30℃，后续步骤同实施例2，得到的产品为氨基葡萄糖盐酸盐。
对比例1
参照申请号为CN2016112278411的专利申请中分离D-氨基葡萄糖盐酸盐的方法，收集在储罐中的50m 3浓度为102kg/m 3的乙酰氨基葡萄糖溶液，通入蒸汽使溶液升温至95℃，泵 入装填有强酸性阳离子树脂交换柱中，保持温度为90℃，使乙酰氨基葡萄糖与阳离子树脂反应240min，反应结束后通入纯水洗涤阳离子树脂，收集含有乙酸的洗涤液40m 3，洗涤完成后再通入12％盐酸洗脱阳离子交换柱，洗脱过程的流速为1.5BV/h。收集到42m 3解析液，氨基葡萄糖盐酸盐的浓度为103kg/m 3，该步骤得率为88.6％。向解析液中添加0.2％活性炭脱色，再泵入至三效蒸发浓缩器中，再经结晶、离心和干燥，得到4410kg氨基葡萄糖盐酸盐，总回收率为80.2％。由于乙酰氨基葡萄糖与阳离子树脂反应温度较高，反应时间长，反应过程中易产生色素，且对离子交换树脂的损耗增加，生产每吨氨基葡萄糖盐酸盐消耗浓盐酸1100kg，120kg阳离子树脂，产生30m 3高无机酸含量和高COD的废水，生产过程中无法回收乙酸。
表1 采用不同阳离子交换方式时氨基葡萄糖盐的提取效果
Figure PCTCN2020111623-appb-000001
发明人还尝试对酶解、分离和提纯工艺参数进行调整，在实施例1优选参数的范围内，离子交换单元的产品回收率可达到99％，填料损耗量控制在20kg/吨的范围内，酸溶液用量控制在600kg/吨产品的范围内，废水产生量低于10m 3/吨产品的效果。
对比例2
参照CN2013106719979中公开的方法，区别在于，将原料由氨基葡萄糖盐酸盐母液替换为含有氨基葡萄糖的酶反应溶液，略去将阳离子交换柱的解析液输送至阴离子交换柱的操作步骤。将收集到的脱乙酰酶反应液和超滤膜透析后的膜透析液共50m 3(含有氨基葡萄糖和乙酸)于储罐中，氨基葡萄糖的浓度为89kg/m 3的溶液，泵入装填有强酸性阳离子树脂交换柱中，保持温度为32℃使氨基葡萄糖吸附在阳离子树脂上，通入纯水清洗阳离子树脂，收集到含有乙酸的下柱液共60m 3，清洗完成后再通入0.3mol/L盐酸溶液洗脱阳离子交换柱，洗脱过程的流速为1.5BV/h。收集到31m 3含有126kg/m 3的氨基葡萄糖盐酸盐溶液，得率为87.8％。通过加热使所得的解析液升温到60℃，添加1％粉末活性炭脱色，过滤后得30.5m 3含有滤液125kg/m 3的氨基葡萄糖盐酸盐溶液，再泵入至三效蒸发浓缩器中，再经结晶、离心和干燥，得到3585kg氨基葡萄糖盐酸盐晶体，晶体呈微黄色，总得率为80.6％。该方法先脱色后浓缩， 活性炭消耗量大，生产每吨氨基葡萄糖盐酸盐消耗浓盐酸900kg，20kg阳离子树脂，产生30m 3高COD含量的废水。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (15)

  1. 一种氨基葡萄糖盐的生产和分离纯化方法，其特征在于，所述方法包括如下步骤：
    (1)以含有氨基葡萄糖的澄清溶液为原料，可选地，当含有氨基葡萄糖的溶液为浑浊溶液时，用超滤膜过滤，以过滤后的澄清的含氨基葡萄糖的溶液为原料；所述超滤膜的截留分子量为5-200kDa；用阳离子交换树脂对原料进行吸附，使阳离子树脂吸附氨基葡萄糖；
    (2)用酸性洗脱液洗脱步骤(1)阳离子交换树脂，获得含有氨基葡萄糖盐的解析液；
    用阴离子交换树脂吸附步骤(1)经过阳离子交换树脂的阳柱下柱液，使阴离子树脂吸附乙酸根离子；将经过阴离子交换树脂的含有乙酰氨基葡萄糖的阴柱下柱液回用于制备氨基葡萄糖；
    (3)用碱性洗脱液洗脱阴离子吸附树脂，所得的解析液中富含乙酸钠，可用于污水处理厂的脱氮除磷工艺，也可以用作生产制造的原料。
  2. 酶法生产氨基葡萄糖盐的方法，其特征在于，所述方法包括：
    以含有乙酰氨基葡萄糖的溶液为原料，采用脱乙酰酶或含脱乙酰酶的制剂进行催化水解，得到酶水解产物；
    将酶水解产物直接用于阳离子交换处理；或，将酶水解产物用膜过滤，所得膜截留液回收脱乙酰酶，所得膜透析液用于阳离子交换处理；
    所述酶水解产物或膜透析液经过阳离子交换及酸性洗脱液洗脱，得到氨基葡萄糖盐。
  3. 根据权利要求2所述的方法，其特征在于，所述酶解是以浓度为40-150g/L的乙酰氨基葡萄糖溶液为原料，按照10-40U/g乙酰氨基葡萄糖的比例加入脱乙酰酶；酶解反应的pH范围为4-8，反应温度为25-55℃，搅拌反应10-40min。
  4. 根据权利要求2或3所述的方法，其特征在于，还将阳离子交换过程的阳柱下柱液再通过阴离子交换及碱性洗脱液洗脱，回收乙酸盐，将阴离子交换过程的阴柱下柱液浓缩后循环送回至脱乙酰酶参与的催化水解过程。
  5. 根据权利要求2或3所述的方法，其特征在于，所述膜可以是陶瓷材质的膜组件，也可以是有机材质的膜组件；所述超滤膜的截留分子量为5-200kDa。
  6. 根据权利要求1所述的方法，其特征在于，用于所述阳离子交换的树脂包括但不限于带有磺酸基的阳离子交换树脂；
    所述酸性洗脱液可以是浓度为0.3-4.0mol/L的盐酸、硫酸、磷酸、丙酮酸或柠檬酸。
  7. 根据权利要求1所述的方法，其特征在于，用于所述阴离子交换的树脂包括但不限于带有季铵基的阴离子交换树脂；
    所述碱性洗脱液可以是浓度为0.30-3.0mol/L的NaOH溶液或KOH溶液。
  8. 根据权利要求6或7所述的方法，其特征在于，用于阳离子交换和/或阴离子交换的设 备可以是离子交换固定床，也可以是离子交换连续移动床，也可以是离子交换模拟移动床。
  9. 根据权利要求8所述的方法，其特征在于，离子交换过程的吸附或洗脱温度为20-75℃，进料流速为2.0-10.0BV/h；洗脱液的流速为1.0-8.0BV/h。
  10. 根据权利要求2,3,6,7,9任一所述的方法，其特征在于，将所得的氨基葡萄糖盐经过浓缩、结晶和干燥，得到高纯度的氨基葡萄糖盐。
  11. 根据权利要求10所述的方法，其特征在于，所述浓缩可选用蒸发浓缩；
    所述蒸发浓缩可以为单效蒸发浓缩或多效蒸发浓缩，末效蒸发器的真空度为80-98kPa；
    所述结晶的温度为5-40℃；
    所述干燥可以是真空低温干燥，也可以是闪蒸干燥；所述真空低温干燥的温度为40-80℃，真空度为70-95kPa；所述闪蒸干燥的热风温度为120-300℃。
  12. 根据权利要求10所述的方法，其特征在于，对结晶母液进行脱色；所述的脱色方法为活性炭吸附脱色；经脱色后的结晶母液循环回用于浓缩过程；
    可选地，所述的脱色方法中，活性炭用量为原料液的0.01-2％；可选地，所述的活性炭可以是碳棒、碳柱或颗粒状活性炭。
  13. 根据权利要求2～12任一所述的方法，其特征在于，包括如下步骤：
    (1)以浓度为40-150g/L的乙酰氨基葡萄糖溶液为原料，加入含有脱乙酰酶的制剂，酶反应的pH范围为4-8，反应温度为25-55℃，搅拌反应10-90min；
    (2)将步骤(1)反应后的酶水解产物用超滤膜或纳滤膜过滤，可得含氨基葡萄糖的超滤膜透析液和膜浓缩液；将膜浓缩液的酶液回用于步骤(1)，参与下一批次的酶反应或废弃；
    (3)用阳离子交换树脂对步骤(2)所得的超滤膜透析液进行吸附，阳离子交换树脂采用酸性洗脱液进行连续洗脱，可得含有氨基葡萄糖盐的解析液；用去离子水清洗阳离子交换树脂后可得含有N-乙酰氨基葡萄糖和乙酸的阳柱下柱液；
    (4)用阴离子交换树脂对步骤(3)经过阳离子交换树脂的阳柱下柱液进行吸附，阴离子吸附树脂采用碱性洗脱液洗脱，可分离出含乙酸盐的解析液；
    (5)将步骤(4)经过阴离子交换树脂的阴柱下柱液循环用于步骤(1)进行下一批次的酶反应过程；或先将步骤(4)经过阴离子交换树脂的阴柱下柱液浓缩，再循环用于步骤(1)进行下一批次的酶反应过程；浓缩方法可以是真空浓缩、纳滤膜或反渗透膜过滤浓缩，也可以是多效蒸发浓缩。
  14. 根据权利要13所述的方法，其特征在于，步骤(5)中，所述纳滤膜为陶瓷膜，孔径为0.5-2nm；所述的反渗透膜为有机卷式膜或者陶瓷膜。
  15. 权利要求3～13任一所述方法在制备氨基葡萄糖盐衍生产品或含氨基葡萄糖盐的产品 中的应用。
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Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111518857A (zh) * 2020-06-11 2020-08-11 江苏海飞生物科技有限公司 酶法生产氨基葡萄糖盐及其提纯方法
CN113248553A (zh) * 2021-06-07 2021-08-13 江苏海飞生物科技有限公司 一种d-氨基葡萄糖盐酸盐的制备方法
CN113373134B (zh) * 2021-07-30 2024-02-20 江苏海飞生物科技有限公司 一种n-乙酰氨基葡萄糖脱乙酰酶的提取方法
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060210582A1 (en) * 2000-10-27 2006-09-21 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A & B
CN101365785A (zh) * 2002-07-01 2009-02-11 阿基昂生命科学公司,以生物技术资源部的名义经营 用于生产葡糖胺和n-乙酰氨基葡糖的方法和物质
CN107267579A (zh) * 2016-04-05 2017-10-20 孙镧 微生物发酵生产n‑乙酰‑d‑氨基葡萄糖和/或d‑氨基葡萄糖盐的方法
CN109666663A (zh) * 2019-01-22 2019-04-23 华东理工大学 一种利用n-乙酰氨基葡萄糖合成氨基寡糖的方法及其专用酶
CN110714042A (zh) * 2018-07-13 2020-01-21 中国科学院天津工业生物技术研究所 氨基葡萄糖的酶法制备
CN111518857A (zh) * 2020-06-11 2020-08-11 江苏海飞生物科技有限公司 酶法生产氨基葡萄糖盐及其提纯方法

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100340187C (zh) * 2003-07-16 2007-10-03 甲阳化学制品股份公司 呈味性改善的氨基葡萄糖盐水溶液及其制备方法，以及氨基葡萄糖盐组合物和食品
JP4738856B2 (ja) * 2005-03-23 2011-08-03 焼津水産化学工業株式会社 Ｎ−アセチルグルコサミン含有組成物の製造方法
CN102167714A (zh) * 2011-03-02 2011-08-31 中国药科大学 快速、高效的硫酸氨基葡萄糖氯化钾复盐制备方法
CN103626809B (zh) * 2013-12-10 2016-01-20 安徽丰原发酵技术工程研究有限公司 一种氨基葡萄糖盐酸盐母液的纯化方法
CN105440088B (zh) * 2015-11-20 2019-02-12 南方科技大学 氨基葡萄糖混合锶盐及其制备方法和应用
WO2017174036A1 (zh) * 2016-04-05 2017-10-12 孙镧 微生物发酵生产n-乙酰-d-氨基葡萄糖和/或d-氨基葡萄糖盐的方法
CN106496289A (zh) * 2016-10-19 2017-03-15 大连皮米海洋生物研究院 一种d‑氨基葡萄糖硫酸盐的制造工艺
CN106831894B (zh) * 2016-12-27 2019-08-30 王惠 一种脱乙酰基耦合吸附分离d-氨基葡萄糖盐酸盐的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060210582A1 (en) * 2000-10-27 2006-09-21 Novartis Vaccines And Diagnostics, Inc. Nucleic acids and proteins from streptococcus groups A & B
CN101365785A (zh) * 2002-07-01 2009-02-11 阿基昂生命科学公司,以生物技术资源部的名义经营 用于生产葡糖胺和n-乙酰氨基葡糖的方法和物质
CN107267579A (zh) * 2016-04-05 2017-10-20 孙镧 微生物发酵生产n‑乙酰‑d‑氨基葡萄糖和/或d‑氨基葡萄糖盐的方法
CN110714042A (zh) * 2018-07-13 2020-01-21 中国科学院天津工业生物技术研究所 氨基葡萄糖的酶法制备
CN109666663A (zh) * 2019-01-22 2019-04-23 华东理工大学 一种利用n-乙酰氨基葡萄糖合成氨基寡糖的方法及其专用酶
CN111518857A (zh) * 2020-06-11 2020-08-11 江苏海飞生物科技有限公司 酶法生产氨基葡萄糖盐及其提纯方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JAMES S. DAVIES; DAVID COOMBES; CHRISTOPHER R. HORNE; F. GRANT PEARCE; ROSMARIE FRIEMANN; RACHEL A. NORTH; RENWICK C. J. DOBSON: "Functional and solution structure studies of amino sugar deacetylase and deaminase enzymes from Staphylococcus aureus", FEBS LETTERS, vol. 593, no. 1, 26 November 2018 (2018-11-26), NL , pages 52 - 66, XP071256661, ISSN: 0014-5793, DOI: 10.1002/1873-3468.13289 *
WANG, CHAO: "A study on recovery and purification conditions of glucosamine hydrochloride mother liquor", BIOTECH WORLD, 31 December 2016 (2016-12-31), pages 243, XP055878168 *

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