Enzymatic production of glucosamine salts and methods for purifying the same

Abstract

The application discloses an enzyme method for producing glucosamine salt and a purification method thereof, and belongs to the technical field of bioengineering. The application takes N-acetyl glucosamine as raw material, obtains glucosamine and acetic acid through deacetylase hydrolysis, separates the glucosamine salt through an acid solution elution cation exchange column, and recovers byproduct sodium acetate through anion exchange. The obtained glucosamine salt is concentrated, crystallized, decolorized and dried to obtain high-purity glucosamine salt crystals. The application combines the enzyme recycling process, residual substrate recycling process and acetic acid recovery process, improves the conversion rate of N-acetyl glucosamine and the total yield of the glucosamine salt product, recycles the enzyme, fully converts the residual substrate, recycles the acetic acid, and through normal-temperature operation conditions, the resin loss rate is low, the amount of hydrochloric acid waste liquid is extremely small, and the economic benefits of energy saving and consumption reduction and the environmental protection and safety effects are realized.

Description

TECHNICAL FIELD

[0001] The present application relates to enzymatic production of glucosamine salt and its purification method, belonging to the field of bioengineering technology. BACKGROUND

[0002] Glucosamine (GlcNAc) is an important amino hexose, which is formed by replacing a hydroxyl group of glucose with an amino group, and is easily soluble in water and hydrophilic solvents. It is widely present in nature, and its chemical name is 2-amino-2-deoxy-D-glucose. It usually exists in the form of N-acetyl derivative (such as chitin) or N-sulfate and N-acetyl-3-O-lactate ether (muramic acid) in microbial, animal-derived polysaccharides and conjugated polysaccharides. Glucosamine hydrochloride has a molecular weight of 215.5 Da, is white crystalline, has no odor, has a slight sweetness, is easily soluble in water, is slightly soluble in methanol, and is insoluble in organic solvents such as ethanol. Glucosamine molecules are not very stable and are prone to oxidation or degradation. Glucosamine can be prepared into salts such as glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, and glucosamine pyruvate, and the stability of which can be significantly improved. Glucosamine has important physiological functions for the human body, participates in liver and kidney detoxification, plays an anti-inflammatory, liver-protecting and kidney-nourishing role, has good curative effect on the treatment of rheumatoid arthritis and gastric ulcer, is a main raw material for the synthesis of antibiotics and anticancer drugs, and can also be applied to food, cosmetics and feed additives.

[0003] At present, there are mainly three types of production methods for GlcNAc: chemical method, enzymatic method and microbial method. Natural raw materials such as shrimp and crab shells and fungal cell walls contain relatively rich chitin, which can be obtained after acid hydrolysis or enzymatic hydrolysis. Enzymatic method mainly involves the specific hydrolysis of chitin by chitinase, and the enzymes involved mainly include endochitinase, exochitinase, β-N-acetylhexosaminidase and deacetylase. Chitin can be obtained after enzymatic hydrolysis.

[0004] With the rapid development of genetic engineering, metabolic engineering and synthetic biology, recombinant microorganisms can directly biosynthesize GlcNAc using glucose as a substrate, and the product concentration can even exceed 100 g / L, which lays a good foundation for large-scale production of GlcNAc. The microbial fermentation method for producing glucosamine has the advantages of high conversion rate, high product concentration and short production cycle. However, at present, the raw materials for extracting glucosamine are mainly shrimp and crab shells and microbial fermentation broth. Whether it is fermentation broth or enzymatic hydrolysis product, after the reaction, the main product is obtained, accompanied by the generation of various by-products and unreacted residues. Therefore, for different raw materials, corresponding glucosamine extraction processes need to be developed. However, the extraction methods in the actual production process often have the disadvantages of complex process route, low separation efficiency, high energy consumption, and large environmental pollution.

[0005] When using acetylglucosamine-rich liquid or chitin hydrolysate as raw material, the first step of extraction is to remove the acetyl group in the molecule. The main methods for deacetylation are acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis consumes a large amount of inorganic acid, and a large amount of alkali solution needs to be added in the subsequent extraction process to neutralize the previously added inorganic acid solution, resulting in a large amount of salt in the extraction process. The consumption of acid and alkali in the extraction process is large, and a large amount of high-salt wastewater is generated. The enzymatic deacetylation method does not require the use of a large amount of acid and alkali solution, so it is more and more favored.

[0006] ZL2016112278411 (Publication No. CN 106831894 B) discloses a method for deacetyl coupling adsorption separation of D-glucosamine hydrochloride. The method uses acetylglucosamine fermentation broth as the starting material, removes microbial cells by ceramic membrane separation, and then removes residual salts in the culture medium by activated carbon decolorization and ion exchange resin. Acetylglucosamine is obtained, and then deacetylation and adsorption are realized at 91℃ by using an acid cation exchange column. The reaction time is about 120 min, and glucosamine hydrochloride is obtained after elution with hydrochloric acid. Since the reaction temperature involved exceeds 90℃, pigment substances are easily produced during processing, and ion exchange resins are easily broken and wasted.

[0007] ZL2013106719979 (Publication No. CN 103626809 B) discloses a method for purifying glucosamine hydrochloride mother liquor. The method uses glucosamine hydrochloride mother liquor as raw material, and adsorbs glucosamine on the cation column by using an acid cation exchange column. After eluting the cation column with hydrochloric acid solution, the obtained elution liquid is passed through an anion exchange column to remove acetic acid, chloride ions and other anions, thereby obtaining glucosamine instead of glucosamine hydrochloride. Since glucosamine cannot be stored for a long time, the method has limited industrial application value. SUMMARY

[0008] In view of the defects of high energy consumption and high pollution in the prior art, the present application provides a new method for producing, separating and purifying glucosamine salt. The method uses acetylglucosamine-rich fermentation broth or enzyme hydrolysate of chitin as raw material, and obtains glucosamine salt by cation exchange, and then obtains acetate by anion exchange and recovers unreacted acetylglucosamine. The method improves the yield of target product, and makes by-products resource utilization, reduces the consumption of raw and auxiliary materials, wastewater and solid waste discharge, and achieves the goals of energy saving and consumption reduction and environmental protection and safety. The method can also obtain high-purity crystals of various glucosamine salts by simply adjusting the type of acid eluent of cation exchange resin in industrial production scale.

[0009] The first object of the present application is to provide a method for producing and separating and purifying glucosamine salt, which comprises the following steps:

[0010] (1) taking a clear solution containing glucosamine as raw material, or, when the solution containing glucosamine is a turbid solution, filtering it with an ultrafiltration membrane, and taking the filtered clear solution containing glucosamine as raw material; the ultrafiltration membrane has a molecular weight cut-off of 5-200 kDa;

[0011] (2) adsorbing the solution containing glucosamine of step (1) with a cation exchange resin, so that the cation resin adsorbs glucosamine;

[0012] (3) eluting the cation exchange resin of step (2) with an acidic eluent, to obtain an eluate containing glucosamine salt;

[0013] adsorbing the under-column liquid of the cation exchange resin of step (2) with an anion exchange resin, so that the anion resin adsorbs acetate ions; and recycling the under-column liquid of the anion exchange resin containing acetylglucosamine for the preparation of glucosamine;

[0014] (4) eluting the anion adsorption resin with an alkaline eluent, and using the obtained eluate rich in sodium acetate for the denitrification and dephosphorization process of sewage treatment plants, or as raw material for chemical reactions, etc.

[0015] In one embodiment, the solution containing glucosamine of step (1) is the reaction product of N-acetylglucosamine after deacetylation by biological or chemical methods, or a solution containing glucosamine from other sources.

[0016] In one embodiment, the ultrafiltration membrane of step (1) can be a membrane assembly made of ceramic material, or a membrane assembly made of organic material.

[0017] In one embodiment, the solution containing glucosamine of step (1) is prepared by taking a solution containing N-acetylglucosamine as raw material, and using deacetylase extract or deacetylase preparation as catalyst for catalytic reaction.

[0018] In one embodiment, the recycling for the preparation of glucosamine of step (3) is used as the enzymatic raw material for the preparation of glucosamine, for deacetylation catalyzed by deacetylase.

[0019] The second object of the present application is to provide a method for preparing glucosamine salt, which comprises first enzymatically removing acetyl groups from a solution containing acetylglucosamine, and then separating and purifying according to the separation and purification method.

[0020] In one embodiment, the acetyl glucosamine-containing solution can be obtained by microbial fermentation, or by enzymatic hydrolysis of chitin-containing biological raw materials, or by chemical hydrolysis of chitin-containing raw materials.

[0021] In one embodiment, the enzymatic hydrolysis is carried out using an acetyl glucosamine solution with a concentration of 40-150 g / L as the raw material, and the deacetylase is added at a ratio of 10-40 U / g acetyl glucosamine; the pH range of the enzymatic reaction is 4-8, the reaction temperature is 25-55°C, and the stirring reaction time is 10-40 min; the acetyl glucosamine solution is obtained by microbial fermentation or chitin hydrolysis.

[0022] In one embodiment, the deacetylase can be derived from microorganisms and obtained by microbial fermentation, or extracted from other organisms; the microorganisms can be naturally screened microorganisms or genetically engineered recombinant microorganisms.

[0023] In one embodiment, the enzymatic hydrolysis is carried out by deacetylase to specifically remove the acetyl group in the N-acetyl glucosamine molecule, and the reaction produces an enzymatic hydrolysis product mainly composed of glucosamine and acetic acid.

[0024] In one embodiment, the method comprises the following steps:

[0025] (1) using an acetyl glucosamine solution with a concentration of 80-150 g / L as the raw material, and adding deacetylase at a ratio of 10-40 U / g acetyl glucosamine, the pH range of the enzymatic reaction is 4-8, the reaction temperature is 25-55°C, and the stirring reaction time is 10-90 min;

[0026] (2) the enzymatic hydrolysis product after step (1) can be directly used in step (3), or subjected to ultrafiltration membrane filtration to obtain an amino glucosamine-containing ultrafiltration membrane dialysis solution and a membrane concentrated solution, and the enzyme solution of the membrane concentrated solution is recycled to step (1) to participate in the next batch of enzymatic reaction process; the ultrafiltration membrane has a molecular weight cut-off of 5-200 kDa;

[0027] (3) using cation exchange resin to adsorb the amino glucosamine membrane dialysis solution obtained in step (2), and using an acidic eluent to continuously elute the cation exchange resin to obtain an elution solution containing amino glucosamine salt;

[0028] (4) using anion exchange resin to adsorb the effluent from the cation exchange resin in step (3), and using an alkaline eluent to elute the anion exchange resin, and the separated elution solution rich in sodium acetate can be used in the denitrification and dephosphorization process of a sewage treatment plant;

[0029] (5) The liquid under the column of step (4) is filtered by a nanofiltration membrane or a reverse osmosis membrane; the concentrated liquid of the nanofiltration membrane or the reverse osmosis membrane is recycled for the next batch of enzyme reaction process in step (1).

[0030] In an embodiment, the equipment for step (3) 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, sulfuric acid, phosphoric acid, pyruvic acid or citric acid; the corresponding glucosamine salt obtained by elution is glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate or glucosamine citrate, respectively; the concentration of the acidic eluent is 0.30-3.0 mol / L.

[0031] In an embodiment, the adsorption and elution temperature of step (3) cation exchange is 20-70℃; the adsorption and elution temperature of step (4) anion exchange is 20-65℃; the feed flow rate of the above two-stage ion exchange chromatography is 2.0-10.0 BV / h; the flow rate of the eluent is 1.0-8.0 BV / h.

[0032] In an embodiment, the equipment for step (4) anion exchange chromatography can be a fixed bed, a continuous ion exchange bed or an ion exchange simulated moving bed; the basic eluent can be NaOH solution or KOH solution; the concentration of the basic eluent is 0.30-3.0 mol / L; sodium acetate or potassium acetate can be recovered from the elution liquid after elution of the anion exchange column, which can be collected by pipeline and transported to the wastewater treatment workshop for use as a supplemental carbon source for denitrification and phosphorus removal in the wastewater treatment process, or as a raw material for other chemical reaction processes.

[0033] In an embodiment, the nanofiltration membrane of step (5) is a ceramic membrane, the pore size of the nanofiltration membrane is between 0.5-2 nm, and the operating pressure is 2-5 atm; the reverse osmosis membrane is an organic spiral 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.

[0034] In an embodiment, after step (5), concentration, crystallization and drying are sequentially performed.

[0035] In an embodiment, the concentration is evaporation concentration; the evaporation concentration can be single-effect evaporation, double-effect evaporation or multi-effect evaporation.

[0036] In an embodiment, the crystallization temperature is 5-40℃.

[0037] In one embodiment, the crystallization mother liquor is also subjected to decolorization; the decolorization method is activated carbon adsorption decolorization; the decolorized crystallization mother liquor is recycled back to the concentration process; in the decolorization method, the activated carbon dosage is 0.01-2.0% (w / v) of the raw material liquid.

[0038] In one embodiment, the drying is vacuum drying or flash drying; the flash drying has an air inlet temperature of 110-290°C and an air outlet temperature of 70-90°C; the vacuum low-temperature drying has a temperature of 40-80°C and a vacuum degree of 70-95 kPa.

[0039] In one embodiment, the multi-effect evaporation concentration is three-effect evaporation, and the temperatures are 80°C, 70°C and 60°C, respectively. The vacuum degree of the last-effect evaporator is 80-98 kPa.

[0040] Advantages:

[0041] Compared with the prior art, the present application has the following advantages:

[0042] (1) The present application realizes the enzymatic production and efficient purification of multiple 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 safety, and the product recovery rate is more than 95%, and the purity of the produced glucosamine salt can be more than 99.5%;

[0043] (2) The enzyme reaction conditions adopted by the present application are mild, and the reaction rate is efficient, and a recycling process of deacetylase is designed. The enzyme and the product are effectively separated by ultrafiltration membrane filtration, and on the other hand, the enzyme is recycled, thereby reducing the use cost of the enzyme;

[0044] (3) The present application forms a recycling process through two-stage ion exchange and reverse osmosis membrane / nanofiltration membrane combination, which effectively recycles the by-product acetate and the incompletely reacted substrate acetylglucosamine, effectively improves the conversion rate of the substrate and the extraction recovery rate of the product, and effectively reduces the consumption of resin and eluent in the ion exchange process, thereby achieving the triple benefits of low consumption, energy saving and environmental protection.

[0045] (4) The ion exchange process of the present application is carried out through continuous moving bed and simulated moving bed, which can improve the separation efficiency of ion exchange and improve the continuous and automatic operation level of the process;

[0046] (5) The ion exchange reaction conditions of the present application are mild, which can reduce the resin consumption by about 60% compared with the commonly used fixed bed, reduce the consumption of acid and alkali required for resin regeneration by about 50%, and greatly reduce the amount of wastewater generated.

[0047] (6) The process of the present application is suitable for the production of various glucosamine salts, and can obtain the corresponding acid glucosamine salt by changing the type of acidic eluent of the cation exchange resin, and has wide industrial application value. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 Extraction process route of glucosamine salt. DETAILED DESCRIPTION

[0049] Technical terms:

[0050] Glucosamine salt: refers to the glucosamine salt product obtained after eluting the cation exchange column with different acids, including but not limited to any one of glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate and glucosamine citrate.

[0051] Membrane dialysis liquid: liquid obtained by passing through the membrane material in the membrane filtration process.

[0052] Membrane concentrated liquid: liquid that cannot pass through the membrane material and is intercepted in the membrane filtration process.

[0053] Elution liquid: liquid obtained after the elution liquid (acid or alkali) flows through the saturated ion exchange resin in the ion exchange process.

[0054] Down-column liquid: liquid that is not adsorbed by the ion exchange resin and directly flows out and is washed out by deionized water in the raw material liquid in the ion exchange process.

[0055] The deacetylase activity unit is defined as: 1U = 1 mmol / min, that is, 1 mmol of glucosamine is obtained in 1 min reaction, which is 1U of enzyme activity unit.

[0056] Purification recovery rate of glucosamine salt: due to the enzyme reaction and ion exchange units involved in the production of glucosamine salt by enzyme method and its purification process, the molecular structure of the reactants will change. The recovery rate is calculated based on the mass of glucosamine.

[0057] The quantification of glucosamine salt and acetylglucosamine was performed by HPLC analysis. The liquid chromatograph was Agilent 1260 series, the chromatographic column was Thermo ODS-2 Hypersil C18 column (250 mm x 4.0 mm), and the sample of glucosamine salt to be tested was filtered through a 0.22 μm microfiltration membrane before being injected into the chromatographic column at a sample injection amount of 10 μl. The analysis of acetylglucosamine and acetic acid was performed by using a chromatographic column HPX-87H column (Bio-Rad, USA). The detector was a differential refractometer detector. The mobile phase was 5 mM H2SO4, the flow rate was 0.6 ml / min, and the detection temperature was 40 °C.

[0058] Example 1

[0059] According to the process route shown below, the operation steps are as follows: Figure 1

[0060] (1) Using an acetylglucosamine solution with a concentration of 40-150 g / L as the raw material, a deacetylase solution or deacetylase preparation is added at a proportion of 10-30 U / g acetylglucosamine, the pH range of the enzyme reaction is 4.0-8.0, the reaction temperature is 25-45 °C, and the stirring reaction is performed for 10-40 min;

[0061] This step specifically removes the acetyl group in the N-acetylglucosamine molecule, and the enzyme hydrolysate mainly containing glucosamine and acetic acid is obtained by the reaction, and the pH of the enzyme reaction is preferably 7.0-8.0;

[0062] (2) The enzyme hydrolysate after the reaction of step (1) is transported to an ultrafiltration membrane device, the molecular weight cut-off of the ultrafiltration membrane is 5-200 kDa, and preferably 5-30 kDa; the membrane concentrate is concentrated by water dialysis, and the ultrafiltration membrane dialysate containing glucosamine and the membrane concentrate containing deacetylase are collected respectively, and the recovery rate of deacetylase after the completion of membrane dialysis is 80-90%;

[0063] (3) The enzyme solution after the membrane concentration of step (2) is returned to the deacetylase solution tank to participate in the enzyme reaction process of the next batch;

[0064] The ultrafiltration membrane dialysate containing glucosamine obtained in step (2) is continuously pumped into the positive column filled with acidic resin (such as cation exchange resin with sulfonic acid group) in the simulated moving bed, and the flow rate of the feed is 2.0-10.0 BV / h, so that the glucosamine in the dialysate is adsorbed on the positive column;

[0065] The positive column is washed with deionized water to obtain the negative column effluent containing N-acetylglucosamine and acetic acid;

[0066] ​The positive column is continuously eluted with acid eluent such as hydrochloric acid, sulfuric acid, phosphoric acid, pyruvic acid, citric acid with a concentration of 0.30-3.0 mol / L, and the elution temperature is 20-70°C, preferably 25°C, to obtain an elution solution containing the corresponding glucosamine salt, which is used in the subsequent concentration, crystallization and drying processes;

[0067] (4) The under-column liquid of the positive column in step (3) is further fed into a negative column filled with basic anion exchange resin (for example, anion exchange resin with quaternary ammonium salt), and the feeding flow rate is 2.0-10.0 BV / h; the adsorption temperature and the elution temperature are controlled to be 20-65°C, preferably 25°C; and acetic acid is adsorbed on the negative column;

[0068] The negative column is washed with deionized water to obtain the under-column liquid of the negative column containing N-acetylglucosamine;

[0069] The negative column is continuously eluted with alkaline eluent such as 0.30-3.0 mol / L NaOH or KOH at a speed of 1.0-8.0 BV / h to separate an elution solution rich in acetate, which can be fed into a wastewater treatment workshop as a supplemental carbon source for the denitrification and phosphorus removal process, or can be used as a chemical raw material;

[0070] (5) The under-column liquid of the negative column in step (4) is fed into a nanofiltration membrane or reverse osmosis membrane concentration device;

[0071] The nanofiltration membrane is a ceramic membrane, and the pore size of the nanofiltration membrane is 0.5-2 nm, and the operating pressure is 2-5 atm;

[0072] The reverse osmosis membrane is an organic spiral membrane or a ceramic membrane, and the molecular weight cut-off of the reverse osmosis membrane is 50-100 Da, and the operating pressure is 4-10 atm;

[0073] The final concentration of N-acetylglucosamine after membrane concentration can reach 10-15% (w / v), and the membrane concentrated liquid is recycled to the N-acetylglucosamine storage tank for the enzyme reaction process in step (1) of the next batch;

[0074] (6) The elution solution of the positive column in step (3) is pumped into a multi-effect evaporator for evaporation concentration, and the multi-effect evaporation concentration is three-effect evaporation, and the gradient control temperature is used, for example, the temperatures of the first-effect, second-effect and third-effect evaporators are set to 80°C, 70°C and 60°C respectively, and the vacuum degree of the third-effect evaporator is 80-98 kPa;

[0075] (7) The material from step (6) is fed into a crystallizer, and the crystallization temperature is controlled at 5-40°C by controlling the jacket temperature of the crystallizer. The crystal suspension produced by the crystallizer is fed into a solid-liquid separation device for separation, and the crystallization mother liquor and the glucosamine salt slurry are obtained respectively. The separation device can be a centrifuge with continuous centrifugation function or a horizontal scraper unloading centrifuge;

[0076] (8) The crystallization mother liquor obtained in step (7) is decolorized by activated carbon, and then is fed back to the multi-effect evaporation device in step (6). The glucosamine salt slurry is fed into a drying device to obtain dried glucosamine salt crystal. The recovery rate of the concentration, crystallization and drying unit can reach 98%.

[0077] Example 2

[0078] According to the process route shown, the operation steps are as follows: Figure 1

[0079] (1) Collect 350 m 3 of acetyl glucosamine solution with a concentration of 102 kg / m 3 in a storage tank, pump the acetyl glucosamine solution into an enzyme reaction tank, and add deacetylase liquid at a ratio of 10-25 U / g acetyl glucosamine. The pH of the enzyme reaction is 7.0-8.0, the reaction temperature is 37°C, and the stirring reaction is carried out for 30 min.

[0080] (2) Pump the enzyme hydrolysate after the reaction in step (1) into an ultrafiltration membrane device, and the molecular weight cut-off of the ultrafiltration membrane is 5000 Da. Add 60 m 3 of pure water for dialysis membrane concentrate, and collect the ultrafiltration membrane dialysate and the membrane concentrate respectively. A total of 380 m 3 of glucosamine dialysate with a content of 56.3 kg / m 3 is obtained. 30 m 3 of the membrane concentrate is returned to the deacetylase liquid storage tank to participate in the enzyme reaction process of the next batch.

[0081] (3) Continuously pump the glucosamine dialysate obtained in step (2) into a simulated moving bed filled with 001 x 7 strong acid styrene resin, and the feed flow rate is 4.0 BV / h. The temperature of the feed and elution is 25°C. The glucosamine in the dialysate is adsorbed on the column, and the column is washed with deionized water to obtain a column effluent containing neutral sugar and acetic acid.

[0082] The column is continuously eluted with 2 mol / L hydrochloric acid solution, and the elution flow rate is 3.0 BV / h, thereby obtaining 91.7 m 3 of glucosamine hydrochloride with a concentration of 252 kg / m 3 . ​

[0083] (4) The lower column liquid of the positive column in step (3) is further transported to the negative column filled with anion exchange resin in a simulated moving bed at a flow rate of 4.0 BV / h, and the negative column is washed with deionized water to obtain a negative column lower column liquid with a N-acetyl glucosamine concentration of about 3%;

[0084] The negative column is continuously eluted with 1.5 mol / L NaOH solution, and 80.6 m 3 of sodium acetate (107.5 kg / m 3 ) enriched eluate is separated; wherein the negative column filler is 201x7 strong basic styrene resin;

[0085] (5) The 514.6 m 3 lower column liquid of the negative column in step (4) is transported to a nanofiltration membrane concentration device, the pore size of the nanofiltration membrane is 1 nm, and the operating pressure is 0.5-1.0 MPa, and a total of 89.3 m 3 of N-acetyl glucosamine concentration of 125 g / L nanofiltration membrane concentrated liquid is concentrated; the nanofiltration membrane concentrated liquid is recycled and recovered to the acetyl glucosamine storage tank for use in the enzyme reaction process of the next batch;

[0086] 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%.

[0087] The 89.3 m 3 concentrated liquid obtained by nanofiltration is recycled to the acetyl glucosamine solution storage tank, and the enzyme liquid of the membrane concentrated liquid in step (2) is returned to the enzyme reaction tank for the second cycle reaction, and 45 m 3 of glucosamine hydrochloride solution containing 238 kg / m 3 is recovered, and the total recovery rate of glucosamine hydrochloride reaches 97%.

[0088] Continue the foregoing process, and the eluate of the glucosamine hydrochloride obtained in step (3) of the first batch and the second batch is combined and concentrated, crystallized and dried, as follows:

[0089] (6) The combined solution containing glucosamine hydrochloride is pumped into a three-effect evaporator, the feed flow rate is controlled to be 6 m 3 / h, the vacuum degree of the last effect condenser is 90 kPa; the cooling water inlet temperature is 8-15℃, and the product concentration of the outlet is 720 g / L;

[0090] (7) The outlet of the three-effect evaporator flows into a crystallizer, and the crystallization temperature is controlled at 40℃ by controlling the jacket temperature of the crystallizer. The crystal suspension produced by the crystallizer is sent into a horizontal screw centrifuge for separation, and crystalline mother liquor and glucosamine hydrochloride crystal mud are separated;

[0091] (8) The crystallization mother liquor is sent to an activated carbon decoloring column at a flow rate of 0.5 m 3 / h for decoloring, and after decoloring, is returned to the storage tank before the three-effect evaporation device; the glucosamine hydrochloride crystal slurry obtained by the horizontal screw centrifuge separation is sent to a flash dryer by a screw conveyor, the inlet air temperature of the flash drying is 150°C, and the outlet air temperature is 80°C, to obtain glucosamine hydrochloride crystals.

[0092] The total recovery rate of the concentration, crystallization and drying units of steps (6)-(8) can reach 98%.

[0093] In the entire production process, 500 kg of concentrated hydrochloric acid solution, 550 L of 30% NaOH solution, 10 tons of pure water are consumed, and 9.7 tons of wastewater is generated, and about 10 kg of cation exchange resin and anion exchange resin are lost for producing 1 ton of glucosamine hydrochloride.

[0094] Example 3

[0095] According to the process route shown in Figure 1 , the difference is that, on the basis of Example 2, the nanofiltration membrane concentration device is replaced by a reverse osmosis ceramic membrane device, that is, the anion column underflow liquid obtained in step (4) is collected to the reverse osmosis ceramic membrane device, the pore size of the reverse osmosis membrane is 1 nm, and the operating pressure is 0.5-1.0 MPa, and the liquid concentrated by the reverse osmosis ceramic membrane device is returned to the enzyme reaction tank to participate in the enzyme reaction process of the next batch.

[0096] In this production process, 11 tons of pure water are consumed, and 10.5 tons of wastewater is generated for producing 1 ton of glucosamine hydrochloride.

[0097] Example 4

[0098] According to the process route shown in Figure 1 , the difference is that, on the basis of Example 2, sulfuric acid solution is used instead of hydrochloric acid solution in step (3), that is, 1 mol / L sulfuric acid solution is used to elute the glucosamine adsorbed on the anion column in step (3), and the product obtained is glucosamine sulfate.

[0099] Example 5

[0100] According to the process route shown in Figure 1 , the difference is that, on the basis of Example 2, phosphoric acid solution is used instead of hydrochloric acid solution in step (3), that is, 1 mol / L phosphoric acid solution is used to elute the glucosamine adsorbed on the anion column in step (3), and the product obtained is glucosamine phosphate.

[0101] Example 6

[0102] According to the process route shown in Figure 1The process route shown is different in that, based on Example 2, citric acid solution is used instead of hydrochloric acid solution in step (3), that is, 1 mol / L citric acid solution is used in step (3) to elute the glucosamine adsorbed on the positive column, and the obtained product is glucosamine citrate.

[0103] Example 7

[0104] according to Figure 1 The process route shown is different in that, based on Example 2, pyruvic acid solution is used instead of hydrochloric acid solution in step (3), that is, 2 mol / L pyruvic acid solution is used in step (3) to elute the glucosamine adsorbed on the positive column, and the obtained product is glucosamine pyruvate.

[0105] Example 8

[0106] according to Figure 1 The process route shown in Example 2 differs in that the simulated moving bed in step (3) is replaced by a continuous moving bed apparatus. The fillers for the positive and negative columns remain unchanged, with the positive column filler being 0.01×7 strongly acidic styrene-based cationic resin and the negative column filler being 2.01×7 strongly basic styrene-based anionic resin. The production of 1 ton of product requires 16 kg of cation exchange resin and 14 kg of anion exchange resin, 600 kg of concentrated hydrochloric acid, and 650 L of 30% sodium hydroxide solution, generating 15 tons of wastewater.

[0107] Example 9

[0108] according to Figure 1 The process route shown in Example 2 differs in that the simulated moving bed in step (3) is replaced by a fixed-bed ion exchange apparatus, the positive column is filled with 201×7 strongly basic styrene-based anion exchange resin, and the negative column is filled with 001×7 strongly acidic styrene-based cation exchange resin. Each ton of product produced consumes 30 kg of cation exchange resin and 28 kg of anion exchange resin, 1300 kg of concentrated hydrochloric acid and 1400 L of 30% sodium hydroxide solution, and generates 50 tons of wastewater.

[0109] Example 10

[0110] according to Figure 1 The process route shown in the figure is different in that, based on Example 4, the triple-effect evaporation concentration and crystallization are omitted, and the desorption solution rich in glucosamine sulfate eluted from the simulated moving bed column is directly transported to the spray drying equipment with a feed flow rate of 5m 3 / h, and the inlet air temperature of the spray drying was 150°C. After drying, 41.5 tons of glucosamine sulfate powder was obtained, and the product purity reached 99%.

[0111] Example 11

[0112] according to Figure 1 The process route shown is different in that, based on Example 2, the enzyme reaction solution obtained in step (1) is continuously pumped into the positive column described in step (3), the feed flow rate is 3.0 BV / h, the feed and elution temperatures are 30°C, and the subsequent steps are the same as in Example 2, and the obtained product is glucosamine hydrochloride.

[0113] Comparative Example 1

[0114] Referring to the method for separating D-glucosamine hydrochloride in the patent application number CN2016112278411, 50m 3 The concentration is 102kg / m 3 The acetyl glucosamine solution was heated to 95°C by steam, and then pumped into a column filled with a strong acidic cationic resin. The temperature was maintained at 90°C, and the acetyl glucosamine and cationic resin were reacted for 240 minutes. After the reaction, pure water was passed to wash the cationic resin, and 40m of washing liquid containing acetic acid was collected. 3 After washing, 12% hydrochloric acid was passed through the cation exchange column, and the flow rate of the elution process was 1.5BV / h. 42m 3 The concentration of glucosamine hydrochloride in the analytical solution is 103 kg / m 3 , the yield of this step is 88.6%. 0.2% activated carbon is added to the analytical solution for decolorization, and then pumped into a triple-effect evaporation concentrator. After crystallization, centrifugation and drying, 4410 kg of glucosamine hydrochloride is obtained, with a total recovery rate of 80.2%. Due to the high reaction temperature and long reaction time of acetylglucosamine and cationic resin, pigments are easily produced during the reaction process, and the loss of ion exchange resin is increased. The production of each ton of glucosamine hydrochloride consumes 1100 kg of concentrated hydrochloric acid, 120 kg of cationic resin, and 30 m 3 Wastewater with high inorganic acid content and high COD cannot recover acetic acid during the production process.

[0115] Table 1 Extraction effect of glucosamine salt using different cation exchange methods

[0116]

[0117] The inventors also tried to adjust the process parameters of enzymatic hydrolysis, separation and purification. Within the range of the preferred parameters of Example 1, the product recovery rate of the ion exchange unit can reach 99%, the filler loss is controlled within the range of 20kg / ton, the acid solution dosage is controlled within the range of 600kg / ton product, and the wastewater generation is less than 10m 3 / ton of product effect.

[0118] Comparative Example 2

[0119] Referring to the method disclosed in CN2013106719979, the difference is that the raw material is replaced by an enzyme reaction solution containing glucosamine, and the operation step of transporting the eluate of the cation exchange column to the anion exchange column is omitted. The collected deacetylase reaction solution and the membrane dialysis solution after ultrafiltration membrane dialysis are 50m 3 (contains glucosamine and acetic acid) in the storage tank, the concentration of glucosamine is 89kg / m 3 The solution is pumped into a strong acid cation exchange column filled with cation resin, the temperature is maintained at 32℃, and the glucosamine is adsorbed on the cation resin. Pure water is introduced to wash the cation resin, and 60m 3 of the lower column liquid containing acetic acid is collected. After washing, 0.3mol / L hydrochloric acid solution is introduced to elute the cation exchange column, and the flow rate during the elution process is 1.5BV / h. 31m 3 of the solution containing 126kg / m 3 of glucosamine hydrochloride is collected, and the yield is 87.8%. The obtained eluate is heated to 60℃, 1% powdered activated carbon is added for decolorization, and 30.5m 3 of the filtrate containing 125kg / m 3 of glucosamine hydrochloride solution is collected, and then pumped into a three-effect evaporation concentrator, and then crystallized, centrifuged and dried to obtain 3585kg of glucosamine hydrochloride crystals, which are slightly yellow, and the total yield is 80.6%. This method first decolorizes and then concentrates, and the consumption of activated carbon is large, 900kg of concentrated hydrochloric acid and 20kg of cation resin are consumed per ton of glucosamine hydrochloride, and 30m 3 of wastewater with high COD content is generated.

[0120] Although the present application has been disclosed with reference to the preferred embodiments above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. Enzymatic production of glucosamine salts and methods for their purification, characterized in that, The method comprises: The method comprises: The method comprises: The method comprises: The method comprises:

2. The method of claim 1, wherein, The method comprises: The method comprises: The method comprises:

3. The method of claim 1, wherein, The method comprises:

4. The method of claim 1, wherein, The method comprises:

5. The method of claim 1, wherein, The method comprises:

6. The method according to any one of claims 1 to 5, characterized in that, The method comprises:

7. The method of claim 6, wherein, The method comprises: The method comprises: The method comprises: The method comprises:

8. The method of claim 7, wherein, The method comprises:

9. The method of claim 8, wherein, The method comprises:

10. The method of claim 9, wherein, The method comprises:

11. The method according to any one of claims 1 to 10, characterized in that, The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: The method comprises: (3) using cation exchange resin to adsorb the ultrafiltration membrane dialysate obtained in step (2), and using acid eluent to continuously elute the cation exchange resin, so as to obtain an elution liquid containing glucosamine salt; using deionized water to clean the cation exchange resin, so as to obtain a column effluent containing N-acetylglucosamine and acetic acid; (4) using anion exchange resin to adsorb the column effluent of the cation exchange resin in step (3), and using alkaline eluent to elute the anion exchange resin, so as to separate an elution liquid containing acetate; (5) recycling the column effluent of the anion exchange resin in step (4) to the enzyme reaction process in step (1) for the next batch of enzyme reaction process; or first concentrating the column effluent of the anion exchange resin in step (4), and then recycling it to the enzyme reaction process in step (1) for the next batch of enzyme reaction process; the concentration method includes vacuum concentration, nanofiltration membrane, reverse osmosis membrane filtration concentration or multi-effect evaporation concentration.

12. The method of claim 11, wherein, In step (5), the nanofiltration membrane is a ceramic membrane with a pore size of 0.5-2 nm; the reverse osmosis membrane is an organic spiral membrane or a ceramic membrane.

13. Use of the method according to any one of claims 1-12 in the preparation of glucosamine salt derivative products or products containing glucosamine salt.

Images