阅读说明：本技术 酶法生产氨基葡萄糖盐及其提纯方法 (Enzyme method for producing glucosamine salt and purification method thereof ) 是由 丁春华 章文劼 于 2020-06-11 设计创作，主要内容包括：本发明公开了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。本发明以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。本发明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。(The invention discloses a glucosamine salt produced by an enzyme method and a purification method thereof, belonging to the technical field of biological engineering. The invention takes N-acetyl glucosamine as raw material, glucosamine and acetic acid are obtained by hydrolysis of deacetylase, glucosamine salt is obtained by separation of cation exchange column eluted by acid solution, and byproduct sodium acetate is obtained by anion exchange recovery. The obtained glucosamine salt is concentrated, crystallized, decolored and dried to obtain the glucosamine salt crystal with high purity. The method combines the enzyme recycling process, the residual substrate recycling process and the acetic acid recycling process, improves the conversion rate of N-acetylglucosamine and the total yield of glucosamine salt products, recycles the enzyme, recycles the residual substrate, recycles the acetic acid, has low resin loss rate and extremely low hydrochloric acid waste liquid generation amount through the operation condition at normal temperature, and realizes the effects of energy conservation, consumption reduction, economic benefit, environmental protection and safety.)

1. The method for producing glucosamine salt by an enzyme method and the purification method thereof are characterized in that the method comprises the following steps:

taking a solution containing acetylglucosamine as a raw material, and carrying out catalytic hydrolysis by adopting deacetylase or a preparation containing the deacetylase to obtain an enzyme hydrolysate;

directly using the enzymatic hydrolysate for cation exchange treatment; or, filtering the enzyme hydrolysate with a membrane, recovering the deacetylase from the obtained membrane retentate, and using the obtained membrane dialysate for cation exchange treatment;

carrying out cation exchange and elution by acidic eluent on the enzymatic hydrolysate or membrane dialysate to obtain glucosamine salt;

and (3) eluting the cation column lower column liquid in the cation exchange process by anion exchange and alkaline eluent, recovering acetate, concentrating the anion column lower column liquid in the anion exchange process, and circularly sending the concentrated anion column lower column liquid back to the catalytic hydrolysis process in which the deacetylase participates.

2. The method according to claim 1, wherein the membrane is a ceramic membrane module or an organic membrane module; the molecular weight cut-off of the ultrafiltration membrane is 5-200 kDa.

3. The method of claim 1, wherein the resin used for the cation exchange includes, but is not limited to, cation exchange resins with sulfonic acid groups;

the acidic eluent can be hydrochloric acid, sulfuric acid, phosphoric acid, pyruvic acid or citric acid with the concentration of 0.3-4.0 mol/L.

4. The method of claim 1, wherein the resin used for the anion exchange includes, but is not limited to, anion exchange resins with quaternary ammonium groups;

the alkaline eluent can be NaOH solution or KOH solution with the concentration of 0.30-3.0 mol/L.

5. A method according to claim 3 or 4, characterized in that the apparatus for cation exchange and/or anion exchange is a fixed bed ion exchange bed, a continuous moving bed ion exchange bed or a simulated moving bed ion exchange bed;

optionally, the adsorption or elution temperature in the ion exchange process is 20-75 ℃, and the feed flow rate is 2.0-10.0 BV/h; the flow rate of the eluent is 1.0-8.0 BV/h.

6. The method according to any one of claims 1 to 5, wherein the glucosamine salt obtained is concentrated, crystallized and dried to obtain a glucosamine salt with high purity.

7. The method of claim 6, wherein the concentration is optionally by evaporative concentration;

the evaporation concentration can be single-effect evaporation concentration or multi-effect evaporation concentration, and the vacuum degree of a last-effect evaporator is 80-98 kPa;

the crystallization temperature is 5-40 ℃;

the drying can be vacuum low-temperature drying or flash evaporation drying; the temperature of the vacuum low-temperature drying is 40-80 ℃, and the vacuum degree is 70-95 kPa; the temperature of the hot air for flash drying is 120-300 ℃;

optionally, decolorizing the crystallization mother liquor; the decoloring method is activated carbon adsorption decoloring; the decolored crystallization mother liquor is recycled for the concentration process;

optionally, in the decoloring method, the amount of the activated carbon is 0.01-2% of the raw material liquid; alternatively, the activated carbon may be a carbon rod, a carbon column, or granular activated carbon.

8. A method according to any one of claims 1 to 7, comprising the steps of:

(1) taking an acetylglucosamine solution with the concentration of 40-150g/L as a raw material, adding a preparation containing deacetylase, and carrying out an enzyme reaction at the pH range of 4-8 and the reaction temperature of 25-55 ℃ for 10-90min under stirring;

(2) filtering the enzymatic hydrolysate reacted in the step (1) by using an ultrafiltration membrane or a nanofiltration membrane to obtain ultrafiltration membrane dialysate and membrane concentrate containing glucosamine; recycling the enzyme solution of the membrane concentrated solution in the step (1) to participate in the enzyme reaction of the next batch or to be discarded;

(3) adsorbing the ultrafiltration membrane dialysate obtained in the step (2) by using cation exchange resin, and continuously eluting the cation exchange resin by using acidic eluent to obtain an analytic solution containing glucosamine salt; washing the cation exchange resin with deionized water to obtain cation column lower liquid containing N-acetylglucosamine and acetic acid;

(4) adsorbing the lower column liquid of the positive column which passes through the positive ion exchange resin in the step (3) by using an anion exchange resin, and eluting the anion exchange resin by using an alkaline eluent to separate an analytic liquid containing acetate;

(5) recycling the anion column lower column liquid which passes through the anion exchange resin in the step (4) for the enzyme reaction process of the next batch in the step (1); or concentrating the lower column liquid of the anion exchange resin in the step (4) and recycling the lower column liquid for the enzyme reaction process of the next batch in the step (1); the concentration method can be vacuum concentration, nanofiltration membrane or reverse osmosis membrane filtration concentration, and also can be multi-effect evaporation concentration.

9. The method as claimed in claim 8, wherein in the 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 roll-type membrane or a ceramic membrane.

10. Use of the method according to any one of claims 1 to 9 for the preparation of glucosamine salt derived products or glucosamine salt containing products.

Technical Field

The invention relates to an enzyme method for producing glucosamine salt and a purification method thereof, belonging to the technical field of biological engineering.

Background

Glucosamine (GlcNAc) is an important hexosamine formed by the substitution of one hydroxyl group of glucose with an amino group, and is readily soluble in water and hydrophilic solvents. Widely existing in nature, chemical name is: 2-amino-2-deoxy-D-glucose is usually present in polysaccharides of microbial, animal origin and in conjugated polysaccharides in the form of N-acetyl derivatives, such as chitin, or N-sulfate esters and N-acetyl-3-O-lactate ether (muramic acid). Glucosamine hydrochloride, with a molecular weight of 215.5Da, is white crystal, odorless, slightly sweet, easily soluble in water, slightly soluble in methanol, and insoluble in organic solvents such as ethanol. Glucosamine molecules are not very stable and are easily oxidized or degraded. Can be prepared into glucosamine salt, such as glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate and the like, and the stability of the glucosamine salt can be obviously improved. Glucosamine has important physiological functions for human bodies, participates in detoxification of liver and kidney, plays roles of resisting inflammation, protecting liver and tonifying kidney, has good curative effect on treating rheumatic arthritis and gastric ulcer, is a main raw material for synthesizing antibiotics and anticancer drugs, and can also be applied to food, cosmetics and feed additives.

Currently, there are three main types of GlcNAc production methods: chemical, enzymatic and microbiological methods. Natural raw materials such as shrimp and crab shells and fungal cell walls contain abundant chitin, and glucosamine monomers can be obtained by acid hydrolysis or enzyme hydrolysis. The enzyme method is mainly characterized in that chitin is specifically hydrolyzed by chitinase, and related enzymes mainly comprise endo-chitinase, exo-chitinase, beta-N-acetylhexosaminidase and deacetylase. The glucosamine monomer can be obtained by hydrolyzing chitin with enzyme.

With the rapid development of genetic engineering, metabolic engineering and synthetic biology, the recombinant microorganism can be used for directly biosynthesizing the GlcNAc by taking glucose as a substrate, and the product concentration can even exceed 100g/L, thereby laying a good foundation for the large-scale production of the GlcNAc. The glucosamine produced by the microbial fermentation method has the advantages of high conversion rate, high product concentration, short production period and the like. However, the raw materials for extracting glucosamine mainly comprise shrimp and crab shells and microbial fermentation broth, and the fermentation broth or enzyme hydrolysate is accompanied by the generation of various byproducts and residues which are not completely reacted while obtaining a main product after the reaction. Therefore, the corresponding glucosamine extraction process needs to be developed for different raw materials. However, the extraction method in the actual production process at present has the defects of complex process route, low separation efficiency, high energy consumption, large environmental pollution and the like.

When liquid rich in acetylglucosamine or chitin hydrolysate is used as raw material, the removal of acetyl groups from the molecule is the first step of extraction, and the deacetylation method mainly comprises acid hydrolysis method and enzyme hydrolysis method. Acid hydrolysis consumes a large amount of inorganic acid, and a large amount of alkali liquor is required to be added in the subsequent extraction process to neutralize the inorganic acid liquor added before, so that a large amount of salt is generated in the extraction process. The acid and alkali consumption in the extraction process is large, and a large amount of high-salinity wastewater which is difficult to treat is generated. On the other hand, the method of enzymatic deacetylation is more and more favored because it does not require the use of a large amount of an acid or alkali solution.

ZL2016112278411 (publication No. CN 106831894B) discloses a method for separating D-glucosamine hydrochloride by deacetylation coupling adsorption, which comprises taking acetylglucosamine fermentation broth as a starting material, removing microbial thallus by ceramic membrane separation, decolorizing with active carbon, removing residual salt in culture medium with ion exchange resin to obtain acetylglucosamine, performing deacetylation reaction and adsorption at 91 deg.C with acidic cation exchange column for about 120min, and eluting with hydrochloric acid to obtain glucosamine hydrochloride. Because the reaction temperature exceeds 90 ℃, pigment substances are easy to generate in the treatment process, and the ion exchange resin is easy to break and lose.

ZL2013106719979 (publication No. CN 103626809B) discloses a purification method of glucosamine hydrochloride mother liquor, which takes the glucosamine hydrochloride mother liquor as a raw material, and makes glucosamine adsorbed on an anode column through an acidic cation exchange column. After the positive column is eluted by hydrochloric acid solution, the obtained analytic solution passes through an anion exchange column again to remove anions such as acetic acid, chloride ions and the like, so that glucosamine is obtained instead of glucosamine hydrochloride. Since glucosamine cannot be stably stored for a long period of time, the method has a limited industrial application value.

Disclosure of Invention

Aiming at the defects of high energy consumption, high pollution and the like in the prior art, the invention provides a novel glucosamine salt production, separation and purification method, which takes fermentation liquor rich in acetylglucosamine or enzyme hydrolysis liquid of chitin as a raw material, obtains acetate by anion exchange on the basis of obtaining glucosamine salt by cation exchange, and recovers unreacted acetylglucosamine completely. The method can also obtain corresponding glucosamine salt by simply adjusting the type of the acidic eluent of the cation exchange resin, and extract and obtain high-purity crystals of various glucosamine salts in industrial production scale.

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

(1) taking a clear solution containing glucosamine as a raw material, and optionally, when the solution containing glucosamine is a turbid solution, filtering by using an ultrafiltration membrane, and taking the filtered clear solution containing glucosamine as the raw material; the molecular weight cut-off of the ultrafiltration membrane is 5-200 kDa;

(2) adsorbing the glucosamine-containing solution obtained in the step (1) by using a cation exchange resin, so that the glucosamine is adsorbed by the cation exchange resin;

(3) eluting the cation exchange resin in the step (2) by using an acidic eluent to obtain an analytic solution containing glucosamine salt;

adsorbing the solution passing through the cation column of the cation exchange resin in the step (2) by using anion exchange resin to make the anion resin adsorb acetate ions; recycling the anion column lower column liquid containing the acetylglucosamine which passes through the anion exchange resin for preparing the glucosamine;

(4) the anion adsorption resin is eluted by alkaline eluent, and the obtained desorption solution is rich in sodium acetate, can be used for the nitrogen and phosphorus removal process of a sewage treatment plant, and can also be used as a raw material of chemical reaction and other suitable purposes.

In one embodiment, the glucosamine-containing solution in step (1) is a reaction product of N-acetylglucosamine which has been deacetylated by a biological method or a chemical method, and can also be a glucosamine-containing solution from other sources.

In one embodiment, the ultrafiltration membrane in step (1) may be a ceramic membrane module or an organic membrane module.

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

In one embodiment, the recycling of glucosamine in step (3) is used as a raw material for the enzymatic hydrolysis of glucosamine, and is used for deacetylation reaction catalyzed by deacetylase.

The second purpose of the invention is to provide a preparation method of glucosamine salt, which comprises the steps of firstly carrying out enzymolysis on a solution containing acetylglucosamine to remove acetyl, and then carrying out separation and purification according to the separation and purification method.

In one embodiment, the acetylglucosamine-containing solution may be obtained by microbial fermentation, enzymatic hydrolysis of a biological material containing chitin, or chemical hydrolysis of a material containing chitin.

In one embodiment, the enzymolysis is to take an acetylglucosamine solution with the concentration of 40-150g/L as a raw material, and add deacetylase according to the proportion of 10-40U/g acetylglucosamine; the pH range of the enzymolysis reaction is 4-8, the reaction temperature is 25-55 ℃, and the stirring reaction is carried out for 10-40 min; the acetylglucosamine solution is a raw material solution containing acetylglucosamine obtained by microbial fermentation or chitin hydrolysis.

In one embodiment, the deacetylase can be derived from a microorganism and obtained by fermentation of the microorganism, or can be extracted from other organisms; the microorganism may be a microorganism screened in nature, or a recombinant microorganism engineered by genetic engineering.

In one embodiment, the enzymatic hydrolysis is carried out by an enzymatic reaction with a deacetylase to specifically remove acetyl groups from the N-acetylglucosamine molecule, resulting in an enzymatic hydrolysate containing glucosamine and acetic acid as major components.

In one embodiment, the method comprises the steps of:

(1) taking an acetylglucosamine solution with the concentration of 80-150g/L as a raw material, adding deacetylase according to the proportion of 10-40U/g of acetylglucosamine, wherein the pH range of the enzymatic reaction is 4-8, the reaction temperature is 25-55 ℃, and stirring for reaction for 10-90 min;

(2) directly performing the step (3) on the enzymatic hydrolysate reacted in the step (1), or performing ultrafiltration membrane filtration to respectively obtain ultrafiltration membrane dialysate and membrane concentrate containing glucosamine, and reusing the enzyme solution of the membrane concentrate in the step (1) to participate in the enzyme reaction process of the next batch; the molecular weight cut-off of the ultrafiltration membrane is 5-200 kDa;

(3) adsorbing the glucosamine membrane dialysate obtained in the step (2) by using cation exchange resin, and continuously eluting the cation exchange resin by using acidic eluent to obtain an analytic solution containing glucosamine salt;

(4) adsorbing the lower column liquid of the cation exchange resin passing through the cation exchange resin in the step (3) by using anion exchange resin, eluting the anion exchange resin by using alkaline eluent, and using the separated sodium acetate-rich desorption liquid for a nitrogen and phosphorus removal process of a sewage treatment plant;

(5) filtering the lower column liquid of the negative column in the step (4) by using a nanofiltration membrane or a reverse osmosis membrane; and (3) recycling the concentrated solution of the nanofiltration membrane or the reverse osmosis membrane for the next batch of enzyme reaction process in the step (1).

In one embodiment, the device used in step (3) for cation exchange chromatography may be a fixed bed, an ion exchange continuous bed, or an ion exchange simulated moving bed; the acid eluent can be hydrochloric acid, sulfuric acid, phosphoric acid, pyruvic acid or citric acid; the corresponding glucosamine salts obtained by elution are glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate and glucosamine citrate respectively; the concentration of the acidic eluent is 0.30-3.0 mol/L.

In one embodiment, the adsorption and elution temperatures for the cation exchange of step (3) are 20-70 ℃; the adsorption temperature and the elution temperature of the anion exchange in the step (4) are 20-65 ℃; the feeding flow rate of the 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.

In one embodiment, the device used in step (4) for anion exchange chromatography may be a fixed bed, an ion exchange continuous bed, or an ion exchange simulated moving bed; the alkaline eluent can be NaOH solution or KOH solution, and the concentration of the alkaline eluent is 0.30-3.0 mol/L; sodium acetate or potassium acetate can be respectively recovered from the desorption solution after the anion exchange column is eluted, and the sodium acetate or the potassium acetate can be collected by a pipeline and conveyed to a sewage treatment workshop for supplementing a carbon source for nitrogen and phosphorus removal in the sewage treatment process and also can be used as a raw material in other chemical reaction processes.

In one embodiment, the nanofiltration membrane in the step (5) is a ceramic membrane, the pore diameter of the nanofiltration membrane is between 0.5 and 2nm, and the operating pressure is 2 to 5 atm; the reverse osmosis membrane is an organic roll-type membrane or a ceramic membrane, the molecular weight cut-off of the reverse osmosis membrane is 50-100Da, and the operating pressure is 4-10 atm.

In one embodiment, the step (5) is further followed by concentrating, crystallizing and drying in this order.

In one embodiment, the concentration is evaporative concentration; the evaporation concentration can be single-effect evaporation, double-effect evaporation or multi-effect evaporation.

In one embodiment, the temperature of the crystallization is from 5 to 40 ℃.

In one embodiment, the crystallization mother liquor is also decolorized; the decoloring method is activated carbon adsorption decoloring; the decolored crystallization mother liquor is recycled for the concentration process; in the decoloring method, the using amount of the active carbon is 0.01-2.0% (w/v) of the raw material liquid.

In one embodiment, the drying is vacuum drying or flash drying; the air inlet temperature of the flash drying is 110-290 ℃, and the air outlet temperature is 70-90 ℃; the temperature of the vacuum low-temperature drying is 40-80 ℃, and the vacuum degree is 70-95 kPa.

In one embodiment, the multi-effect evaporation is concentrated to three-effect evaporation at 80 ℃, 70 ℃ and 60 ℃. The vacuum degree of the last effect evaporator is 80-98 kPa.

Has the advantages that:

compared with the prior art, the invention has the following advantages:

(1) the invention realizes the enzyme production and high-efficiency purification of various glucosamine salts in industrial production scale. The production process has the advantages of high conversion rate, high recovery rate, low raw material consumption, environmental protection and safety, the product recovery rate is over 95 percent, and the purity of the produced glucosamine salt can be over 99.5 percent;

(2) the enzyme reaction conditions adopted by the invention are mild, the reaction rate is high, and a recycling process of the deacetylase is designed, so that the enzyme and the product are effectively separated on one hand, the enzyme is recycled on the other hand, and the use cost of the enzyme is reduced;

(3) according to the invention, through the combination of two-stage ion exchange and the reverse osmosis membrane/nanofiltration membrane, a circulation process is formed, so that the byproduct acetate and the incompletely reacted substrate acetylglucosamine are effectively recycled, the conversion rate of the substrate and the extraction recovery rate of the product are effectively improved, 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.

(4) The invention carries out the ion exchange process through the continuous moving bed and the simulated moving bed, and can improve the separation efficiency of the ion exchange and simultaneously improve the continuity and automation level of the process operation;

(5) the ion exchange reaction condition of the invention is mild, compared with the common fixed bed, the consumption of the resin can be reduced by about 60 percent, the consumption of the acid liquor and the alkali liquor required by the resin regeneration can be reduced by about 50 percent, and the generation amount of the waste water can be greatly reduced.

(6) The process is suitable for the production of various glucosamine salts, can obtain the glucosamine salt corresponding to acid by changing the type of the acidic eluent of the cation exchange resin, and has wide industrial application value.

Drawings

FIG. 1 is a process scheme for the extraction of glucosamine salts.

Detailed Description

Technical terms:

glucosamine salts: refers to glucosamine salt products obtained after different acid washing and cation exchange column removal, including but not limited to any one of glucosamine hydrochloride, glucosamine sulfate, glucosamine phosphate, glucosamine pyruvate and glucosamine citrate.

Membrane dialysate: the liquid obtained during membrane filtration permeates the membrane material.

Membrane concentrate: the liquid which cannot permeate the membrane material to be trapped in the membrane filtration process.

Analysis solution: in the ion exchange process, the eluent (acid solution or alkali solution) flows through the saturated ion exchange resin to obtain liquid.

Column liquid: in the ion exchange process, the raw material liquid directly flows out without being adsorbed by ion exchange resin and is washed out by deionized water.

The deacetylase activity unit is defined as: 1U is 1mmol/min, namely 1min reaction, 1mmol of glucosamine is obtained as 1U enzyme activity unit.

And (3) purifying and recovering rate of glucosamine salt: as the enzyme method for producing glucosamine salt and the purification process thereof involve units such as enzyme reaction, ion exchange and the like, the molecular structure of reactants can be changed. The recovery rates were calculated based on the mass of glucosamine.

The quantitative analysis of glucosamine salt and acetylglucosamine adopts HPLC analysis method, the liquid chromatograph is Agilent 1260 series, the chromatographic column is Thermo ODS-2 Hypersil C18 column (250mM × 4.0.0 mM), the glucosamine salt sample to be detected is firstly filtered by 0.22 μm microfiltration membrane and then injected into the chromatographic column with 10 μ l sample injection quantity, the analysis of acetylglucosamine and acetic acid adopts chromatographic column HPX-87H column (Bio-Rad, USA), the detector adopts differential refractometer detector, the mobile phase is 5mM H₂SO₄The flow rate was 0.6ml/min and the detection temperature was 40 ℃.