Method for preparing and separating L-asparaginic acid and by-product ammonium sulfate thereof by enzyme method
By immobilizing L-aspartase in resin and preparing enzyme-immobilized resin particles, the problem of low enzyme recycling rate was solved, achieving efficient separation of L-aspartic acid and ammonium sulfate and improving product quality.
Patent Information
- Application Number
- CN202511398788.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-28
- Publication Date
- 2025-12-09
AI Technical Summary
In the existing technology, enzymes are difficult to recover during the production of L-aspartic acid, resulting in low utilization and affecting product quality.
L-Aspartase was immobilized in resin to prepare L-Aspartase immobilization resin. The enzyme preparation was carried out by continuous flow circulation reaction. By controlling the temperature and speed of the stirring tank, enzyme resin particles with a particle size of 5-8 mm were formed to enhance mechanical strength and prevent breakage.
It improves enzyme utilization, reduces enzyme residue in products, enhances product quality, and achieves efficient separation of L-aspartic acid and ammonium sulfate.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of chemical industry, and particularly relates to a method for preparing and separating L-aspartic acid and byproduct ammonium sulfate by enzyme method. BACKGROUND
[0002] L-Aspartic Acid is an important alpha-amino acid, and its molecular formula is C4H7NO4 and its molecular weight is 133.10. It is one of the 20 basic amino acids constituting proteins, and belongs to acid amino acid together with glutamic acid. High-purity L-Aspartic Acid is usually colorless to white crystal or crystalline powder, tastes slightly sour, and has a melting point above 230 DEG C. It is soluble in water, but hardly soluble in organic solvents such as ethanol and diethyl ether.
[0003] At present, the process for producing L-Aspartic Acid is as follows: firstly, preparing ammonium fumarate aqueous solution from fumaric acid, and then mixing with L-aspartic acid enzyme for reaction. In this process, the enzyme is directly dispersed in the reaction liquid, and although the reaction speed is fast, the enzyme is difficult to be recovered, the utilization rate is low, and the enzyme fermentation liquid enters the reaction liquid, which affects the product quality. SUMMARY
[0004] The present application aims to provide a method for preparing and separating L-Aspartic Acid and byproduct ammonium sulfate by enzyme method. In the method, L-Aspartic acid enzyme is fixed in resin to prepare L-Aspartic acid enzyme fixed resin, and the utilization rate of L-Aspartic acid enzyme is increased without affecting the reaction speed.
[0005] The technical scheme of the present application is as follows: The present application provides a method for preparing and separating L-Aspartic Acid and byproduct ammonium sulfate by enzyme method, which comprises the following steps: Step 1: reacting fumaric acid with ammonia to prepare ammonium fumarate aqueous solution; Step 2: continuously pumping the ammonium fumarate solution into a reactor filled with L-Aspartic acid enzyme resin particles, and performing continuous flow circulation reaction in the reactor.
[0006] Step 3: after detecting the reaction end point, decolorizing the reaction liquid, adding sulfuric acid to adjust pH, precipitating L-Aspartic acid, and performing solid-liquid separation to obtain solid and mother liquor, wherein the solid is L-Aspartic acid crude product, and the L-Aspartic acid crude product is continuously refined; Step 4: recovering ammonium sulfate from the mother liquor, The L-Aspartic acid enzyme resin particles are prepared by the following method: A: inoculating the strain in a shake flask culture medium, culturing, then gradually expanding the culture to the scale of a fermentation tank, centrifuging, and collecting L-Aspartic acid enzyme solid; the strain is a strain capable of efficiently expressing L-Aspartic acid enzyme after screening.
[0007] B Add L-aspartase solid and resin into water, stir to form sol; C Disperse the sol in a stirred tank containing water, control the temperature of the stirred tank at 0~5°C, and the stirring speed at 48~55 rpm to obtain L-aspartase resin particles with a particle size of 5~8 mm.
[0008] Further, in the step 1, the concentration of the prepared ammonium fumarate aqueous solution is 180~220 g / L, and the pH value is 8.5~10.
[0009] Further, in the step 2, the temperature of the continuous flow circulation reaction is 35~40°C, and the circulation time is 10~16 hours.
[0010] Further, in the step 3, the end point of the reaction is that the concentration of fumaric acid is less than 5 g / L, and the pH value is 2.8~3.2.
[0011] Further, in the step 3, the purification of the crude L-aspartate includes dissolving the crude product in dilute aqueous ammonia to form a 15~20% solution, adjusting the pH to about 5.0, adding 1~2% activated carbon, stirring at 70~80°C to decolorize, filtering while hot, cooling the filtrate, and crystallizing at 5°C overnight, filtering the crystals, and drying to obtain high-purity L-aspartate product.
[0012] Further, in the step 3, the recovery of ammonium sulfate from the mother liquor includes concentrating the mother liquor in a four-effect evaporator to 1 / 6~1 / 5 of the initial volume, crystallizing at 10°C or below, filtering, and drying to obtain ammonium sulfate solid.
[0013] Further, in the A, the culture medium includes corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide, and desalted water.
[0014] Further, in the B, the resin is one of unsaturated polyester resin, epoxy resin, or carrageenan, and the ratio of the L-aspartase solid, the resin, and water is 1:1~2:20.
[0015] Further, the L-aspartase resin particle preparation method further includes soaking the L-aspartase resin particles with a particle size of 5~8 mm in a 0.3~0.5 mol / L KCl solution to solidify and increase the mechanical strength.
[0016] Further, the L-aspartase resin particle preparation method further includes soaking the L-aspartase resin particles with a particle size of 5~8 mm in a 0.25~0.5% glutaraldehyde solution to cross-link and increase the mechanical strength.
[0017] The present application has the following beneficial effects: The application provides an enzyme method for preparing and separating L-aspartic acid and byproduct ammonium sulfate, wherein L-aspartic acid enzyme and resin are formed into sol in water, dispersed in a water stirring tank, and particles with a particle size of 5-8 mm are obtained by controlling the water temperature (0-5°C) and stirring speed (48-55 r / min) in the stirring tank. Through experiments, it is verified that the L-aspartic acid enzyme resin particles are filled in a reactor, and can be recycled up to 30 times under the condition of meeting the process requirements (reaction rate and conversion rate). The application further studies a method for enhancing the mechanical strength of the L-aspartic acid enzyme resin particles, and the mechanical strength is increased by soaking the L-aspartic acid enzyme resin particles in a 0.3-0.5 mol / L KCl solution for solidification or in a 0.25-0.5% glutaraldehyde solution for crosslinking, so that the L-aspartic acid enzyme resin particles are prevented from being broken and lost under the scouring of the reaction liquid.
[0018] The application provides an enzyme method for preparing and separating L-aspartic acid and byproduct ammonium sulfate, and the method does not need to directly contact the enzyme fermentation liquor with the reaction liquid, so that the enzyme residue in the product is reduced and the product quality is increased. DETAILED DESCRIPTION
[0019] The technical solutions in the embodiments of the application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the application but not all the embodiments. Based on the embodiments in the application, all the other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the application.
[0020] The application provides an enzyme method for preparing and separating L-aspartic acid and byproduct ammonium sulfate, and the method does not need to directly contact the enzyme fermentation liquor with the reaction liquid, so that the enzyme residue in the product is reduced and the product quality is increased. Step 1: preparing an ammonium fumarate aqueous solution by reacting fumaric acid with ammonia, wherein the concentration of the obtained ammonium fumarate aqueous solution is 180-220 g / L (determined by a transmittance photometer method), and the pH value is 8.5-10; Step 2: continuously pumping the ammonium fumarate solution into a reactor filled with L-aspartic acid enzyme resin particles, and performing a continuous flow circulation reaction in the reactor, wherein the reaction temperature is 35-40°C, and the circulation time is 10-16 hours.
[0021] Step 3 After the reaction end point (fumaric acid concentration less than 5 g / L) is detected, the reaction solution is decolorized, sulfuric acid is added to adjust the pH to 2.8-3.2, L-aspartic acid is precipitated, and solid-liquid separation is performed, the solid is L-aspartic acid crude product, and the L-aspartic acid crude product is continuously refined, and the mother liquor is recovered with ammonium sulfate, the L-aspartic acid crude product refining comprises the following steps: the crude product is dissolved into a 15-20% solution with dilute ammonia water, the pH is adjusted to about 5.0, 1-2% activated carbon is added, and stirring is performed at 70-80°C to decolorize; after hot filtration, the filtrate is cooled and crystallized at 5°C overnight, the crystals are filtered and dried, and high-purity L-aspartic acid finished product is obtained; Step 4 The mother liquor is concentrated to 1 / 6-1 / 5 of the initial volume in a four-effect evaporator, and ammonium sulfate is recovered by crystallization at 10°C or lower, filtration and drying.
[0022] The L-aspartase enzyme resin particles are prepared by the following method: A The strain is inoculated in a shake flask medium, cultured, and then gradually expanded to the scale of a fermentation tank, centrifuged, and L-aspartase enzyme solids are collected, and the medium comprises corn steep powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water; B The L-aspartase enzyme solids and resin are added to water, stirred to form a sol, the resin is one of unsaturated polyester resin, epoxy resin or carrageenan, and the ratio of the L-aspartase enzyme solids, resin and water is 1:1-2:20; C The sol is dispersed in a stirred tank containing water, the temperature of the stirred tank is controlled at 0-5°C, and the stirring speed is 48-55 revolutions / minute, to obtain L-aspartase enzyme resin particles with a particle size of 5-8 mm.
[0023] In order to increase the mechanical strength of the L-aspartase enzyme resin particles prepared above, the L-aspartase enzyme resin particles with a particle size of 5-8 mm are soaked in a 0.3-0.5 mol / L KCl solution for solidification or soaked in a 0.25-0.5% glutaraldehyde solution for cross-linking.
[0024] The raw materials involved in the present application can be purchased from conventional reagent companies.
[0025] Example 1 The present embodiment provides a preparation method of L-aspartase enzyme resin particles, comprising the following steps: A The strain is inoculated in a shake flask medium (a mixture of corn steep powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), cultured, and then gradually expanded, centrifuged, and L-aspartase enzyme solids are collected; B Take L-aspartase solid and carrageenan resin into water, wherein the ratio of L-aspartase solid, carrageenan resin and water is 1:1:20, stir, form sol; C Disperse the sol in a stirring tank filled with water, control the temperature of the stirring tank at 0~5°C, the stirring speed at 48 rpm, filter, obtain L-aspartase resin particles with a particle size of 5~8 mm.
[0026] Example 2 The present embodiment provides a preparation method of L-aspartase resin particles, comprising the following steps: A Inoculate the strain into a flask culture medium (a mixture of corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), culture, then gradually enlarge the culture, centrifuge, collect L-aspartase solid; B Take L-aspartase solid and unsaturated polyester resin into water, wherein the ratio of L-aspartase solid, unsaturated polyester resin and water is 1:1.5:20, stir, form sol; C Disperse the sol in a stirring tank filled with water, control the temperature of the stirring tank at 0~5°C, the stirring speed at 50 rpm, filter, soak the solid particles in a 0.3~0.5 mol / L KCl solution for 10 hours, obtain L-aspartase resin particles with a particle size of 5~8 mm.
[0027] Example 3 The present embodiment provides a preparation method of L-aspartase resin particles, comprising the following steps: A Inoculate the strain into a flask culture medium (a mixture of corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), culture, then gradually enlarge the culture, centrifuge, collect L-aspartase solid; B Take L-aspartase solid and carrageenan resin into water, wherein the ratio of L-aspartase solid, carrageenan resin and water is 1:1:20, stir, form sol; C Disperse the sol in a stirring tank filled with water, control the temperature of the stirring tank at 0~5°C, the stirring speed at 55 rpm, filter, soak the solid particles in a 0.25~0.5% glutaraldehyde solution for 12 hours, obtain L-aspartase resin particles with a particle size of 5~8 mm.
[0028] Example 4 The present embodiment provides a preparation method of L-aspartase resin particles, comprising the following steps: A inoculate the strain into a flask culture medium (a mixture of corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), culture, then gradually expand the culture, centrifugal, collect L-aspartase solid; B take L-aspartase solid and carrageenan resin to add water, wherein the ratio of L-aspartase solid, carrageenan resin and water is 1:3:20, stir, form sol; C disperse the sol in a stirred tank containing water, control the temperature of the stirred tank 0~5°C, stirring speed 47 revolutions / minute, filter, get L-aspartase resin particles with particle size of 8~12mm.
[0029] Example 5 The preparation method of L-aspartase resin particles includes the following steps: A inoculate the strain into a flask culture medium (a mixture of corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), culture, then gradually expand the culture, centrifugal, collect L-aspartase solid; B take L-aspartase solid and carrageenan resin to add water, wherein the ratio of L-aspartase solid, carrageenan resin and water is 1:0.5:20, stir, form sol; C disperse the sol in a stirred tank containing water, control the temperature of the stirred tank 0~5°C, stirring speed 47 revolutions / minute, filter, get L-aspartase resin particles with particle size of 4~6mm.
[0030] Example 6 The preparation method of L-aspartase resin particles includes the following steps: A inoculate the strain into a flask culture medium (a mixture of corn syrup dry powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide and desalted water), culture, then gradually expand the culture, centrifugal, collect L-aspartase solid; B take L-aspartase solid and carrageenan resin to add water, wherein the ratio of L-aspartase solid, carrageenan resin and water is 1:0.5:10, stir, form sol; C disperse the sol in a stirred tank containing water, control the temperature of the stirred tank 0~5°C, stirring speed 47 revolutions / minute, filter, get L-aspartase resin particles with irregular shape and different size.
[0031] Example 7 The example provides a method for preparing and separating L-aspartate and its byproduct ammonium sulfate by enzyme method, including the following steps: Step 1: Fumaric acid is reacted with ammonia to prepare an aqueous ammonium fumarate solution, and the obtained aqueous ammonium fumarate solution has a concentration of 200 g / L and a pH value of 8.5; Step 2: The ammonium fumarate solution is continuously pumped into a reactor filled with the L-aspartase resin particles prepared in the examples, and the reactor is maintained at 37°C to perform a continuous flow circulating reaction for 10 hours.
[0032] Step 3: After detecting the reaction endpoint (fumaric acid concentration less than 5 g / L), the reaction solution is added to activated carbon for decolorization, and sulfuric acid is added to adjust the pH value, and L-aspartate is precipitated, centrifuged, and the solid and mother liquor are obtained, the solid is the crude L-aspartate product, the crude product is dissolved in 15-20% ammonia water solution, the pH value is adjusted to about 5.0, 2% activated carbon is added, and the mixture is stirred at 70-80°C for decolorization, then filtered hot, the filtrate is cooled and crystallized at 5°C overnight, the crystals are filtered and dried, and the high-purity L-aspartate product is obtained; Step 4: The mother liquor is concentrated to 1 / 5 of the initial volume in a four-effect evaporator, and the ammonium sulfate solid is obtained by crystallization below 10°C, filtration and drying.
[0033] Experimental Example 1000 grams of the L-aspartase resin particles prepared in Examples 1-5 after air-drying are respectively loaded into a fixed bed reactor with a height of 60 cm and a diameter of 10 cm, and a 3 mm aperture is provided at the upper and lower ends of the reactor, and the L-aspartase resin particles are loaded between the two partitions to prevent leakage. 10 kg of the previously prepared 200 g / L aqueous ammonium fumarate solution is added to the fixed bed reactor, an external circulating pump is used to circulate the reaction solution between the bed layers, and the reaction is maintained at a temperature of 37°C. After 10 hours of reaction, the ammonium fumarate concentration is detected. Respectively recorded as Experimental Examples 1-6.
[0034] Comparative Experimental Example Another 20-liter reaction kettle is prepared, 10 kg of 200 g / L aqueous ammonium fumarate solution is added, and 1.5 kg of L-aspartase aqueous solution (1% concentration) is directly added, stirred, and reacted at 37°C. After 10 hours of reaction, the ammonium fumarate concentration is detected.
[0035] The detection results are shown in Table 1:
[0036] Table 1: Ammonium fumarate concentration (transmittance photometer method, first batch) The L-aspartase resin particles prepared in Examples 1 to 3 have a particle size of 5 to 8 mm, which is moderate, and are packed in a fixed bed reactor to be in sufficient contact with the ammonium fumarate reaction solution. If the particle size is too large (8 to 12 mm in Example 4), the specific surface area of the L-aspartase resin particles is small, and the active enzyme sites are in less contact with the reaction solution, resulting in a slow reaction rate. If the particle size is too small (4 to 6 mm in Example 5), the specific surface area is large, but the bed resistance is large, resulting in a small flow rate using the same circulating pump, and a long reaction solution circulation period, and a slow reaction rate. In comparison with the reaction of the L-aspartase aqueous solution in a 20 L reactor, the reaction rates of Examples 1 to 3 are comparable, or even slightly higher. The L-aspartase resin particles prepared in Example 6 have the worst reaction effect due to the irregular shape and the different sizes.
[0037] In Examples 1 to 3, the ratio of the L-aspartase solid, the resin and water is 1:1 to 2:20, which is moderate, and the L-aspartase resin particles prepared have a regular shape and a uniform pore size, and the reaction solution can be in sufficient contact with the L-aspartase wrapped in the resin, resulting in a fast reaction rate.
[0038] The fixed bed reactors packed with the L-aspartase resin particles of Examples 1 to 5 were subjected to 30 repeated L-aspartate preparation experiments, and the experimental conditions were maintained. The ammonium fumarate concentration detected at the completion of the 30th experiment is shown in Table 2.
[0039] Table 2 Ammonium fumarate concentration (transmittance photometer method, 30th time) The L-aspartase resin particles in the fixed bed reactors of Examples 1 to 5 were removed after the completion of the reaction, and the completeness of the resin particles (the ratio of the complete resin particles to the total mass of the resin particles) was observed and estimated, as shown in Table 3.
[0040] Table 3 Completeness of L-aspartase resin particles The L-aspartase resin particles prepared in Examples 2 and 3 were solidified in a 0.3 to 0.5 mol / L KCl solution or crosslinked in a 0.25 to 0.5% glutaraldehyde solution, and had a high mechanical strength and a low breakage rate. It was also verified that the ammonium fumarate concentration g / L of Examples 2 and 3 in Table 2 was also low, and the conversion rate was high. In Comparative Example 2, the resin content in the sol was low (1:0.5:20) during preparation, the particle size was small, the bed resistance was large, the breakage rate of the resin particles was high, and the amount of L-aspartase loss was large, so the ammonium fumarate content was high after the completion of the 30th reaction.
[0041] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims
1. A method for the enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate, comprising the following steps: Step 1: Prepare an aqueous solution of ammonium fumarate by reacting fumaric acid with ammonia; Step 2: The ammonium fumarate solution is continuously pumped into a reactor filled with L-aspartate enzyme resin particles, and a continuous flow circulation reaction is carried out in the reactor. Step 3: After detecting the reaction endpoint, the reaction solution is decolorized, and sulfuric acid is added to adjust the pH, precipitating L-aspartic acid. Solid-liquid separation is performed to obtain a solid and a mother liquor. The solid is crude L-aspartic acid, which is further purified. Step 4: Ammonium sulfate is recovered from the mother liquor. The L-aspartic acid enzyme resin particles are prepared by the following method: A. The bacterial strain was inoculated into a shake flask culture medium, cultured, and then the culture was gradually scaled up to the scale of a fermenter. After centrifugation, the L-aspartate enzyme solid was collected. B. Add L-aspartase solid and resin to water and stir to form a sol; C. The sol is dispersed in a mixing tank containing water, and the temperature of the mixing tank is controlled at 0~5°C and the stirring speed is 48~55 rpm to obtain L-aspartic acid enzyme resin particles with a particle size of 5~8 mm.
2. The method for preparing and separating L-aspartic acid and its byproduct ammonium sulfate by enzymatic method according to claim 1, characterized in that: In step 1, the concentration of the prepared ammonium fumarate aqueous solution is 180~220g / L, and the pH value is 7.5~10.
3. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In step 2, the temperature of the continuous flow cyclic reaction is 35~40°C, and the cycle time is 10~16 hours.
4. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In step 3, the reaction endpoint is when the fumaric acid concentration is less than 5 g / L, and the pH is 2.8~3.
2.
5. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In step 3, the purification of crude L-aspartic acid includes dissolving the crude product in dilute ammonia water to form a 15-20% solution, adjusting the pH to about 5.0, adding 1-2% activated carbon, stirring and decolorizing at 70-80℃, filtering while hot, cooling the filtrate and crystallizing overnight at 5℃, filtering out the crystals, drying, and obtaining the high-purity L-aspartic acid product.
6. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In step 3, the recovery of ammonium sulfate from the mother liquor includes concentrating the mother liquor to 1 / 6 to 1 / 5 of its initial volume in a four-effect evaporator, crystallizing it below 10°C, filtering it, and drying it to obtain solid ammonium sulfate.
7. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In A, the culture medium includes corn steep liquor powder, magnesium sulfate, potassium dihydrogen phosphate, glucose, sodium hydroxide, and desalinated water.
8. The method for enzymatic preparation and separation of L-aspartic acid and its byproduct ammonium sulfate according to claim 1, characterized in that: In B, the resin is one of unsaturated polyester resin, epoxy resin or carrageenan, and the ratio of L-aspartate enzyme solid, resin and water is 1:1 to 2:
20.
9. The method for preparing and separating L-aspartic acid and its byproduct ammonium sulfate by enzymatic method according to claim 1, characterized in that: The method for preparing L-aspartase resin particles further includes immersing L-aspartase resin particles with a particle size of 5-8 mm in a 0.3-0.5 mol / L KCl solution for curing to increase mechanical strength.
10. The method for preparing and separating L-aspartic acid and its byproduct ammonium sulfate by enzymatic method according to claim 1, characterized in that: The method for preparing L-aspartase resin particles further includes immersing L-aspartase resin particles with a particle size of 5-8 mm in a 0.25-0.5% glutaraldehyde solution for cross-linking to increase mechanical strength.