Method for preparing decolorized beta-alanine and application of decolorized beta-alanine
Through the method of pretreatment of enzyme liquid by activated carbon, the problem of pigment influence in biological β-alanine production is solved, efficient decolorization and low-cost production are achieved, and production efficiency and product quality are improved.
Patent Information
- Application Number
- CN202411614364.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-02-18
Smart Images
Figure CN119464404A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of biochemical engineering and relates to a method for preparing decolorized beta-alanine and application thereof. Background Art
[0002] β-Alanine, also known as 3-aminopropionic acid, is the only β-amino acid that exists in nature. Microorganisms and plants can synthesize β-alanine by themselves, while mammals need to take it from the external environment. As an important precursor or intermediate for the production of food additives, drugs and nitrogen-containing chemicals, β-alanine can be widely used in food, feed, chemical industry and medicine. β-Alanine exists in almost all organisms and is one of the key components for the synthesis of pantothenate in organisms, which is an indispensable vitamin precursor for the synthesis of coenzyme A (CoA). Coenzyme A participates in more than 100 metabolic reactions in organisms and plays an important role in life activities. In addition, β-alanine also plays an important role in the regulation of the immune and central nervous systems of eukaryotic organisms.
[0003] At present, chemical and biological methods can be used in industry to produce β-alanine. The chemical method has high efficiency, short cycle and low cost; however, the reaction conditions are relatively extreme, and it needs to be carried out under high temperature, high pressure, strong acid and strong alkali conditions, and there are many by-products and high energy consumption. The biological method includes fermentation and enzyme methods, which are low-cost, mild, safe and green, and are the main research direction at present. The natural culture medium used in the biological method has complex components, containing a large amount of corn syrup, yeast extract and sucrose, etc. The reaction liquid is brown, especially the reaction liquid obtained by the transformation of growing cells is concentrated after heat treatment, and the color becomes darker. The presence of these pigments affects the analysis of amino acids and causes analytical errors. At the same time, if the pigments are not completely treated, it will also affect the finished product and reduce the quality and purity of the target product. A variety of methods can be used to remove pigments from the conversion liquid, such as ion exchange resin method, adsorption resin method, nanofiltration, ultrafiltration, supercritical fluid extraction, etc. Among them, supercritical fluid extraction and other methods are expensive, cumbersome to operate and difficult to scale production, so they are difficult to promote and apply.
[0004] In summary, the development of an efficient, low-cost, and simple-to-operate method for preparing decolorized alanine is of great significance for the field of β-alanine production. Summary of the invention
[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a method for preparing decolorized β-alanine and its application, in order to achieve efficient decolorization with a low amino acid loss rate, while reducing costs and simplifying the process.
[0006] To achieve this object, the present invention adopts the following technical solutions:
[0007] In a first aspect, the present invention provides a method for preparing decolorized β-alanine, the method for preparing decolorized β-alanine comprising:
[0008] mixing the aspartate decarboxylase solution with activated carbon to obtain pretreated aspartate decarboxylase;
[0009] The pre-treated aspartate decarboxylase is mixed with aspartic acid to react to obtain decolorized β-alanine, or,
[0010] The aspartate aminotransferase solution is mixed with activated carbon to obtain pretreated aspartate aminotransferase, and the pretreated aspartate decarboxylase is mixed with pretreated aspartate aminotransferase, fumaric acid and ammonia water to react to obtain decolorized β-alanine.
[0011] In the present invention, a completely new method for preparing decolorized beta-alanine is designed, and the enzyme solution required for beta-alanine production is first added to activated carbon for pretreatment, and the enzyme molecules are adsorbed in the activated carbon pores. The enzyme pretreated with the activated carbon has higher thermal stability and more durable and efficient catalytic activity. The enzyme pretreated with the activated carbon can react at a relatively higher temperature, reducing the risk of bacterial contamination in the conversion process. The enzyme pretreated with the activated carbon is more stable and not easy to decompose, and can reduce the generation of pigments; at the same time, the activated carbon can also timely adsorb the pigments generated in the conversion process. In addition, the activated carbon first pretreats the enzyme, and less alanine products are adsorbed in the conversion process, which can reduce the loss rate of the product in the decolorization process, and finally realize that the conversion and decolorization processes are carried out simultaneously, which can shorten the entire production cycle, improve production efficiency, and realize efficient decolorization under the condition of low amino acid loss rate, while reducing costs and simplifying processes.
[0012] Preferably, the activated carbon comprises activated carbon granules or activated carbon powder.
[0013] Preferably, the particle size of the activated carbon particles is 0.1-3 mm, for example, it can be 0.2, 0.3, 0.4, 0.5, 0.6, 1, 1.2, 1.5, 1.8, 2, 2.2, 2.4, 2.6, 2.7, 2.8 or 2.9 mm, etc.
[0014] Preferably, the particle size of the activated carbon powder is 200-400 mesh, for example, 250, 300 or 350 mesh.
[0015] Preferably, the aspartate decarboxylase solution is mixed with activated carbon at a temperature of 4-8°C, such as 5, 6 or 7°C, for 12-24h, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23h.
[0016] Preferably, the aspartate aminotransferase solution is mixed with activated carbon at a temperature of 4-8°C, such as 5, 6 or 7°C, and a time of 12-24h, such as 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23h.
[0017] Preferably, the mass ratio of the activated carbon to the aspartate decarboxylase solution is 1:(2-10), for example, it can be 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9, and the concentration of aspartate decarboxylase in the aspartate decarboxylase solution is 0.3-10 mg / L, for example, it can be 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 mg / L, etc.
[0018] Preferably, the mass ratio of the activated carbon to the aspartate aminotransferase solution is 1:(2-10), for example, it can be 1:3, 1:4, 1:5, 1:6, 1:7, 1:8 or 1:9, and the concentration of aspartate aminotransferase in the aspartate aminotransferase solution is 0.3-10 mg / L, for example, it can be 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 or 9.5 mg / L, etc.
[0019] Preferably, the ratio of the pretreatment enzyme to aspartic acid is 100-500 U / g dry substrate, for example, it can be 101, 102, 103, 104, 110, 120, 150, 200, 250, 300, 350, 400, 450, 460, 470, 480, 490, 491, 492, 495, 496, 497, 498 or 499 U / g dry substrate, etc.
[0020] Preferably, the aspartic acid is added in a feeding or batch addition manner.
[0021] In the present invention, the substrate is designed to be added in a flow-addition or batch-wise manner rather than being added all at once, thereby reducing the pigment generated by the discoloration of the substrate itself.
[0022] Preferably, the flow addition includes uniform flow addition.
[0023] Preferably, the ratio of the pretreated aspartate aminotransferase to fumaric acid is 100-500 U / g dry substrate, for example, it can be 101, 102, 103, 104, 110, 120, 150, 200, 250, 300, 350, 400, 450, 460, 470, 480, 490, 491, 492, 495, 496, 497, 498 or 499 U / g dry substrate, etc.
[0024] Preferably, the ratio of the pretreated aspartate decarboxylase to fumaric acid is 5-50 U / g dry substrate, for example, it can be 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 46, 47, 48 or 49 U / g dry substrate, etc.
[0025] Preferably, the molar ratio of fumaric acid to aqueous ammonia is 1:1.
[0026] Preferably, the reaction temperature is 30-70°C, for example, 31, 32, 33, 34, 35, 36, 40, 45, 50, 55, 60, 65, 66, 67, 68 or 69°C, and the reaction time is 48-96h, for example, 49, 50, 55, 60, 65, 70, 75, 80, 8, 5, 90, 91, 92, 93, 94 or 95h, etc.
[0027] As a preferred technical solution, the method for preparing decolorized β-alanine comprises the following steps:
[0028] (1) mixing an aspartate decarboxylase solution with activated carbon at 4-8° C. for 12-24 hours to obtain pretreated aspartate decarboxylase, and / or mixing an aspartate aminotransferase solution with activated carbon at 4-8° C. for 12-24 hours to obtain pretreated aspartate aminotransferase;
[0029] (2) adding aspartic acid to 100-500 U / g dry substrate of the pretreated aspartate decarboxylase, reacting at 30-70° C. for 48-96 hours to obtain decolorized β-alanine, or,
[0030] The pretreated aspartate decarboxylase at 5-50 U / g of dry substrate is mixed with the pretreated aspartate aminotransferase at 100-500 U / g of dry substrate, fumaric acid and ammonia water are added, and the mixture is reacted at 30-70° C. for 48-96 hours to obtain decolorized β-alanine.
[0031] Compared with the prior art, the present invention has at least the following beneficial effects:
[0032] The invention designs a new method for preparing decolorized beta-alanine. The enzyme solution required for the production of beta-alanine is firstly added with activated carbon for pretreatment. The enzyme pretreated with activated carbon has higher thermal stability and more durable high-efficiency catalytic activity, can reduce the generation of pigments, can react at a relatively higher temperature, can reduce the risk of bacterial contamination in the conversion process, can reduce the loss rate of the product in the decolorization process, and finally realizes that the conversion and decolorization processes are carried out simultaneously, can shorten the entire production cycle, improve production efficiency, realize high-efficiency decolorization under the condition of low amino acid loss rate, and at the same time reduce costs and simplify the process.
[0033] Instruction Manual
[0034] Figure 1 This is the result diagram of the optimal reaction temperature of aspartate decarboxylase pretreated with activated carbon;
[0035] Figure 2 This is the result diagram of the optimal reaction temperature of untreated aspartate decarboxylase;
[0036] Figure 3 This is the thermal stability result of aspartate decarboxylase pretreated with activated carbon;
[0037] Figure 4 This is the result of thermal stability of untreated aspartate decarboxylase;
[0038] Figure 5 This is the thermal stability result of aspartate aminotransferase pretreated with activated carbon;
[0039] Figure 6 This is the thermal stability result of untreated aspartate aminotransferase. DETAILED DESCRIPTION
[0040] The technical solution of the present invention is further described below by specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.
[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.
[0042] In the enzymatic production of β-alanine, the obtained β-alanine-containing conversion liquid contains a large amount of impurities such as bacterial fragments, sugars, enzymes and biomacromolecules, miscellaneous proteins or colloid substances. After the eluate is concentrated and crystallized, it still contains a large amount of pigments and protein impurities, which are yellow turbid liquid after dissolution. The present invention designs a new β-alanine preparation method combining conversion and decolorization, which is overall synergistic, can shorten the entire production cycle, improve production efficiency, achieve efficient decolorization under low amino acid loss rate, and reduce costs and simplify the process.
[0043] In a specific embodiment of the present invention, activated carbon powder was purchased from Sinopharm Group, activated carbon granules were purchased from Sinopharm Group, aspartate decarboxylase was purchased from Bio-Labo, aspartate aminotransferase was purchased from Bio-Labo, and the enzyme solution concentration was 0.3-10 mg / L.
[0044] Example 1
[0045] This embodiment provides a method for preparing decolorized β-alanine.
[0046] The aspartate decarboxylase solution was mixed with activated carbon powder with a mass of 30% of the enzyme solution, and the mixture was stirred at 4°C for 20 hours to obtain pretreated aspartate decarboxylase. The pretreated aspartate decarboxylase was analyzed. The results were as follows: Figure 1-Figure 4 As shown, the enzyme pretreated with activated carbon has higher thermal stability and more lasting and efficient catalytic activity.
[0047] Take 0.5 M aspartic acid, pre-treat aspartate decarboxylase (500 U / g dry substrate), stir at 50° C., 50 rpm for 72 h, and collect the filtrate by filtration.
[0048] Example 2
[0049] This embodiment provides a method for preparing decolorized β-alanine.
[0050] After mixing the aspartate decarboxylase solution with 30% of the activated carbon powder by mass of the enzyme solution, the mixture was placed at 4° C. and stirred for 20 h to obtain pretreated aspartate decarboxylase. The pretreated aspartate decarboxylase (500 U / g dry substrate) was stirred and mixed with water, and 1.7% of the total amount of aspartic acid was added per hour at a uniform rate. The final aspartic acid concentration was 0.5 M. After stirring at 50° C. and 50 rpm for 72 h, the filtrate was collected by filtration.
[0051] Example 3
[0052] This embodiment provides a method for preparing decolorized β-alanine.
[0053] After mixing the aspartate decarboxylase solution with 30% of the activated carbon powder by mass of the enzyme solution, the mixture was placed at 4° C. and stirred for 20 hours to obtain pretreated aspartate decarboxylase. After mixing the aspartate transaminase solution with 30% of the activated carbon powder by mass of the enzyme solution, the mixture was placed at 4° C. and stirred for 20 hours to obtain pretreated aspartate transaminase. The pretreated aspartate decarboxylase (10 U / g dry substrate) and the pretreated aspartate transaminase (500 U / g dry substrate) were stirred and mixed with water. Fumaric acid was added at a uniform rate of 1.5% of the total amount of fumaric acid per hour, and an equimolar amount of ammonia water was added at a uniform rate. The final concentration of fumaric acid was 0.5 M. After stirring at 50° C. and 50 rpm for 80 hours, the filtrate was collected by filtration.
[0054] The pre-treated aspartate aminotransferase was analyzed and the results were as follows Figure 5-Figure 6 As shown, the enzyme pretreated with activated carbon has higher thermal stability and more lasting and efficient catalytic activity.
[0055] Example 4
[0056] This embodiment provides a method for preparing decolorized β-alanine.
[0057] After mixing the aspartate decarboxylase solution with 30% of the mass of the enzyme solution activated carbon particles, the mixture was placed at 4°C and stirred for 20 hours to obtain pretreated aspartate decarboxylase. After mixing the aspartate transaminase solution with 30% of the mass of the enzyme solution activated carbon powder, the mixture was placed at 4°C and stirred for 20 hours to obtain pretreated aspartate transaminase. The pretreated aspartate decarboxylase (5U / g dry substrate) and the pretreated aspartate transaminase (500U / g dry substrate) were stirred and mixed with water. Fumaric acid was added at a uniform rate of 1.5% of the total amount of fumaric acid per hour, and an equimolar amount of ammonia water was added at a uniform rate. The final concentration of fumaric acid was 0.5M. The conversion process was carried out at 50°C, 50rpm, and the reaction was stirred for 80 hours, and the filtrate was collected by filtration.
[0058] Example 5
[0059] This embodiment provides a method for preparing decolorized β-alanine.
[0060] The aspartate decarboxylase solution was mixed with activated carbon powder in an amount of 50% by mass of the enzyme solution, and the mixture was stirred at 8° C. for 12 hours to obtain pretreated aspartate decarboxylase.
[0061] Take 0.5 M aspartic acid, pre-treat aspartate decarboxylase (100 U / g dry substrate), stir at 70°C, 50 rpm for 48 h, and collect the filtrate by filtration.
[0062] Example 6
[0063] After mixing the aspartate decarboxylase solution with activated carbon powder accounting for 10% of the mass of the enzyme solution, the mixture was placed at 6° C. and stirred for 18 hours to obtain pretreated aspartate decarboxylase. After mixing the aspartate transaminase solution with activated carbon powder accounting for 30% of the mass of the enzyme solution, the mixture was placed at 7° C. and stirred for 10 hours to obtain pretreated aspartate transaminase. The pretreated aspartate decarboxylase (50 U / g dry substrate) and the pretreated aspartate transaminase (100 U / g dry substrate) were stirred and mixed with water. Fumaric acid was added at a uniform rate of 1.5% of the total amount of fumaric acid per hour. At the same time, an equimolar amount of ammonia water was added at a uniform rate. The final concentration of fumaric acid was 0.5 M. After stirring at 30° C. and 50 rpm for 96 hours, the filtrate was collected by filtration.
[0064] Comparative Example 1
[0065] This comparative example provides a method for preparing decolorized β-alanine.
[0066] Take 0.5M aspartic acid and aspartate decarboxylase (500U / g dry substrate), stir at 50°C, 50rpm for 72h, add activated carbon powder with a mass of 30% of the enzyme for decolorization, stir at 50°C, 50rpm for 1h, and filter to collect the filtrate.
[0067] Comparative Example 2
[0068] This comparative example provides a method for preparing decolorized β-alanine.
[0069] Take 0.5M fumaric acid, 0.5M ammonia water, aspartate decarboxylase (10U / g dry substrate), aspartate aminotransferase (500U / g dry substrate), stir at 50°C, 50rpm for 80h, add powdered activated carbon with a mass percentage of 30% of the enzyme for decolorization, stir at 50°C, 50rpm for 1h, and filter to collect the filtrate.
[0070] Test Case
[0071] This test example analyzes and tests the samples obtained by the decolorization methods of each embodiment and comparative example.
[0072] Determination of decolorization effect: The transmittance of β-alanine prepared in each example and comparative example was measured at 420 nm by spectrophotometry.
[0073] Determination of β-alanine yield: HPLC was used to determine the concentration of β-alanine in the collected filtrate (g / L), multiplied by the volume of the collected filtrate (L), and divided by the total mass (g) of the substrate (aspartic acid or fumaric acid) used to obtain the yield (%) of β-alanine.
[0074] The specific results are shown in Table 1. It can be seen from Table 1 that the present invention is designed to pretreat the enzyme solution required for the production of β-alanine, so that an enzyme with higher thermal stability and more durable high-efficiency catalytic activity can be obtained, and the conversion and decolorization processes can be carried out simultaneously, thereby shortening the entire production cycle, improving production efficiency, and achieving high-efficiency decolorization with a low amino acid loss rate, while reducing costs and simplifying the process. In addition, controlling the addition method of the substrate can further improve the decolorization effect and the yield of β-alanine.
[0075] Table 1
[0076] Experimental Group 420nm transmittance β-Alanine yield Example 1 85.4％ 92％ Example 2 93％ 92.7％ Example 3 88％ 90.2％ Example 4 87.1％ 91.7％ Example 5 89％ 92％ Example 6 82％ 90.5％ Comparative Example 1 74％ 82.8％ Comparative Example 2 69.9％ 78％
[0077] In summary, the present invention designs a new method for preparing decolorized β-alanine, wherein the enzyme solution required for β-alanine production is first added with activated carbon for pretreatment, and the enzyme pretreated with activated carbon has higher thermal stability and more durable and efficient catalytic activity, can reduce the generation of pigments, can react at a relatively higher temperature, reduce the risk of contamination during the conversion process, can reduce the loss rate of the product during the decolorization process, and finally realizes that the conversion and decolorization processes are carried out simultaneously, which can shorten the entire production cycle, improve production efficiency, achieve efficient decolorization under the condition of low amino acid loss rate, and reduce costs and simplify the process.
[0078] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.
Claims
1. A method for preparing decolorized β-alanine, characterized in that: The method for preparing decolorized β-alanine comprises: mixing the aspartate decarboxylase solution with activated carbon to obtain pretreated aspartate decarboxylase; The pretreated aspartate decarboxylase is mixed with aspartic acid to react to obtain decolorized β-alanine, or, The aspartate aminotransferase solution is mixed with activated carbon to obtain pretreated aspartate aminotransferase, and the pretreated aspartate decarboxylase is mixed with pretreated aspartate aminotransferase, fumaric acid and ammonia water to react to obtain decolorized β-alanine.
2. The method for preparing decolorized β-alanine according to claim 1, characterized in that: The activated carbon includes activated carbon particles or activated carbon powder.
3. The method for preparing decolorized β-alanine according to claim 2, characterized in that: The particle size of the activated carbon particles is 0.1-3 mm.
4. The method for preparing decolorized β-alanine according to claim 2, characterized in that: The particle size of the activated carbon powder is 200-400 meshes.
5. The method for preparing decolorized β-alanine according to any one of claims 1 to 4, characterized in that: The aspartate decarboxylase solution and activated carbon are mixed at a temperature of 4-8°C for 12-24 hours; Preferably, the temperature of the aspartate aminotransferase solution and the activated carbon is 4-8°C and the time is 12-24h; Preferably, the mass ratio of the activated carbon to the aspartate decarboxylase solution is 1:(2-10), and the concentration of aspartate decarboxylase in the aspartate decarboxylase solution is 0.3-10 mg / L; Preferably, the mass ratio of the activated carbon to the aspartate aminotransferase solution is 1:(2-10), and the concentration of aspartate aminotransferase in the aspartate aminotransferase solution is 0.3-10 mg / L.
6. The method for preparing decolorized β-alanine according to any one of claims 1 to 5, characterized in that: The ratio of the pretreated aspartate decarboxylase to aspartic acid is 100-500 U / g dry substrate; Preferably, the aspartic acid is added in a feeding or batch addition manner; Preferably, the flow addition includes uniform flow addition.
7. The method for preparing decolorized β-alanine according to any one of claims 1 to 6, characterized in that: The ratio of the pretreated aspartate aminotransferase to fumaric acid is 100-500 U / g dry substrate.
8. The method for preparing decolorized β-alanine according to any one of claims 1 to 7, characterized in that: The ratio of the pretreated aspartate decarboxylase to fumaric acid is 5-50 U / g dry substrate; Preferably, the molar ratio of fumaric acid to aqueous ammonia is 1:
1.
9. The method for preparing decolorized β-alanine according to any one of claims 1 to 8, characterized in that: The reaction temperature is 30-70°C and the reaction time is 48-96h.
10. The method for preparing decolorized β-alanine according to any one of claims 1 to 9, characterized in that: The method for preparing decolorized β-alanine comprises the following steps: (1) mixing an aspartate decarboxylase solution with activated carbon at 4-8° C. for 12-24 hours to obtain pretreated aspartate decarboxylase, and / or mixing an aspartate aminotransferase solution with activated carbon at 4-8° C. for 12-24 hours to obtain pretreated aspartate aminotransferase; (2) adding aspartic acid to 100-500 U / g dry substrate of the pretreated aspartate decarboxylase, reacting at 30-70° C. for 48-96 hours to obtain decolorized β-alanine, or, The pretreated aspartate decarboxylase at 5-50 U / g of dry substrate is mixed with the pretreated aspartate aminotransferase at 100-500 U / g of dry substrate, fumaric acid and ammonia water are added, and the mixture is reacted at 30-70° C. for 48-96 hours to obtain decolorized β-alanine.