A method for extracting L-histidine using simulated moving bed chromatography

Abstract

This invention belongs to the technical field of L-histidine production and discloses a method for extracting L-histidine using simulated moving bed chromatography, which includes a fermentation step and an extraction step. This method can achieve continuous production of L-histidine and has the advantages of high fermentation efficiency, short production cycle, low production cost, low pollution, and high extraction yield.

Description

Technical Field

[0001] The invention relates to the technical field of L-histidine production, and in particular to a method for extracting L-histidine by utilizing simulated moving bed chromatography. Background Art

[0002] L-histidine is a basic amino acid containing an imidazole nucleus in its molecule. It is a semi-essential amino acid, not essential for adults, but particularly important for the growth of infants, young children, and animals. Its synthesis in the human body is slow. It has multiple physiological functions and can be used as a biochemical reagent and pharmaceutical agent, including in medications for heart disease, anemia, rheumatoid arthritis, and other conditions. Histidine is also an essential raw material for the synthesis of several pharmaceutical intermediates. Due to its significant nutritional benefits, its market prospects are promising, and its role in medical research is gaining increasing attention.

[0003] Currently, L-histidine is primarily extracted domestically from pig blood meal hydrolysate using an ion exchange process. Alternatively, it can be extracted from defatted soybean hydrolysate. However, due to high costs, high hydrolysis losses, and environmental pollution, these methods are unsuitable for large-scale industrial production. This extraction method, however, enables continuous production of L-histidine, offering advantages such as a short production cycle, low costs, minimal pollution, and high extraction yield. It offers numerous advantages over protein hydrolysis extraction methods. Summary of the Invention

[0004] To solve the above problems and overcome the shortcomings of the existing technology, the present invention provides a method for extracting L-histidine using simulated moving bed chromatography. This extraction method can achieve continuous production of L-histidine and has the advantages of short production cycle, low production cost, low pollution, and high extraction yield.

[0005] The purpose of the present invention is achieved through the following technical solutions.

[0006] A method for extracting L-histidine using simulated moving bed chromatography is characterized in that the method includes the following fermentation steps and extraction steps.

[0007] Specifically, the fermentation process includes the following:

[0008] 1) Inoculate the Serratia marcescens seed solution into a fermentation tank containing fermentation medium. Control the liquid volume in the fermentation tank to 50 L and the fermentation conditions to be 33°C, pH 7.0, rotation speed 80 rpm, and pressure 0.05 MPa.

[0009] 2) Throughout the fermentation process, potassium hydrogen phosphate aqueous solution was fed at a rate of 20 ml / h until the fermentation was completed. After 6 h of fermentation, a fermentation promoter was added at a rate of 0.5 L / h until the fermentation was completed, and the histidine fermentation broth was collected. The total fermentation time was 52 h.

[0010] Specifically, the extraction process includes the following: step 1) ceramic membrane filtration, step 2) simulated moving bed chromatography, step 3) primary decolorization, step 4) concentration and re-dissolution, step 5) secondary decolorization, step 6) purification and concentration, and step 7) crystallization and centrifugation.

[0011] Furthermore, the fermentation promoter contains 100-1000 g / L of glucose and 10-100 mg / L of calcium acetate.

[0012] Furthermore, the fermentation medium components are: glucose 70g / L, yeast powder 5g / L, betaine 1.5g / L, dipotassium hydrogen phosphate 5g / L, corn steep liquor 40ml / L, ammonium sulfate 4g / L, magnesium sulfate 0.5g / L, biotin 0.15mg / L, vitamin B1 0.15mg / L.

[0013] Furthermore, the concentration of the dipotassium hydrogen phosphate aqueous solution is 100 g / L

[0014] Furthermore, the extraction process includes the following:

[0015] Step 1) Ceramic membrane filtration: The temperature of the histidine fermentation broth is controlled to 70-80°C, the pH is adjusted to 5.0-5.5, and the broth is filtered through a ceramic membrane to collect the clear liquid;

[0016] Step 2) Simulated Moving Bed Chromatography: The ceramic membrane clear solution obtained in step 1) is passed through a simulated moving bed chromatography equipped with a cation exchange resin and eluted with aqueous ammonia to obtain an L-histidine eluate;

[0017] Step 3) primary decolorization: the L-histidine eluate obtained in step 2) is passed through a decolorization membrane to remove some pigments and small molecular impurities, thereby obtaining a decolorized solution;

[0018] Step 4) Concentration and re-dissolution: The decolorized solution obtained in step 3) is concentrated to an L-histidine content of 220-250 g / L using a four-effect evaporator, cooled to 10°C with condensed water, and crystallized for 6 hours. The resulting mother liquor and crude histidine are collected, and the crude histidine is redissolved, the pH is adjusted to 6.5-7.0, and the solution is collected;

[0019] Step 5) Secondary decolorization: The solution obtained in step 4) was decolorized with activated carbon at a temperature of 50° C. for 60 min, and the filtrate was collected by filtration;

[0020] Step 6) Purification and concentration: The filtrate obtained in step 5) is concentrated to a histidine content of 220-250 g / L using a four-effect evaporator, cooled to 10°C with condensed water, crystallized for 6 hours, and filtered to obtain a wet product;

[0021] Step 7) Crystallization and centrifugation: The wet product obtained in step 6) is centrifuged and dried under low temperature vacuum to obtain the L-histidine product.

[0022] Furthermore, the fermentation promoter contains 100-1000 g / L of glucose, 10-100 mg / L of calcium acetate, and 10-50 mg / L of malonic acid.

[0023] Preferably, the fermentation promoter comprises the following components: 500 g / L glucose, 100 mg / L calcium acetate, and 50 mg / L malonic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :Effect of calcium acetate on histidine production in fermentation broth;

[0025] Figure 2 : Effect of malonate on histidine production in fermentation broth.

[0026] The beneficial effects achieved by the present invention mainly include but are not limited to the following aspects:

[0027] The activated cultured Serratia marcescens of the present invention is used to produce histidine. During the fermentation process, glucose and dipotassium hydrogen phosphate are fed in a stream, thereby effectively supplementing nitrogen sources and nutrients required for strain growth, maintaining the growth vitality of the strain, significantly improving fermentation acid production performance, reducing glucose consumption by bacterial growth, and increasing the sugar-acid conversion rate. Testing shows that the histidine yield and sugar-acid conversion rate are significantly improved by this method.

[0028] The acid production mechanisms and tolerance to stimulating factors of different strains vary greatly and are not of reference value. Although the existing technology has conducted extensive research on the acid production mechanism of glutamate, for example, adding an appropriate amount of sodium citrate to the fermentation medium can increase the amount of histidine produced by Corynebacterium glutamicum, sodium citrate has no obvious stimulating effect on Serratia marcescens.

[0029] The fermentation process of histidine does not rely on the TCA pathway. While appropriate weakening of the TCA pathway can increase metabolic flux into the histidine synthesis pathway, the TCA cycle maintains normal bacterial cell proliferation and metabolism and should not be excessively weakened. Malonic acid acts as a TCA cycle inhibitor. By weakening the TCA cycle, it can increase metabolic flux into the histidine synthesis pathway, thereby increasing histidine production.

[0030] L-histidine is an intermediate product of the HMP pathway. The HMP pathway also produces acetic acid by-products, resulting in a waste of carbon metabolic flow. By adding calcium acetate, a certain inhibitory effect is exerted on the by-products, thereby allowing more metabolic flow of the HMP pathway to enter the histidine synthesis pathway, providing more prerequisite substances for the biosynthesis of histidine, thereby increasing the production of histidine.

[0031] The present invention chooses to start feeding in the middle of fermentation because a large amount of histidine synthesis occurs in the middle of fermentation, and the initial stage of fermentation is mainly based on strain proliferation. At this time, weakening the TCA pathway will reduce the activity of the strain.

[0032] The invention adopts simulated moving bed chromatography separation technology to extract L-histidine, adopts color reaction during chromatographic column loading and elution interception, reduces interception loss, improves chromatographic yield, utilizes the principle of physical adsorption based on the different times that different ions pass through the chromatographic resin, separates other impurities generated in the histidine fermentation broth, and thus improves the purity of the histidine.

[0033] The present invention can effectively remove other miscellaneous acids and impurities in the fermentation liquid at one time, thereby improving product quality. At the same time, it reduces the amount of acid and alkali used, reduces the amount of wastewater discharged, and achieves the goals of clean production, energy saving and environmental protection.

[0034] The present invention adopts a four-effect evaporator, and the evaporation process is carried out under vacuum, which not only ensures the hygiene requirements of the materials, but also meets the environmental protection requirements, and greatly reduces the evaporation temperature, with the advantages of energy saving, low steam consumption, and low cooling water circulation.

[0035] The invention performs decolorization twice, wherein a decolorization membrane is used for one decolorization without using activated carbon, so the amount of activated carbon used is reduced, the amount of fixed waste generated is reduced accordingly, and the pollution to the environment is reduced. DETAILED DESCRIPTION

[0036] In order to enable people skilled in the art to better understand the technical solution in this application, the technical solution will be fully described below in conjunction with the specific embodiments of this application.

[0037] Example 1

[0038] A method for extracting L-histidine using simulated moving bed chromatography comprises the following steps:

[0039] 1. Fermentation process:

[0040] 1) Place Serratia marcescens (ATCC31026) in an activation medium and incubate at 33°C in a constant temperature incubator for 24 hours. The activation medium consists of 1g / L anhydrous glucose, 10g / L peptone, 10g / L beef extract, 5g / L yeast extract, 2.5g / L sodium chloride, and 20g / L agar. Before transplanting the strain, add water to the seed tank to a fixed volume of 5L. Directly heat the tank with steam to 119-122°C and a pressure of 0.13-0.14 MPa. Hold the mixture for 30 minutes for sterilization.

[0041] 2) The activated culture solution was added to the sterilized seed culture tank by pressure difference method. The liquid volume was 5L. The culture conditions were controlled, with an initial temperature of 33°C, pH 7.0, a stirring motor speed of 80 rpm, and an air volume of 70 m 3 / h, pressure 0.05MPa, continuous culture for 16h. The controlled liquid composition is glucose 35g / L, yeast powder 5g / L, betaine 1.5g / L, dipotassium hydrogen phosphate 1.5g / L, corn steep liquor 30ml / L, ammonium sulfate 5g / L, magnesium sulfate 0.4g / L, biotin 0.15mg / L, vitamin B1 0.15mg / L.

[0042] 3) At the beginning of fermentation, potassium hydrogen phosphate was fed into the fermentation at a concentration of 100 g / L and a flow rate of 1.5 ml / h. After 5 h of fermentation, glucose was fed at a concentration of 500 g / L and a flow rate of 0.1 L / h until the end of fermentation, with continuous culture for 16 h.

[0043] 4) After the seed irrigation OD value reaches 20.3, transplant the seeds and inoculate them into the fermentation tank at a rate of 10%. The liquid volume in the fermentation tank is 50L, and the fermentation conditions are controlled at a temperature of 33℃, pH 7.0, a stirring motor speed of 80rpm, and an air volume of 400m 3 / h, pressure 0.05MPa.

[0044] 5) The fermentation medium composition was controlled to include 70 g / L glucose, 5 g / L yeast extract, 1.5 g / L betaine, 5 g / L dipotassium hydrogen phosphate, 40 ml / L corn steep liquor, 4 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.15 mg / L biotin, and 0.15 mg / L vitamin B1. Dipotassium hydrogen phosphate was fed into the fermentation medium at the start of fermentation to a concentration of 100 g / L at a flow rate of 20 ml / h. After 6 h of fermentation, a fermentation promoter (500 g / L glucose, 100 mg / L calcium acetate, and 50 mg / L malonic acid) was added at a flow rate of 0.5 L / h until the end of fermentation. The total fermentation time was 52 h, and the resulting L-histidine fermentation broth produced 38.7 g / L of histidine.

[0045] 2. Extraction process:

[0046] 1) Ceramic membrane filtration: The temperature of the L-histidine fermentation broth is controlled at 70-80°C, the pH is adjusted to 5.0-5.5 with sulfuric acid, 60% water is added, and the mixture is stirred evenly. The mixture is filtered through a ceramic membrane to remove bacterial proteins and other particulate impurities, and the clear liquid from the ceramic membrane is collected.

[0047] 2) Simulated moving bed chromatography: The ceramic membrane clear liquid is passed through a simulated moving bed chromatography equipped with a cation exchange resin and eluted with ammonia water of a certain concentration to obtain an L-histidine eluate;

[0048] 3) Primary decolorization: The obtained L-histidine eluate is passed through a decolorization membrane with a pore size of 300-500 molecular weight to remove some pigments and small molecular impurities to obtain a decolorized solution;

[0049] 4) Concentration and re-dissolution: The decolorized solution was concentrated to an L-histidine content of 220-250 g / L using a four-effect evaporator. The solution was cooled to 10°C with condensed water and allowed to grow for 6 hours. The resulting solution was centrifuged and the mother liquor and crude product were collected. The mother liquor was recycled and reused. The crude product was purified and re-dissolved at 60 g / L. The pH was adjusted to 6.5-7.0, and the resulting solution was collected.

[0050] 5) Secondary decolorization: The obtained solution was decolorized with activated carbon at a temperature of 50°C for 60 minutes. After decolorization with activated carbon, the light transmittance of the solution was 98%, and the filtrate was collected.

[0051] 6) Purification and concentration: The filtrate was concentrated to a histidine content of 220-250 g / L using a four-effect evaporator, cooled to 10°C with condensed water, and crystallized for 6 hours before filtration to obtain the wet product.

[0052] 7) Crystallization and centrifugation: The wet product is centrifuged and dried under low temperature vacuum to obtain the L-histidine product; after testing, the purity reaches over 97%.

[0053] In summary, the present invention proposes a method for extracting L-histidine using simulated moving bed chromatography, which utilizes ceramic membrane filtration for sterilization, decolorization membrane for impurity removal and decolorization, and simulated moving bed chromatography for separation to improve product quality, further increase yield, and increase production, thereby providing a cost-effective product for the L-histidine market. Meanwhile, the method reduces wastewater discharge, reduces environmental pollution, and increases social benefits. Therefore, the technology has great market prospects.

[0054] Comparative Example 1

[0055] A method for extracting L-histidine using simulated moving bed chromatography comprises the following steps:

[0056] 1. Fermentation process:

[0057] 1) Place Serratia marcescens (ATCC31026) in an activation medium maintained at 33°C in a constant temperature incubator for 24 hours. The activation medium consists of 1g / L anhydrous glucose, 10g / L peptone, 10g / L beef extract, 5g / L yeast extract, 2.5g / L sodium chloride, and 20g / L agar. Before transplanting the strain, add water to the seed tank to a constant volume of 5L. Directly heat the medium with steam to 119-122°C and a pressure of 0.13-0.14 MPa. Hold for 30 minutes for sterilization.

[0058] 3) The activated culture solution was added to the sterilized seed culture tank by pressure difference method. The liquid volume was 5L. The culture conditions were controlled, with an initial temperature of 33°C, pH 7.0, a stirring motor speed of 80 rpm, and an air volume of 70 m 3 / h, pressure 0.05MPa, continuous culture for 16h. The controlled liquid composition is glucose 35g / L, yeast powder 5g / L, betaine 1.5g / L, dipotassium hydrogen phosphate 1.5g / L, corn steep liquor 30ml / L, ammonium sulfate 5g / L, magnesium sulfate 0.4g / L, biotin 0.15mg / L, vitamin B1 0.15mg / L.

[0059] 4) At the start of fermentation, feed potassium dihydrogen phosphate to a concentration of 100 g / L at a rate of 1.5 ml / h. After 5 hours of fermentation, feed glucose to a concentration of 500 g / L at a rate of 0.1 L / h until the end of fermentation, continuing for 16 hours. After the seed OD value reaches 20.3, transplant the seedlings and inoculate the fermentation tank at a 10% inoculum rate.

[0060] The liquid volume in the fermentation tank is 50L, and the fermentation conditions are controlled at 33℃, pH 7.0, stirring motor speed 80rpm, and air volume 400m 3 / h, pressure 0.05MPa.

[0061] 4) The fermentation medium composition was controlled to include 70 g / L glucose, 5 g / L yeast extract, 1.5 g / L betaine, 5 g / L dipotassium hydrogen phosphate, 40 ml / L corn steep liquor, 4 g / L ammonium sulfate, 0.5 g / L magnesium sulfate, 0.15 mg / L biotin, and 0.15 mg / L vitamin B1. Dipotassium hydrogen phosphate was fed into the fermentation system at the start of fermentation at a concentration of 100 g / L and a flow rate of 20 ml / h. After 6 h of fermentation, a fermentation promoter (500 g / L glucose) was added at a flow rate of 0.5 L / h until the end of fermentation. The total fermentation time was 52 h, and the histidine yield reached 22.1 g / L.

[0062] 2. The extraction process is the same as in Example 1.

[0063] Example 2

[0064] 1. The process is the same as that of Comparative Example 1. On the basis of Comparative Example 1, the fermentation promoter is optimized and different concentrations of calcium acetate (horizontally) are set to 0, 20, 40, 60, 80, 100, 120, and 140, respectively, in mg / L. Figure 1As shown in the figure, with the increase of calcium acetate concentration, the histidine content in the fermentation broth (vertical axis, g / L) also increased. When the calcium acetate concentration reached 100 mg / L, the histidine concentration reached its peak. Continuing to increase the calcium acetate concentration had no obvious effect on the histidine yield. The sugar-acid conversion rate and the histidine trend were consistent, indicating that calcium acetate mainly increased the histidine yield by improving the sugar-acid conversion rate of Serratia marcescens. The possible reason is that L-histidine is an intermediate product of the HMP pathway, and the HMP pathway also produces acetic acid by-products, thereby causing a waste of carbon metabolic flow. By adding an appropriate concentration of calcium acetate, a certain inhibitory effect is exerted on the by-products, thereby allowing more metabolic flow of the HMP pathway to enter the histidine synthesis pathway, providing more prerequisite substances for the biosynthesis of histidine, thereby increasing the histidine yield. In addition, calcium ions are also activators of the enzymes required for histidine synthesis.

[0065] 2. Select the concentration of calcium acetate as 100 mg / L and continue to evaluate the effect of malonate on the histidine production in the fermentation broth. Set different concentrations of calcium acetate, 0, 10, 20, 30, 40, 50, 60, 70, in mg / L. Figure 2 As shown in the figure, as the malonate concentration increases, the histidine content in the fermentation broth also increases. When the calcium acetate concentration reaches 50 mg / L, the histidine concentration approaches its peak. Further addition of calcium acetate has no substantial effect on histidine production, and the sugar-acid conversion rate and histidine trends remain essentially the same. This may be because the fermentation process of histidine does not rely on the TCA pathway. Appropriate weakening of the TCA pathway can increase the metabolic flux entering the histidine synthesis pathway. However, the TCA cycle maintains normal proliferation and metabolism of bacterial cells and cannot be excessively weakened. Therefore, it is necessary to start feeding in the middle of the fermentation. This is because a large amount of histidine synthesis occurs in the middle of the fermentation, and in the early stage of fermentation, the main focus is on strain proliferation. At this time, weakening the TCA pathway will reduce strain viability. Appropriate amounts of malonate can act as an inhibitor of the TCA cycle. By weakening the TCA cycle, the metabolic flux of the histidine synthesis pathway can be increased, thereby increasing the sugar-acid conversion rate and histidine production.

[0066] Although the above describes the specific implementation methods of the present invention in combination with the embodiments, it does not limit the scope of protection of the present invention. Those skilled in the art should understand that it should be clear to those skilled in the art that these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection of the present invention.

Claims

1. A method for extracting L-histidine using simulated moving bed chromatography, characterized in that: The method includes a fermentation process and an extraction process; The fermentation process is as follows: 1) Place Serratia marcescens ATCC31026 in an activation medium and culture in a constant temperature incubator maintained at 33°C for 24 hours. The activation medium consists of 1 g / L anhydrous glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2.5 g / L sodium chloride, and 20 g / L agar strips to obtain an activated culture solution. Before transplanting the strain, add water to the seed tank after adding the materials to the fixed volume of 5 L. Directly connect the steam to heat to 119-122°C and a pressure of 0.13-0.14 MPa, keep warm for 30 minutes, and sterilize. 2) The activated culture solution obtained in step 1) was added to the sterilized seed culture tank by pressure differential method. The liquid volume was 5 L. The culture conditions were controlled to maintain the initial temperature of 33 ° C, pH 7.0, stirring motor speed of 80 rpm, and air volume of 70 m 3 / h, pressure 0.05MPa, continuous culture for 16h, the feed liquid composition is controlled to be glucose 35g / L, yeast powder 5g / L, betaine 1.5g / L, potassium hydrogen phosphate 1.5g / L, corn steep liquor 30ml / L, ammonium sulfate 5g / L, magnesium sulfate 0.4g / L, biotin 0.15mg / L, vitamin B1 0.15mg / L; 3) At the beginning of fermentation, potassium hydrogen phosphate was fed at a concentration of 100 g / L and a flow rate of 1.5 ml / h. After 5 h of fermentation, glucose was fed at a concentration of 500 g / L and a flow rate of 0.1 L / h until the end of fermentation, with continuous culture for 16 h. 4) After the seed irrigation OD value reaches 20.3, transplant the seeds and inoculate them into the fermentation tank at a rate of 10%. The liquid volume in the fermentation tank is 50L. The fermentation conditions are controlled at a temperature of 33°C, a pH of 7.0, a stirring motor speed of 80rpm, and an air volume of 400m 3 / h, pressure 0.05MPa; 5) The fermentation medium composition was controlled to be glucose 70 g / L, yeast powder 5 g / L, betaine 1.5 g / L, dipotassium hydrogen phosphate 5 g / L, corn steep liquor 40 ml / L, ammonium sulfate 4 g / L, magnesium sulfate 0.5 g / L, biotin 0.15 mg / L, and vitamin B1 0.15 mg / L. At the beginning of fermentation, dipotassium hydrogen phosphate was fed at a concentration of 100 g / L and a flow rate of 20 ml / h. After 6 h of fermentation, fermentation promoters were added at a flow rate of 0.5 L / h, including glucose 500 g / L, calcium acetate 100 mg / L, and malonic acid 50 mg / L, until the end of fermentation. The total fermentation time was 52 h.

2. The method according to claim 1, characterized in that The extraction process is as follows: Step 1) Ceramic membrane filtration: The temperature of the histidine fermentation broth is controlled to 70-80°C, the pH is adjusted to 5.0-5.5, and the broth is filtered through a ceramic membrane to collect the clear liquid; Step 2) Simulated Moving Bed Chromatography: The ceramic membrane clear solution obtained in step 1) is passed through a simulated moving bed chromatography equipped with a cation exchange resin and eluted with aqueous ammonia to obtain a histidine eluate; Step 3) primary decolorization: the histidine eluate obtained in step 2) is passed through a decolorization membrane to remove some pigments and small molecular impurities, thereby obtaining a decolorized solution; Step 4) Concentration and re-dissolution: The decolorized solution obtained in step 3) is concentrated to a histidine content of 220-250 g / L using a four-effect evaporator, cooled to 10°C with condensed water, and crystallized for 6 hours. The resulting mother liquor and crude histidine are collected, and the crude histidine is redissolved, the pH is adjusted to 6.5-7.0, and the solution is collected; Step 5) Secondary decolorization: The solution obtained in step 4) was decolorized with activated carbon at a temperature of 50° C. for 60 min, and the filtrate was collected by filtration; Step 6) Purification and concentration: The filtrate obtained in step 5) is concentrated to a histidine content of 220-250 g / L using a four-effect evaporator, cooled to 10°C with condensed water, crystallized for 6 hours, and filtered to obtain a wet product; Step 7) Crystallization and centrifugation: The wet product obtained in step 6) is centrifuged and dried under low temperature vacuum to obtain the L-histidine product.

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