Method for purifying histidine from escherichia coli fermentation liquor

Abstract

The invention provides a method for purifying histidine from Escherichia coli fermentation liquor, which comprises the following steps: taking L-histidine fermentation liquor, adjusting the pH value to 4-5 at 60-70 DEG C, and carrying out microfiltration to remove thalli in the fermentation liquor to obtain L-histidine microfiltrate; adding activated carbon into the L-histidine micro-filtrate for decoloration to obtain an L-histidine decoloration solution; carrying out concentration, crystallization and centrifugal separation on the eluted material liquid to obtain an L-histidine crude product and an L-histidine primary mother solution; redissolving the L-histidine crude product to obtain a histidine heavy solution; adding activated carbon into the histidine heavy solution for decoloration to obtain an L-histidine decoloration solution; concentrating, crystallizing and centrifugally separating the decolored L-histidine solution to obtain an L-histidine refined product and an L-histidine secondary mother solution, and drying the L-histidine refined product to obtain a finished product. The L-histidine is purified from the L-histidine fermentation liquor by adopting an ion exchange chromatographic separation technology, so that the overall extraction yield of the L-histidine is improved, and the production cost of the L-histidine is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation, in particular to a method for purifying histidine from Escherichia coli fermentation liquid. Background Art

[0002] L-Histidine, chemically known as L-α-amino-β-imidazole propionic acid, is a basic amino acid containing an imidazole nucleus in its molecule and is an essential amino acid. Histidine was first isolated by German physicist Albrecht Kossel in 1896. In the field of nutrition, histidine is considered an essential amino acid for humans, mainly for children. After adulthood, humans can begin to synthesize histidine themselves. Adding a small amount of histidine to the diet of patients with chronic uremia increases the rate at which the amino acid binds to hemoglobin and alleviates renal anemia, so histidine is also an essential amino acid for patients with uremia.

[0003] L-histidine has multiple physiological functions and is widely used in the pharmaceutical, feed and food industries. In particular, its role in medical research is increasingly valued. At present, L-histidine, L-arginine and L-serine are urgently needed amino acid varieties in the market. Therefore, it is of great practical significance to develop a method for purifying L-histidine to improve the extraction yield of L-histidine. Summary of the invention

[0004] The invention proposes a new idea for purifying histidine, a method for purifying histidine from Escherichia coli fermentation broth, and improves the extraction yield of L-histidine.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] A method for purifying histidine from Escherichia coli fermentation broth, characterized in that it comprises the following steps:

[0007] (1) taking L-histidine fermentation broth, adjusting the pH value to 4-5 at 60-70° C., removing the bacteria in the fermentation broth by microfiltration, and obtaining L-histidine microfiltrate; (2) adding activated carbon to the L-histidine microfiltrate for decolorization, and obtaining L-histidine decolorized liquid; (3) passing the L-histidine decolorized liquid into an ion exchange chromatography system, performing ion exchange chromatography treatment, and collecting the eluted liquid; (4) concentrating, crystallizing, and centrifuging the eluted liquid to obtain L-histidine crude product and L-histidine primary mother liquor; (5) redissolving the L-histidine crude product to obtain a histidine heavy solution; (6) adding activated carbon to the histidine heavy solution for decolorization, and obtaining L-histidine decolorized liquid; (7) concentrating, crystallizing, and centrifuging the L-histidine decolorized liquid to obtain L-histidine fine product and L-histidine secondary mother liquor, and drying the L-histidine fine product to obtain a finished product.

[0008] Preferably, the L-histidine primary mother liquor obtained in step (4) is added to the activated carbon decolorization liquid in step (2), and the decolorization operation is repeated.

[0009] Preferably, the L-histidine secondary mother liquor obtained in step (7) is added to the elution solution in step (4), and the concentration, crystallization and centrifugation operations are repeated.

[0010] Compared with the existing known technologies, the technical solution provided by the present invention has the following significant effects:

[0011] The invention adopts ion exchange chromatography separation technology to purify L-histidine from L-histidine fermentation broth, thereby improving the overall extraction yield of L-histidine and reducing the production cost of L-histidine. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the method for purifying histidine of the present invention. DETAILED DESCRIPTION

[0013] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments.

[0014] A method for purifying histidine from Escherichia coli fermentation broth comprises the following steps:

[0015] Step 1: Take L-histidine fermentation broth with a content of 60-80 g / L, and adjust the pH value to 4-5 with HcL at 60-70°C.

[0016] Step 2: adding activated carbon to the L-histidine microfiltrate for decolorization to obtain an L-histidine decolorized solution;

[0017] In step 2, the microfiltration treatment uses a ceramic membrane with a membrane material of TiO2 / ZrO2; a multi-channel ceramic membrane element with a pore size of 40-60nm, preferably 50nm;

[0018] In order to achieve better separation, during the microfiltration process, the L-histidine fermentation broth is passed through a ceramic membrane, the filtration temperature is controlled at 60-70°C, the feed pressure is 0.2-0.25MPa, and the discharge pressure is 0.15-0.23MPa. When the volume of the concentrate is 35%-40% of the initial volume of the fermentation broth, 30-35% of the initial volume of the fermentation broth is added with deionized water for dialysis to obtain a dialysate, and the dialysate is combined with the microfiltrate to form a microfiltration mixed solution, and then subjected to subsequent treatment.

[0019] Step 3: passing the L-histidine decolorizing solution into an ion exchange chromatography system, performing ion exchange chromatography treatment, and collecting the eluted solution;

[0020] During decolorization, the amount of activated carbon added is 3-5% of the histidine content, the decolorization temperature is 60-70°C, the decolorization time is 30 minutes, and a plate and frame are used for decolorization filtration. The decolorized solution is refluxed in the plate and frame for 10 minutes before discharging. The material can be discharged when T430 is 50%. When T430 is below 50%, appropriate activated carbon (not exceeding 5% of the histidine content) is added for re-decolorization.

[0021] The stationary phase used in the ion exchange chromatography is a cation exchange chromatography of hydrogen ions, and the mobile phase is water. The particle size of the hydrogen ion exchange resin is 0.2-0.3 mm.

[0022] In order to improve the separation effect of chromatographic treatment, the conditions of chromatographic treatment are: chromatographic column temperature 35-65°C, feed flow rate 0.5-1.0BV / h, elution water flow rate 1.0-1.5BV / h, discharge volume 0.5-0.9BV / h, and wastewater output 1.0-1.3BV / h; further preferably, chromatographic column temperature 40-45°C, feed flow rate 0.7BV / h, elution water flow rate 1.5BV / h, discharge volume 0.7BV / h, and wastewater output 1.2BV / h.

[0023] There is no special limit to the scale of ion exchange chromatography separation, which is mainly determined by the scale of the feed liquid to be treated. For example, when the processing volume is small, one ion exchange chromatography column can be used for separation; when industrial large-scale processing is carried out, multiple ion exchange chromatography columns can be connected in series for separation, such as 9, 12, 24, etc. When multiple chromatography columns are connected in series for separation, the feed inlet, elution water inlet, product outlet and wastewater outlet are determined by the commonly used technical means in the field.

[0024] Step 4: Concentrating, crystallizing, and centrifuging the eluted liquid to obtain a crude L-histidine product and a primary L-histidine mother liquor;

[0025] In order to improve the crystallization yield, in step 4, the product liquid is concentrated to a histidine content of 400-450 g / L, then cooled to 15-25°C for crystallization. After the crystallization, a 1250 type vertical scraper centrifuge is used for solid-liquid separation to obtain crude L-histidine and a primary mother liquor of L-histidine. Further optimization is performed, the product liquid is concentrated to a content of 400 g / L, then cooled to 20°C for crystallization. After half an hour, the mixture is stirred at a speed of 20-30 rpm and centrifuged after two hours.

[0026] It is further preferred to use centrifugation to separate the solid and liquid, and add ion-free water equivalent to 5%-10%, preferably 7%, of the volume of the L-histidine crystallization mother liquor to wash the crystals.

[0027] It should be emphasized that the L-histidine primary mother liquor obtained in step 4 can also be added to the activated carbon decolorization liquid in step 2, and the decolorization operation is repeated. Specifically, the amount of activated carbon added when decolorizing the L-histidine primary mother liquor is 3-5% of the histidine content, the decolorization temperature is 60-70°C, the decolorization time is 30 minutes, the decolorization filtration uses a plate and frame, and the decolorized solution is refluxed in the plate and frame for 10 minutes before discharging (T430 has no requirement), and the decolorized solution is added to the microfiltration mixed solution to enter the ion exchange system.

[0028] Step 5: re-dissolving the crude L-histidine to obtain a histidine heavy solution;

[0029] In step 5, the crude histidine is redissolved in water at a temperature of 60-80° C. and a redissolved concentration of 70-90 g / L.

[0030] Step 6: adding the histidine heavy solution to activated carbon for decolorization to obtain L-histidine decolorized solution;

[0031] In step 6, the re-dissolved histidine solution is decolorized, the amount of activated carbon added is 3-5% of the histidine content, the decolorization temperature is 60-70°C, the decolorization time is 30 minutes, and the decolorization filtration uses a plate and frame. The decolorized solution is refluxed in the plate and frame for 10 minutes before discharging. The material can be discharged at T430 of 90%. If T430 is below 90%, appropriate activated carbon (not exceeding 5% of the histidine content) is added for re-decolorization.

[0032] Step 7: Concentrate, crystallize and centrifuge the L-histidine decolorizing solution to obtain L-histidine fine product and L-histidine secondary mother liquor, and dry the L-histidine fine product to obtain a finished product.

[0033] In steps 6 and 7, the decolorized liquid is concentrated to a content of 400-450 g / L and crystallized at 15-25°C. Preferably, the product liquid is concentrated to a content of 400 g / L and then cooled to 20°C for crystallization. After half an hour of crystallization, the liquid is stirred at a speed of 20-30 rpm and centrifuged after two hours. The solid-liquid separation is performed by centrifugation in a 1250 type vertical scraper centrifuge, and 5%-10%, preferably 7% of the volume of the L-histidine crystal mother liquor is added to wash the crystals to obtain L-histidine fine products and L-histidine secondary mother liquor. The L-histidine crystals are dried at 55-65°C to obtain the L-histidine finished product.

[0034] One point that needs to be emphasized is that the L-histidine secondary mother liquor obtained in step 7 can also be added to the elution liquid in step 4, and the concentration, crystallization, and centrifugation operations are repeated, and concentrated together with the elution liquid to achieve the recycling of materials.

[0035] The novel method for purifying histidine proposed by the invention provides a new idea for purifying histidine, and the method for purifying histidine improves the overall extraction yield of L-histidine and reduces the production cost of L-histidine.

Claims

1. A method for purifying histidine from Escherichia coli fermentation broth, characterized in that: The steps include: (1) taking L-histidine fermentation broth, adjusting the pH value to 4-5 at 60-70° C., and removing the bacteria in the fermentation broth by microfiltration to obtain L-histidine microfiltrate; (2) adding activated carbon to the L-histidine microfiltrate for decolorization to obtain an L-histidine decolorized solution; (4) concentrating, crystallizing, and centrifuging the eluted liquid to obtain a crude L-histidine product and a primary L-histidine mother liquor; (5) re-dissolving the crude L-histidine to obtain a histidine heavy solution; (6) adding activated carbon to the heavy histidine solution for decolorization to obtain an L-histidine decolorized solution; (7) The L-histidine decolorizing solution is concentrated, crystallized, and centrifuged to obtain L-histidine fine product and L-histidine secondary mother liquor, and the L-histidine fine product is dried to obtain a finished product.

2. A method for purifying histidine from Escherichia coli fermentation broth according to claim 1, characterized in that: The L-histidine primary mother liquor obtained in step (4) is added to the activated carbon decolorization liquid in step (2), and the decolorization operation is repeated.

3. A method for purifying histidine from Escherichia coli fermentation broth according to claim 1, characterized in that: The L-histidine secondary mother liquor obtained in step (7) is added to the elution solution in step (4), and the concentration, crystallization and centrifugation operations are repeated.

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