Separation method of l-tryptophan by super-high cross-linking adsorption resin

Abstract

The present application relates to the field of separation and purification of L-Trp, and particularly relates to separation of L-Trp by using super-high cross-linking adsorption resin, wherein a mixed solution of L-Trp and L-Glu is introduced into a fixed bed of super-high cross-linking adsorption resin for adsorption, the height-diameter ratio of the fixed bed is 10:1-25:1, and the loading amount is 2mg / g resin-20mg / g resin. Further, L-Trp and L-Glu are eluted, the elution flow rate is 0.5BV / h-3BV / h, L-Trp and L-Glu are collected at the outlet of the fixed bed, respectively, the purity of the obtained L-Trp and L-Glu products is higher than 98%, the yield is higher than 98%, and the resin does not need to be regenerated by using acid or alkali.

Description

Technical Field

[0001] This invention belongs to the field of L-Trp separation and purification, specifically relating to a method for separating L-Trp from fermentation broth using ultra-high cross-linking adsorption resin. Background Technology

[0002] L-Trp is one of the essential amino acids for humans and animals. It has wide applications in the pharmaceutical, food, and feed industries. In the pharmaceutical industry, L-Trp has been widely used in the treatment of depression, insomnia, hypertension, and pellagra. In the food industry, L-Trp is widely used as a food additive, such as an antioxidant, flavoring agent, and preservative, and can also improve the body's utilization efficiency of plant protein. In the feed industry, L-Trp has been recognized by the Ministry of Agriculture of my country as the third largest amino acid feed additive after lysine and methionine. It can regulate fat metabolism in animals, reduce fat accumulation, increase the protein content in the liver and overall body components of livestock and poultry, and reduce the proportion of fat. Furthermore, L-Trp also has a certain effect on improving the immune function of livestock and poultry. In the agricultural field, L-Trp can be used as an insecticide, acting on some caterpillars, causing them to refuse to eat and die. It can also enhance the stability of two main substances in pesticides, cyclic phosphate and salicylic acid, thereby enhancing their insecticidal effect.

[0003] Microbial direct fermentation using inexpensive carbon sources (glucose, corn flour, wheat bran, etc.) is currently the most widely used method for L-Trp production. After pretreatment to remove pigments and proteins, the L-Trp fermentation broth contains high concentrations of soluble salts (mainly sodium chloride and ammonium sulfate), as well as various byproduct amino acids, such as L-glutamic acid (L-Glu), glycine, and aspartic acid, with L-Glu being the most prevalent. The cost of L-Trp separation and purification accounts for approximately 60% of the total production cost, making it a key step restricting the development of the L-Trp industry. Currently, the main methods used for separating and purifying L-Trp from the fermentation broth include ion exchange and reverse micelle extraction. Yang Xujin used reverse micelle extraction technology to separate L-Trp from the fermentation broth pretreated with microfiltration and ultrafiltration membranes, and then prepared an L-Trp product with a content higher than 98% through nanofiltration, crystallization, and drying (Chinese Patent Publication No.: CN 102382030 A). However, reverse micelle extraction is difficult to apply to the large-scale industrial production of L-Trp. He Xina of Fujian Normal University used cation exchange resin D061 to separate L-Trp from fermentation broth, using 2 mol / L ammonia as the eluent. The collected L-Trp product was decolorized with anion exchange resin and concentrated and crystallized to obtain L-Trp with a purity of 98.4%. However, the regeneration of the cation exchange resin used in this process requires a large amount of strong acid and strong base, and the presence of high concentrations of salt in the fermentation broth will greatly reduce the exchange capacity and separation efficiency of the resin (He Xina. Study on the separation and purification of L-tryptophan in fermentation broth. Fuzhou: Fujian Normal University. 2013). Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for separating L-Trp by ultra-high cross-linking adsorption resin.

[0005] The technical solution adopted in this invention is as follows:

[0006] A method for separating L-Trp using ultra-high crosslinked adsorption resin is disclosed. The separation method is adsorption, and the adsorbent used is a weakly polar ultra-high crosslinked adsorption resin. The backbone of the weakly polar ultra-high crosslinked adsorption resin is polystyrene-divinylbenzene, and the functional group is at least one of carbonyl or ester groups, which are weakly polar groups. The weakly polar ultra-high crosslinked adsorption resin has an average particle size of 0.2-1 mm, a water content of 30%-60%, a most probable pore size of 1-10 nm, a specific surface area of ​​800-1800 m² / g, and a pore volume of 0.5-1.4 cm³ / g.

[0007] Furthermore, in the adsorption method, the raw material liquid used is the L-Trp fermentation broth after solids removal, and its main components are L-Trp, L-Glu and inorganic salts.

[0008] Furthermore, the separation process adopts a fixed-bed operation mode with a height-to-diameter ratio of 10:1 to 15:1. The sample loading amount can reach 25 mg / g resin, the pH of the raw material solution can be 3 to 8.5, and sodium hydroxide with a pH between 10 and 12.5 is used as the eluent with an elution flow rate of 0.5 to 2 BV / h.

[0009] The specific process of adsorption is as follows:

[0010] A. Separation of L-Trp by ultra-high cross-linked adsorption resin

[0011] A certain volume of L-Trp feed solution is passed into the chromatography column, and L-Trp is eluted under certain elution flow rate conditions.

[0012] Detection of BL-Trp and L-Glu concentrations

[0013] This invention uses pre-column derivatization high-performance liquid chromatography (HPLC) for the quantitative detection of L-Glu concentration. The pre-column derivatization step is as follows:

[0014] After diluting the sample by a certain factor, accurately measure 10 μL and transfer it to a 2 mL centrifuge tube. Add 200 μL of derivatization buffer solution and 300 μL of derivatizing reagent solution, and mix thoroughly. Place the centrifuge tube in a 65°C water bath in the dark for 1 hour, then remove it and allow it to cool to room temperature. Add volume buffer to bring the volume to 1.2 mL and shake well. Filter the solution using a 0.22 μm microporous membrane and perform separation and determination using high-performance liquid chromatography (HPLC). The derivatization buffer solution used was an aqueous solution of 42 g / L NaHCO3, the derivatizing reagent was an acetonitrile solution of 10 g / L 2,4-dinitrofluorobenzene, and the volume buffer solution was 6.8 g / L K2HPO4·H2O with 0.1 mol / L NaOH as the solvent.

[0015] The testing conditions are:

[0016] (1) Chromatographic column: C18 column (250mm×4.6mm id, 5μm);

[0017] (2) Mobile phase: A: 50% methanol-water solution; B: 4.1 g / L sodium acetate aqueous solution, with pH adjusted to 6.4 by acetic acid.

[0018] The mobile phase gradient elution program is shown in the table below:

[0019] Table 1 Elution gradient of mobile phase

[0020]

[0021] (3) Mobile phase flow rate: 1.0 ml / min;

[0022] (4) Detection wavelength: 360nm;

[0023] (5) Column temperature: 33℃;

[0024] (6) Injection volume: 10 μL.

[0025] Testing steps:

[0026] (1) Column equilibration: The prepared buffer solution and deionized water were filtered through a 0.22 μm aqueous microporous membrane, and methanol was filtered through a 0.45 μm nylon microporous membrane. After filtration, the column was sonicated for 30 min. The high-performance liquid chromatograph was turned on, and the column was flushed with 16% mobile phase A and 84% mobile phase B at a flow rate of 1 ml / min for about 60 min. The equilibration was completed when the pump pressure stabilized and the baseline tended to be horizontal.

[0027] (2) Sample detection: Write the analysis program and injection sequence according to the detection conditions, place the pretreated standard and sample in the corresponding position of the autosampler according to the injection sequence, start the injection and collect the spectral information, and calculate the concentration of the sample based on the peak area.

[0028] In this invention, a UV-Vis spectrophotometer is used to quantitatively detect the concentration of L-Trp at a wavelength of 218 nm.

[0029] In this invention, BV / h refers to a multiple of the bed volume flowing through the resin column per hour, where BV is the bed volume.

[0030] The beneficial effects of this invention are:

[0031] (1) Applying ultra-high cross-linking adsorption resin to the separation of L-Trp has a good separation effect on L-Trp. It does not require a large amount of strong acid and strong alkali to regenerate the resin, which can greatly reduce the consumption of acid and alkali. At the same time, it can improve the purity of the separated product and improve the separation efficiency.

[0032] (2) The ultra-high cross-linking adsorption resin used in this invention can be used not only for the separation process of L-Trp, but also for the separation of other small molecule amphoteric compounds with hydrophobic groups such as L-phenylalanine and p-amino acid benzoic acid. Attached Figure Description

[0033] Figure 1 Elution curves of L-Trp and L-Glu in a fixed bed of ultra-high cross-linked adsorption resin NHP-200. Detailed Implementation

[0034] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the specific material ratios, process conditions, and results described in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as described in detail in the claims.

[0035] Example 1: Fixed-bed separation of L-Trp and L-Glu.

[0036] (1) Column mounting

[0037] The pretreated ultra-high cross-linked adsorption resin NHP-200 was packed into the chromatography column using a wet packing method. The resin packing amount was 10.5 g, and the inner diameter of the chromatography column was 1 cm.

[0038] (2) Sample loading and elution

[0039] The liquid at the top of the resin was allowed to flow out to a depth of approximately 1.5 cm above the resin surface. Then, 20 mL of a mixed solution of L-Trp and L-Glu was loaded into the chromatography column at an injection rate of 0.4 mL / min. Elution was then initiated with sodium hydroxide solution at pH 12 at a flow rate of 0.4 mL / min, with samples collected every 10 min at the column outlet until L-Trp and L-Glu were completely eluted from the resin. The concentration of L-Glu in the sample was analyzed using high-performance liquid chromatography (HPLC), and the concentration of L-Trp was analyzed using UV-Vis spectrophotometry. The elution curves for L-Trp and L-Glu are shown below. Figure 1 As shown, the purity and yield of both L-Trp and L-Glu products were higher than 98%.

[0040] Example 2: Fixed-bed separation of L-Trp and L-Glu.

[0041] (1) Column mounting

[0042] The pretreated ultra-high cross-linked adsorption resin NHP-200 was packed into the chromatography column using a wet packing method. The resin packing amount was 10.5 g, and the inner diameter of the chromatography column was 1 cm.

[0043] (2) Sample loading and elution

[0044] The liquid at the top of the resin was allowed to flow out to a depth of approximately 1.5 cm above the resin surface. Then, 1.5 mL of a mixed solution of L-Trp and L-Glu was loaded into the chromatography column at an injection rate of 0.2 mL / min. Elution was then initiated with sodium hydroxide solution at pH 12 at a flow rate of 0.2 mL / min, with samples collected every 15 min at the column outlet until L-Trp and L-Glu were completely eluted from the resin. The concentration of L-Glu in the samples was analyzed using high-performance liquid chromatography (HPLC), and the concentration of L-Trp was analyzed using UV-Vis spectrophotometry. The purity of both L-Trp and L-Glu products was greater than 99%, and the yield was greater than 98%.

Claims

1. A method for separating L-Trp using ultra-high cross-linked adsorption resin, characterized in that, The separation method used is adsorption, and the adsorbent used is a weakly polar ultra-high cross-linked adsorption resin. The weakly polar ultra-high crosslinked adsorption resin has a polystyrene-divinylbenzene backbone and a carbonyl functional group. The resin has an average particle size of 0.2–1 mm, a water content of 30%–60%, a most probable pore size of 1–10 nm, and a specific surface area of ​​800–1800 m². 2 / g, pore volume 0.5~1.4cm 3 / g; In the adsorption method, the feed liquid used is the L-Trp fermentation broth after solids removal, and its main components are L-Trp, L-Glu and inorganic salts. The adsorption separation process employs a fixed-bed operation mode with a height-to-diameter ratio of 10:1 to 15:

1. The sample loading amount can reach 25 mg / g resin, and the pH of the raw material solution can be 3 to 8.

5. Sodium hydroxide with a pH between 10 and 12.5 is used as the eluent, and the elution flow rate is 0.5 to 2 BV / h.

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