KR102282386B1 - Method for Preparing Natural L-cysteine Crystals by Continuous Chromatography - Google Patents

Abstract

The present application relates to a method for preparing natural L-cysteine crystals and to L-cysteine crystals prepared by the method. Through the method for preparing L-cysteine crystals of the present application, L-cysteine crystals can be obtained with high recovery and/or purity from natural L-cysteine fermentation broth without chemical reaction or use of artificial synthetic compounds.

Description

Method for Preparing Natural L-cysteine Crystals by Continuous Chromatography

The present application relates to a method for preparing L-cysteine crystals and to L-cysteine crystals prepared by the method.

In general, L-cysteine is produced by decomposing L-cystine derived from animals, such as duck down or human hair, or L-cystine, derived from fermentation, using microbial metabolites, into L-cysteine using an electrochemical reduction reaction. there is. In contrast, as a method for producing L-cysteine using a microorganism, a fermentation process (US8802399B, US6946268B) that produces natural L-cysteine using a strain modified with O-acetyl transferase in a medium containing sulfide (US8802399B, US6946268B), a microorganism culture method A process for preparing natural L-cysteine (WO2013/089478 and WO2012/053794) is known by mixing the prepared O-phosphohomoserine with sulfide and inducing an enzyme-catalyzed reaction using O-phosphoserine sulfidylase. .

It is known that L-cysteine prepared by the microbial culture method can be separated through ion exchange chromatography and other known methods, but information such as specific methods, yields, and purity are not described at all (EP1645623A1, EP1298200B1, US20050221453A1, EP1234874A1, EP1571223A2, and EP1650296A).

For example, after adjusting the pH of the fermentation broth containing L-cysteine down to 5 or less, it is contacted with an acidic cation exchanger or a strongly acidic cation exchanger to adsorb L-cysteine to the ion exchanger, and the adsorbed L-cysteine is dissolved in an aqueous hydrochloric acid solution. A method of eluting and purifying with a yield of 90% or more is known (US8088949B).

In addition, the fermentation broth containing L-cysteine having a pH of 6 to 9 is contacted with a basic anion exchanger to adsorb L-cysteine to the ion exchanger, and the adsorbed L-cysteine is eluted in an aqueous hydrochloric acid solution, followed by acidic cations at pH 4 or less. A method of adsorbing L-cysteine to an ion exchanger by contact with an exchanger and eluting the adsorbed L-cysteine in an aqueous hydrochloric acid solution to purify in a yield of 85% or more has been known (US9120729B).

However, in the above method, a chemical reaction proceeds through repeated ion exchange steps, and a large amount of eluent is used as a subsequent process of the ion adsorption process, so a large amount of process water is required, and additional purification steps must be performed. There are disadvantages.

On the other hand, there is a method of purifying by a chemical method using a strong acid solvent (Chinese Patent Publication No. 105274554), but a strong acid solvent must be used in a large amount, and a strong basic solvent for neutralization thereof must also be used in large amount, so it is an environmental problem. can cause

Therefore, there is a continuous need for a purification method for isolating L-cysteine with higher yield and purity.

Under this background, the present inventors have made intensive efforts to increase the purity and yield of L-cysteine crystals, and as a result, a purification method having advantages of not only high yield and purity, but also efficient productivity improvement and low water usage, has been completed.

One object of the present invention is to provide a method for preparing L-cysteine crystals.

Another object of the present invention is to provide an L-cysteine crystal prepared according to the method for preparing the L-cysteine crystal.

Hereinafter, the present invention will be described in more detail.

On the other hand, each description and embodiment disclosed herein may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

One aspect of the present application for solving the above problem is to obtain a separation solution after (a) a fermentation broth of pH 3.0 to 9.0 containing L-cysteine is injected into a continuous chromatography apparatus using a strongly acidic cation exchange resin as a stationary phase. to do;

(b) concentrating the separated solution; and

(c) recovering L- cysteine crystals from the concentrate, it is a method for producing L-cysteine crystals.

As used herein, "L-cysteine" is one of the amino acids constituting a protein, and is the only sulfur-containing amino acid having a thiol group (R-SH) among L-amino acids. L-cysteine may be chemically synthesized or biologically prepared through microbial fermentation, etc., but is not limited thereto. Specifically, in the present application, L-cysteine may be L-cysteine produced biologically through microbial fermentation, and O-phosphohomoserine and sulfide, which are precursors produced through microbial fermentation, are converted into an enzyme in the presence of phosphoserine sulfidylase. It may be a natural L-cysteine obtained by inducing a catalytic reaction. The natural L-cysteine may be L-cysteine that has not undergone a chemical reaction, chemical adsorption, or elution step in the manufacturing process.

As used herein, the term “natural” means not caused by a chemical reaction. As published in EU Flavorings Regulation 1334/2008, only substances obtained by physical, enzymatic or microbial processes are defined as "natural" flavorings. In view of the above, both L-cysteine produced by the electrochemical reduction reaction of L-cystine, regardless of animal-derived and microbial fermentation-derived origin, cannot be called natural.

As used herein, the term "fermentation solution" refers to a culture solution obtained by culturing a microorganism producing L-cysteine, a culture containing a microorganism cultured with the medium, or an enzyme conversion solution containing a precursor and an enzyme capable of producing L-cysteine. means Specifically, the fermentation broth containing L-cysteine may be a culture broth or enzyme conversion solution containing natural L-cysteine. More specifically, it may be an L-cysteine culture or a culture solution biologically produced through microbial fermentation having L-cysteine-producing ability, or O-phosphohomoserine and sulfide, which are precursors produced through microbial fermentation, are mixed with phosphoserine sulfide. It may be an enzymatic conversion solution of natural L-cysteine obtained by inducing an enzyme-catalyzed reaction in the presence of drillase. The L-cysteine crystals prepared by using the fermentation broth as a raw material solution do not undergo a chemical reaction and can be referred to as natural L-cysteine.

Herein, the fermentation broth may be used as a raw material solution for a continuous chromatography process. That is, it may be injected into the continuous chromatography apparatus of step (a).

The pH of the fermentation broth injected into the continuous chromatography apparatus may be different depending on the preparation method, but may be 2.5 to 10.0, 2.5 to 9.5, 3.0 to 9.0, 3.5 to 8.5, 3.5 to 7.5, 4.5 to 7.0, 5.0 to 6.0. may be pH. The fermentation broth itself can also be used as a raw material for a continuous chromatography process, and the fermentation broth containing L-cysteine before step (a) is 2.5 to 9.5, specifically 3.0 to 9.0, more specifically 3.5 to It may further include the step of adjusting the pH to be 8.5, more specifically 3.5 to 7.5, 4.5 to 7.0, or 5.0 to 6.0. However, it is not limited thereto. For example, it may be adjusted by adding an acid such as sulfuric acid or hydrochloric acid, or a base such as sodium hydroxide (caustic soda), ammonia, lithium hydroxide, or potassium hydroxide, but is not limited thereto. The pH adjusting agent may be appropriately selected and used by those skilled in the art as long as crystals of L-cysteine can be finally obtained without affecting the structure of L-cysteine.

The lower the pH of the L-cysteine fermentation broth, the stronger the tendency for L-cysteine to be cationized. Since the stationary phase of the continuous chromatography used herein is a strongly acidic cation exchange resin, it tends to adsorb cations, and when L-cysteine is cationized, some adsorbed to the stationary phase may decrease the recovery rate of the continuous chromatography process. In addition, the higher the pH, the stronger the tendency for L-cysteine to be oxidized and converted to L-cystine. As a result, there may be a tendency to decrease the recovery rate of the continuous chromatography process. Accordingly, the fermentation broth containing L-cysteine is a fermentation broth having a pH of 2.5 to 9.5, specifically 3.0 to 9.0, more specifically 3.5 to 8.5, even more specifically 3.5 to 7.5, 4.5 to 7.0, or 5.0 to 6.0. can

However, the recovery rate of the continuous chromatography process is affected by various process variables such as the flow rate between the resin tower sections of the continuous chromatography process, the process temperature, the composition of the mobile phase, and the continuous chromatography sequence. The recovery rate is not a factor limited only by the pH of the continuous chromatography stock solution.

Herein, the fermentation broth containing L-cysteine before step (a) may further include a step of diluting or concentrating. This step may be performed before or after the step of adjusting the pH described above.

The concentration may be performed in a conventional concentrator (eg, a forced circulation concentrator, a thin film concentrator, or a rotary concentrator).

The L-cysteine concentration of the diluted or concentrated fermentation broth may be adjusted to 10 to 180 g/L, specifically 10 to 150 g/L, but the recovery rate of the continuous chromatography process and the separation obtained through the continuous chromatography process Since it is not a factor that greatly affects the quality of the liquid (specifically, the L-cysteine content of the solids excluding water in the separated liquid), the concentration of the fermentation broth containing L-cysteine used as a raw material for the continuous chromatography process is controlled. It is not an essential process for isolating and purifying L-cysteine. However, when the concentration is adjusted to 180 g/L or more, the concentration of L-cysteine is higher than the solubility of L-cysteine, and low-quality L-cysteine crystals unsuitable for recovery occur, which may cause a decrease in the recovery rate of the continuous chromatography process. . If the concentration of the raw material solution for the continuous chromatography process is high, the amount of water used in the continuous chromatography process can be reduced compared to the amount of L-cysteine treated. Such a characteristic cannot be found in the ion exchange process, where the amount of water used is determined by the maximum adsorption amount of L-cysteine and the ion exchange resin.

As used herein, the term "continuous chromatography" (Continuous Chromatography) refers to a process that is a continuous development of the existing batch chromatography process. Specifically, the solid phase and the liquid phase may be continuously supplied into the chromatography apparatus, and the solid phase and the liquid phase move in opposite directions to cause countercurrent contact, thereby enabling more efficient separation of substances. Herein, it may be used as a concept including True Moving Bed (TMB) chromatography and Simulated Moving Bed (SMB) chromatography. In addition, since the two true moving bed chromatography and the similar moving bed chromatography have the same principle, a person skilled in the art may appropriately select and use it in consideration of productivity and other matters.

When the continuous chromatography process is used herein, since no adsorption/elution process is required, productivity per hour is higher than that of the ion exchange process, and the use of process water can be reduced. In addition, in order to obtain an L-cysteine powder product in high yield from a process solution containing L-cysteine obtained through an ion exchange process or a general chromatography process, a lot of energy is required for the concentration crystallization process. can reduce

As an embodiment of the present application, an SMB chromatography apparatus may be used, and a schematic diagram of the SMB chromatography apparatus is shown in FIG. 2 . Process variables such as the number of resin towers, the volume of the resin tower, the amount of resin filling, the flow rate in each section, whether or not a buffer tank is installed, and the transfer time of the resin tower can be changed, and are not limited to the fixed conditions specified above.

The stationary phase of the continuous chromatography apparatus may be an ion exchange resin, specifically

It may be a strongly acidic cation exchange resin. The functional group of the strongly acidic cation exchange resin may be a sulfuric acid group, but is not limited thereto. In addition, the core material of the strongly acidic cation exchange resin used herein is not limited as long as it can attach a strongly acidic functional group. For example, a styrene-divinylbenzene copolymer may be used, but is not limited thereto. As a specific example, the strongly acidic cation exchange resin may be a styrene sulfate-divinylbenzene copolymer, but is not limited thereto.

In the present application, as another type of stationary phase frequently used in the separation and purification of amino acids in the art, a functional group-free exchange resin such as a functional group-free styrene-divinylbenzene copolymer and a functional group-free methacrylate polymer; strongly basic anion exchange resins such as trimethylamine styrene-divinylbenzene copolymer; weakly basic anion exchange resins such as tertiary amine styrene-divinylbenzene copolymers; Weakly basic cation exchange resins such as carboxyl methacrylate polymers are difficult to purify to 50% (w/w) or more of the L-cysteine content of the solid in the separation solution obtained through the continuous chromatography process. On the other hand, when a strongly acidic cation exchange resin such as styrene sulfate-divinylbenzene copolymer is used, the L-cysteine content of the solid content excluding water in the separation solution obtained through the continuous chromatography process is 80% (w/w) or more , specifically, it can be purified to 90% (w/w) or more.

In the chromatography apparatus, water, caustic soda diluted solution, sulfuric acid diluted solution, phosphoric acid diluted solution, hydrochloric acid diluted solution, potassium hydroxide diluted solution, or their Mixtures may be used, but are not limited thereto. As a specific example, water may be used as the mobile phase of continuous chromatography. When a mobile phase including a chemical compound is used, the chemical compound may remain in the final product, and for this reason, the product may not be sold or distributed as a natural product because it exceeds the residual standard for chemical compound. In addition, as chemical substances other than water are not added to the process water, product cost savings can be expected. In the ion exchange process, in order to elute the adsorbed L-cysteine, a solvent such as hydrochloric acid or sulfuric acid must be used as an eluent, and therefore, an increase in product cost associated with the use and disposal of the chemical is inevitably accompanied.

When the fermentation broth containing L-cysteine in step (a) is injected into a continuous chromatography apparatus, a separation liquid containing L-cysteine is separated according to a continuous chromatography process to obtain a separation liquid. Herein, the separation solution containing the L-cysteine is separated may be briefly expressed as “separation solution”, “chromatographic separation solution”, or “process solution”.

The quality of the separated solution may be evaluated by the content of L-cysteine in the solids except for water in the separated solution. The separation solution of the present application may have an L-cysteine content of 80% (w/w), specifically 85% (w/w), and more specifically 90% (w/w) of solids excluding water. When the L-cysteine content of the solid content excluding water in the separated solution is 80% or more, L-cysteine crystals with a purity of 90% or more can be prepared. Specifically, the L-cysteine content of the solid content excluding water in the separated solution is 90% or more. % or more, L-cysteine crystals having a purity of 98% or more can be prepared.

However, since the purity of L-cysteine crystals is also affected by other detailed process conditions such as concentration, crystallization, and crystal separation, the purity of L-cysteine crystals depends on the L- It is not a factor limited only by the cysteine content.

Herein, step (a) may be expressed as a "continuous chromatography process". "Continuous chromatography process recovery" means the L-cysteine recovery ratio of the obtained separation solution compared to the fermentation broth injected in step (a), and is used to evaluate the efficiency of the process. The recovery of the continuous chromatography process herein may be 50% (w/w), specifically 60% (w/w), more specifically 70% (w/w), more specifically 80% (w/w) or more. However, the present invention is not limited thereto.

Step (b) is a step of concentrating the separation solution for crystallization. In step (b), the separated solution may be concentrated to obtain a concentrated solution. The concentration may be carried out in a conventional concentrator (eg, a forced circulation concentrator, a thin film concentrator, or a rotary concentrator) by the appropriate selection of a person skilled in the art. (b) the concentration of L-cysteine in the step concentrate may be 100 to less than 800 g / L, specifically 200 to less than 800 g / L, more specifically 300 to less than 800 g / L, and even more specifically 300 to less than 800 g / L It may be 500 g/L, but is not limited thereto.

If the concentration of L-cysteine in the concentrate is low, crystallization is not induced during concentration due to the lack of supersaturation required for nucleation and crystal growth, and crystallization may be performed during cooling or crystallization may proceed through an additional process. However, the recovery rate may be low or the crystallization time may be long. When the concentration of L-cysteine in the concentrate is 300 g/L or more, L-cysteine crystal nuclei may be formed during the concentration process. Additionally, cooling the L-cysteine slurry concentrated to 300 g/L or more may increase the recovery rate of the L-cysteine crystallization process. When the L-cysteine concentration of the concentrate is 800 g/L, a solidification phenomenon may occur due to the generation of a large amount of crystal particles, which may make it impossible to stir the crystal slurry and separate the crystals. Preferably, the concentration of L-cysteine in the separation solution is 100 to less than 800 g/L, specifically 200 to less than 800 g/L, more specifically 300 to less than 800 g/L, or 300 g/L to 500 g/L. It can be adjusted in g/L. When the cooling crystallization is performed with the separation solution having the above concentration, the purity of the L-cysteine crystal may be 98% or more. However, since the purity of the L-cysteine crystal is also affected by other detailed process conditions such as concentration, crystallization, and crystal separation, the purity of the L-cysteine crystal is determined by the L-cysteine concentration of the concentrated solution produced by SMB chromatography. It may not be a factor limited to

L-cysteine crystallization may occur during the concentration according to step (b). As used herein, the term “crystallization” refers to a phenomenon in which a liquid or a solid in an amorphous state forms a crystal, and two phenomena of the generation of crystal nuclei and the growth of the crystal nuclei occur concurrently.

The concentrate may form and/or grow crystal nuclei through cooling and/or aging before recovery. In addition, even if L-cysteine crystals do not precipitate in the concentrate, crystals may be formed when the concentrate is cooled and/or aged.

The cooling step may specifically mean cooling to a temperature of -10 to 55 °C over 2 hours to 6 hours, specifically 0 to 45 °C over 2 hours to 6 hours, more specifically It may mean cooling to a temperature of 0 to 30 °C, and more specifically, may mean cooling to a temperature of 0 to 15 °C over 2 to 6 hours.

The aging may mean leaving it to stand without a change in temperature. Herein, it may mean maintaining the cooled temperature constant, or it may mean maintaining the temperature of the concentrate constant even when cooling is not performed. Specifically, it may be aged for 1 to 3 hours.

Step (b) herein may be expressed as a “crystallization process”. The term “crystallization process recovery rate” refers to the L-cysteine recovery rate of the obtained L-cysteine crystal compared to the L-cysteine of the separated solution according to the continuous chromatography process, and is used to evaluate the efficiency of the crystallization process. Herein, the recovery of the continuous chromatography process is 50% (w/w), specifically 60% (w/w), more specifically 70% (w/w), even more specifically 80% (w/w) or more. can

In step (c), precipitated cysteine crystals may be recovered from the concentrate. Specifically, L-cysteine crystals may be recovered from the slurry by solid-liquid separation of the concentrate. This may proceed using a solid-liquid separator such as a reduced pressure membrane filtration device, a pressure membrane filtration device, and a centrifugal separator, but is not limited thereto. The slurry and/or the precipitated cysteine crystals may be additionally washed or dried.

Herein, the filtrate obtained after obtaining L-cysteine crystals in step (c) is the mother liquor in which L-cysteine remains, and in order to improve the final purification recovery rate of L-cysteine, the fermentation broth of step (a) or step (b) It may be added in whole or in part to the separation solution of the, but is not limited thereto.

The purity of the L-cysteine crystals prepared according to the preparation method of the present application may be 95% (w/w) or more, specifically 98% (w/w), more specifically 99% (w/w).

Another aspect of the present invention for solving the above problems is to provide an L-cysteine crystal prepared according to the above-described method for producing an L-cysteine crystal.

The L-cysteine crystal and its preparation method are as described above.

The method for producing L-cysteine crystals of the present application isolates and purifies the L-cysteine fermentation broth in its natural state without the use of chemical reactions or artificial synthetic compounds, and at the same time, L-cysteine crystals can be obtained with high recovery and/or purity. there is. In addition, the manufacturing method of the present application exhibits efficient productivity, and in particular, the amount of water used can be significantly reduced.

1 shows a representative example of a process for preparing L-cysteine crystals using a continuous chromatography process from L-cysteine in a natural state contained in a fermentation broth.
Figure 2 shows the arrangement of the resin tower and the flow rate for each section of the SMB chromatography process used in one embodiment of the present application.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Test Methods

A common analysis method used in the examples of the present application is as follows.

(1) HPLC method for quantitative analysis of L-cysteine

HPLC analysis conditions for analyzing the purity and concentration of L-cysteine herein are as follows:

Instrument: HPLC 1260 Infinity System (Agilent Technology Inc.)

Column: HP C18 (150 mm × 3.9 mm; 5 μm)

Mobile phase: Acetonitrile/Water/Heptafluorobutyric acid (8/92/0.1)

Flow rate: 0.425 mL/min

Temperature: 30 degrees

Detection: UV at 220 nm

Sample injection volume: 2 uL

(2) Method for measuring purity of L-cysteine crystals

Herein, the quality of L-cysteine crystals is evaluated based on the purity of L-cysteine, and the method follows the following sequence:

(a) removing residual moisture by placing the L-cysteine crystals in a vacuum dryer containing silica gel at a vacuum degree of 20 mmHg or less for 24 hours, and cooling the L-cysteine crystals to room temperature;

(b) quantifying 0.5000 g of L-cysteine crystal cooled to room temperature, putting it into a 1 L quantitative flask, and diluting it with 0.2N HCl solution to prepare a sample of 0.5000 g/L;

(c) L-cysteine standard crystal (≥98.5%) was treated in the same manner as in step (a), weighed 0.5000 g, put into a 1 L metering flask, diluted with 0.2N HCl solution, and 0.5000 g/L of sample After confirming the purity of the standard product through the certificate of the standard reagent manufacturer, the step of converting the concentration of L-cysteine in the sample (converted [L-cysteine concentration] is 0.5000 g/L × [standard product] water]); and

(d) using the sample prepared in step (c) as an external standard and analyzing the sample prepared in step (b) by HPLC to analyze the purity of the L-cysteine crystal used in step (a)

(3) Method for analyzing the content of L-cysteine based on the solid content in the fermentation broth or the separation solution according to the chromatography process

Herein, the quality of the fermentation broth or the separation liquid according to the chromatography process is evaluated based on the content of L-cysteine in the solid obtained by removing water from the solution, and the method follows the following sequence:

(a) Deodorize about 5 g of sea sand (10 to 20 mesh; Daejeong Hwageum) in a ceramic container, place it in a forced circulation oven at 105 ° C for 3 hours to remove residual moisture, and then place it in a vacuum dryer containing silica gel for 1 hour. cooling the temperature to room temperature;

(b) putting the solution to be analyzed in the container of step (a), and quantifying [the mass of the solution] using the difference in weight before and after the addition;

(c) After removing the residual moisture by placing the container of step (b) in a forced circulation oven at 105 ° C for 3 hours, place it in a vacuum dryer containing silica gel for 1 hour to cool the temperature to room temperature, and use the weight difference The step of quantifying the amount of water removed and using it to convert the content based on the mass of the solid content of the solution to be measured ([content based on mass of solid content] is ([mass of solution] - [mass of removed water]) / [weight of solution] mass]);

(d) measuring the density of the solution to be analyzed using a specific gravity suspension meter;

(e) measuring the concentration of L-cysteine in the solution to be analyzed using HPLC; and

(f) converting the content of L-cysteine in the solid from which water has been removed from the solution to be analyzed using the content, density, and L-cysteine concentration based on the solid content of the solution ([L- Cysteine content] is [Concentration of L-cysteine] / [Density of solution] / [Content based on mass of solid content of solution])

manufacturing example

(1) Preparation of fermentation broth containing L-cysteine

After culturing a microorganism capable of producing O-phosphoserine in a fermentation medium to obtain an O-phosphoserine fermentation broth, the fermentation broth is subjected to an enzyme conversion reaction with sulfides using O-phosphoserine sulfidylase (OPS sulfhydrase). , a fermentation broth containing L-cysteine was obtained.

Specifically, KCCM 11103P (CA07-0022/pCL-prmf- serA *(G336V) -serC ; Korean Patent Registration No. 10-1381048 in which serB is deleted in Escherichia coli W3110 strain and mutant serA * is introduced to have OPS-producing ability Ho) strains were cultured on MMYE agar medium plates at 33° C. for 24 hours, and 1/10 of the cells on the plate were scraped from one plate and placed in a baffle flask in flask seed medium (10 g/L glucose). , 0.5 g/L magnesium sulfate, 3 g/L potassium dihydrogen phosphate, 10 g/L yeast extract, 0.5 g/L sodium chloride, 1.5 g/L ammonium chloride, 12.8 g/L sodium pyrophosphate , 1 g/L of glycine) and seed cultured at 30° C. at 200 rpm for 6 hours. After the seed culture was completed, the seed culture medium in a volume corresponding to 16% of the main culture medium volume was inoculated into a 1L small fermenter filled with 300 ml of the main culture medium, and the culture was performed at 33 ° C. at pH 7.0 to obtain an OPS fermentation broth. ., 100 mM Na ₂ S, 0.2 mM pyridoxal 5′-phosphate (PLP) in the presence of 50 mM OPS fermentation broth and Mycobacterium tuberculosis H37Rv-derived 50 mg / ml Msm-T enzyme reaction L - A fermentation broth containing cysteine was obtained (Korean Patent Registration No. 10-1381048).

.

The pH of the L-cysteine fermentation broth was 9.3, and the concentration of L-cysteine was 26 g/L. The L-cysteine content of the solid content from which water was removed from the L-cysteine fermentation broth was 26.7%. The pH of the fermentation broth was adjusted down to pH 5.5 using 98% sulfuric acid. This was concentrated with a thin film concentrator to prepare an L-cysteine fermentation broth having an L-cysteine concentration of 120 g/L as the following SMB chromatography stock solution. Concentration conditions are as follows:

Internal pressure: 80 mmHg

Steam pressure: 2 bar

Maximum injection volume: 100 L

Process fluid forced circulation flow rate: 10 L/min

Evaporation rate: Approx. 25 L/hr

(2) Obtaining a separated solution from which L-cysteine was separated using a continuous chromatography apparatus

To obtain a separated solution from which L-cysteine was separated, an SMB chromatography apparatus was used. A schematic diagram of the SMB chromatography apparatus is shown in FIG. 2 .

Specifically, as shown in FIG. 2, it consists of a total of 15 resin towers, the volume of each tower is 1.5 L, and the resin is filled with 95% of the tower volume. The SMB stock solution is injected into the No. 8 resin tower at a flow rate of 15 ml/min. The mobile phase is injected into the resin tower No. 1 at a flow rate of 95 ml/min. The SMB production process liquid (separate liquid) is discharged from the No. 3 resin tower at a flow rate of 50 ml/min. The SMB process waste liquid is discharged from the resin tower No. 12 at a flow rate of 60 ml/min. The liquid discharged from the No. 15 resin tower is mixed with the mobile phase at a flow rate of 65 ml/min and injected into the No. 1 resin tower at a total flow rate of 160 ml/min. A 1L buffer tank was installed between the resin tower No. 7 and No. 8 resin tower to perform automatic control through constant water level control so that the SMB chromatography raw material solution could be introduced into the resin tower at a constant flow rate. The resin tower moved every 8 minutes in the direction of decreasing number, but the resin tower No. 1 moved to the No. 15 resin tower and was driven in a circulating manner.

^{In Preparation Example, TRILITE ®} MCK32L, PUROLITE ^® PCR642, or DIAION ^® UBK555 resin, which is a styrene-divinelbenzene copolymer resin having a strong acidic sulfuric acid group as a functional group, is mounted on the resin tower of the chromatography apparatus, respectively, and SMB chromatography raw material After injecting 0.1 kl or more of the liquid, the device was operated to obtain an SMB production process liquid (separated liquid). The pH of the separation solution was 6.1, 6.3, and 5.9, respectively, and the L-cysteine concentrations were 35.1, 34.8, and 33.9 g/L, respectively.

(3) concentration

The separation solution was concentrated by connecting a thin film concentration tube and a forced circulation concentration tube in series until the L-cysteine concentration reached 400 g/L. L-cysteine crystals were precipitated during the concentration, and the temperature of the L-cysteine crystal slurry immediately after the concentration was 55 °C. Concentration conditions are as follows:

Internal pressure: 80 mmHg

Steam pressure: 2 bar

Maximum injection volume: 100 L

Process fluid forced circulation flow rate: 10 L/min

Evaporation rate: about 10 L/hr

(4) cooling and recovery of L-cysteine crystals

The L-cysteine crystal slurry was cooled at a constant cooling rate to 15° C. for 4 hours in a jacket tank with stirring, and stirred at the same temperature as the cooling completion temperature for 2 hours. Thereafter, L-cysteine crystals were separated from the L-cysteine crystal slurry into solid-liquid using a basket centrifuge. The separation conditions of the basket separator are as follows:

Equipment: 4.5 L basket separator (H-122;Kokusan)

Washing liquid: tertiary distilled water

Filter type: Polyamide multifilament fiber filter fabric

Filter air permeability: 250 L/m2/s (at 2 mbar)

Bowl rotation speed: 3,000 rpm

Bowl rotation time: 20 min

During separation, 20% of washing water was added based on the volume of the L-cysteine crystal slurry. After separation, drying was performed for 2 hours or more using a fluidized bed dryer at 70° C. to lower the residual moisture to 0.2% or less, and finally L-cysteine crystals were prepared.

Accordingly, the yield of the SMB chromatography process, the L-cysteine content (%) of the solid in the separated solution obtained through the SMB chromatography process, and the purity of the L-cysteine crystal were measured. The recovery rate of the SMB chromatography process was calculated as the L-cysteine recovery rate of the separated liquid compared to the fermentation broth injected into the SMB chromatography apparatus. The results are shown in Table 1 below together with the results of Preparation Example. When a styrene-divinylbenzene copolymer resin having a sulfuric acid group as a functional group was used as the stationary phase, all experimental results showed that the yield of the SMB chromatography process was 90%. above, the L-cysteine content of the solids excluding water in the separation solution obtained through the SMB chromatography process was 92.5% or more, and the purity of the L-cysteine crystals was 98.6% or more.

From this, it can be confirmed that L-cysteine crystals can be obtained in higher yield and purity when continuous chromatography using a stationary-phase resin having a strong acidic functional group is used.

Experimental Example 1 - Evaluation according to the type of ion exchange resin

In the above preparation example, L-cysteine crystals were prepared by changing only the stationary phase resin mounted on the SMB chromatography apparatus. Specifically, the resin used as the stationary phase was selected as a prerequisite for mass production that is not unreasonable for industrial use, and based on functional groups, a weakly acidic carboxyl group, a strong basic trimethylamine group, and a weakly basic tertiary amine group. , were selected to include those without functional groups.

Accordingly, the yield of the SMB chromatography process, the L-cysteine content (%) of the solid in the separated solution obtained through the SMB chromatography process, and the purity of the finally recovered L-cysteine crystals were measured. The recovery rate of the SMB chromatography process was calculated as the L-cysteine recovery rate of the separated liquid compared to the fermentation broth injected into the SMB chromatography apparatus. The results are shown in Table 1 below together with the results of the Preparation Example.

Stationary phase type constituent functional group SMB Chromatography process yield (%) L-cysteine content (%) L-cysteine crystal purity (%) TRILITE ^® MCK32L Styrene-divinylbenzene copolymer sulfate group 92.4 93.2 99.2 PUROLITE ^® PCR642 Styrene-divinylbenzene copolymer sulfate group 90.8 92.9 98.6 DIAION ^® UBK555 Styrene-divinylbenzene copolymer sulfate group 91.4 92.5 99.3 DIAION ^® SP850 Styrene-divinylbenzene copolymer does not exist 22.6 37.5 crystallization
not in progress MACRONET ^® MN202 Styrene-divinylbenzene copolymer does not exist 25.4 44.5 crystallization
not in progress AMBERLITE ^® XA1600 Styrene-divinylbenzene copolymer does not exist 25.8 31.8 crystallization
not in progress DIAION ^® HP2MGL methacrylate polymer does not exist 16.2 39.1 crystallization
not in progress DIAION ^® WK10 Methacrylate-divinylbenzene copolymer carboxyl group 15.4 32.7 crystallization
not in progress TRILITE ^® AMP16 Styrene-divinylbenzene copolymer trimethylamine group 13.5 38.9 crystallization
not in progress TRILITE ^® AW90 Styrene-divinylbenzene copolymer tertiary amine group 14.2 32.2 crystallization
not in progress

When a styrene-divinylbenzene copolymer resin having a sulfate group as a functional group was used as the stationary phase, all the experimental results showed that the yield of the SMB chromatography process was 90% or more, the L-cysteine content was 92.5% or more, and the purity of the crystal was It was more than 98.6%. On the other hand, when L-cysteine crystals are obtained using a resin without a weakly acidic carboxyl group, a strong basic trimethylamine group, a weakly basic tertiary amine group, or a functional group, the yield of the SMB chromatography process is 13.5 to 25.8%, and , the L-cysteine content of the solid content excluding water in the separated solution was 31.8 to 44.5%. This was at a level of less than half of the yield and content when a resin having a strong acidic functional group was used as a stationary phase.

From this, when a fermentation broth of L-cysteine is separated and crystallized using continuous chromatography, when a resin having a strong acidic functional group is used as a stationary phase, L-cysteine can be obtained in high yield, concentration and purity. can confirm.

Experimental Example 2 - Evaluation according to pH of fermentation broth containing L-cysteine

In the above preparation example (TRILITE ^® MCK32L was used), only the pH of the fermentation broth containing L-cysteine was changed to prepare L-cysteine crystals. Specifically, after obtaining a fermentation broth containing L-cysteine in the same manner as in Preparation Example, the pH of the fermentation broth was variously adjusted to 2.5 to 9.5 using 98% sulfuric acid or 50% caustic soda solution.

Accordingly, the recovery rate of the SMB chromatography process, the L-cysteine content (%) of the solid in the separated solution obtained through the SMB chromatography process, and the purity of the finally recovered L-cysteine crystals were measured. The recovery rate of the SMB chromatography process was calculated as the L-cysteine recovery rate of the separated liquid compared to the fermentation broth injected into the SMB chromatography apparatus. The results are shown in Table 2 below.

pH of fermentation broth SMB Chromatography process recovery (%) L-cysteine content (%) L-cysteine crystal purity (%) 2.5 30.1 81.3 90.2 3.0 59.4 85.4 93.2 3.5 68.2 90.1 98.1 4.0 76.5 91.2 98.7 4.5 88.2 93.7 99.5 5.0 90.2 94.5 99.6 5.5 92.4 93.2 99.2 6.0 91.8 92.1 98.7 6.5 89.4 92.7 98.7 7.0 86.1 92.2 98.6 7.5 69.2 90.3 98.3 8.0 60.3 88.4 91.1 8.5 58.2 87.9 94.5 9.0 50.4 85.7 92.7 9.5 22.7 58.2 65.3

The yield of the SMB chromatography process was found to be 50% or more in the pH 3.0 to 9.0 section, 85% or more in the pH 4.5 to 7.0 section, and 90% in the pH 5.0 to 6.0 section. In the separation solution obtained through the SMB chromatography process, the L-cysteine content of the solids excluding water was 85% or more in the range of pH 3.0 to 9.0, and 90% or more in the range of pH 3.5 to 7.5.

In addition, the purity of the L-cysteine crystal was 90% or more in the pH 2.5 to 9.0 range, 91% or more in the pH 3.0 to 9.0 range, and in particular, it was found to be 98% or more in the pH 3.5 to 7.5 range.

Even if the pH of the fermentation broth is less than 3.0 and greater than 9.0, the SMB chromatography process using the L-cysteine fermentation broth can be performed, and L-cysteine crystals with high concentration and purity can be obtained. However, it was confirmed that a more industrially effective process can be performed when the pH of the fermentation broth is 3.0 to 9.0.

Experimental Example 3 - Evaluation according to the concentration of the concentrate

In the above preparation example ( ^{using TRILITE ®} MCK32L), L-cysteine crystals were prepared by varying only the concentration of the concentrated solution of the separated solution from 100 to 800 g/L.

Specifically, the concentration of L-cysteine in the separated solution was 35.1 g/L, and by concentrating it, the concentration of L-cysteine was variously adjusted to 100 to 800 g/L, and a total of 8 experiments were performed.

Accordingly, the crystal nucleation time, the purity of the finally recovered L-cysteine crystals, and the recovery rate of the crystallization process were measured. The recovery rate of the crystallization process was calculated as the L-cysteine recovery rate of the obtained L-cysteine crystal compared to the L-cysteine of the separated solution according to the SMB chromatography process. The results are shown in Table 3.

Concentration Concentration (g/L) time of nucleation L-cysteine crystal purity (%) Crystallization process recovery (%) 100 no crystals no crystals 0 200 cooling 99.7 16.7 300 concentrating 99.6 50.4 400 concentrating 99.2 58.6 500 concentrating 98.3 65.8 600 concentrating 95.1 70.3 700 concentrating 91.4 75.2 800 concentrating Inability to separate crystals due to solidification Inability to separate crystals due to solidification

The nucleation of L-cysteine was produced when the concentration was 200 g/L or more, but the crystal nucleation was generated during concentration at the concentration of 300 g/L or more, so that the crystallization could proceed quickly.

The purity of the L-cysteine crystal was 91% or more in the range of 200 to 700 g/L, 95% or more in the range of 200 to 600 g/L, and 98% or more in the range of 200 to 500 g/L. The crystallization process recovery increased in proportion to the concentration concentration. However, when the concentration was 700 g/L, L-cysteine crystals could be obtained with high purity and high recovery rate, but when the concentration was 800 g/L, the L-cysteine crystal slurry was solidified, making stirring and crystal separation impossible. From this, it is expected that L-cysteine crystals can be obtained with high purity and high recovery at less than 800 g/L.

From this, it was confirmed that when the concentration of the SMB chromatography separation solution was 200 g/L or more and less than 800 g/L, it was confirmed that it was easy to obtain L-cysteine crystals. In particular, it was confirmed that when the concentration is 300 to 700 g/L, crystals can be obtained with faster crystallization progress, high purity, and high recovery rate.

Experimental Example 4 - Evaluation according to changes in cooling conditions when the concentration is low

In Example 3, when the concentration of the SMB chromatography separation solution was 100 g/L, the concentrated solution in which L-cysteine crystals were not formed even after cooling to 15 ºC was stirred in a jacket tank to -10 ºC for 2 hours and 30 minutes. After cooling at a constant cooling rate for 12 hours, the mixture was stirred at the same temperature as the cooling completion temperature for 12 hours. As a result, L-cysteine crystals were produced. This was separated into solid and liquid using a reduced pressure membrane filtration device, 100 ml of washing water was added, and dried in an oven dryer at 35 ºC for 12 hours to lower the residual moisture to 12.0% or less, and finally L-cysteine crystals were prepared. The purity of the L-cysteine crystal was 99.7%, and the recovery rate of the crystallization process was 4.2%.

From this, it was confirmed that even if the concentration of the SMB chromatography separation solution is less than 200 g/L, it is possible to obtain L-cysteine crystals by controlling the cooling temperature. However, the recovery rate of the crystallization process was very low for industrial use. In addition, there was a disadvantage in that the cooling and crystallization process had to be carried out up to the harsh conditions below zero, and the crystallization time was lengthened.

Experimental Example 5 - Evaluation according to cooling temperature

In the above preparation, only the cooling temperature of the L-cysteine crystal slurry was changed to prepare L-cysteine crystals. Specifically, the L-cysteine crystal slurry was stirred at 55 ºC for 2 hours without cooling, and the L-cysteine crystal slurry was stirred at a cooling rate of 10 ºC/h from 0 to 45 ºC. A total of 5 crystallization experiments were performed, including various cooling. The initial volume of the L-cysteine slurry used in each experiment was 1 L.

Table 4 shows the purity of the L-cysteine crystal and the recovery rate of the crystallization process. The recovery rate of the crystallization process was calculated as the L-cysteine recovery rate of the obtained L-cysteine crystal compared to the L-cysteine of the separated solution according to the SMB chromatography process. The results are shown in Table 4.

Cooling temperature (ºC) L-cysteine crystal purity (%) Crystallization process recovery (%) 0 98.9 68.2 15 99.2 58.6 30 99.4 45.1 45 99.5 38.3 55 (cooling not in progress) 99.5 34.2

The L-cysteine crystal purity was greater than 98% in all cases, and the crystallization process recovery increased in inverse proportion to the cooling temperature. That is, the lower the cooling temperature, the higher the recovery rate of the crystallization process, and the process recovery rate of 45% or more was confirmed at 30 degrees or less, and the process recovery rate of 50% or more can be expected at less than 25 degrees.

Experimental example 6 - SMB Evaluation according to the concentration of the fermentation broth containing L-cysteine used as a raw material for chromatography

In the above preparation example, L-cysteine crystals were prepared by varying only the concentration of the fermentation broth (pH5.5) containing L-cysteine used as a raw material for SMB chromatography. Specifically, the L-cysteine fermentation broth having a concentration of 26 g/L obtained in Preparation Example was used as it is as the SMB chromatography stock solution, and diluted with water to a L-cysteine concentration of 10 g/L as the SMB chromatography stock solution. A total of 6 experiments were performed, including concentration with a thin film concentrator and use as a raw material solution for SMB chromatography at a L-cysteine concentration of 60 g/L to 150 g/L. When the concentration of L-cysteine was concentrated to 180 g/L, L-cysteine crystals were precipitated and the SMB chromatography process was not performed. The precipitated L-cysteine crystals were precipitated as fine crystals, so it was difficult to separate them, and the purity was very low (50% or less).

The volume of the raw material solution used in each experiment was at least 0.1 kl or more, and the L-cysteine content of the solids excluding water in the process solution produced by SMB chromatography and the yield of the SMB chromatography process is shown in Table 5.

SMB chromatography raw material solution L-cysteine concentration (g/L) SMB Chromatography process yield (%) L-cysteine content (%) 10 90.3 94.2 26 91.5 93.0 60 90.9 93.4 90 91.8 92.8 120 92.4 93.2 150 92.0 93.2

The yield of the SMB chromatography process was 90% or more in all sections, and the L-cysteine content of the solids except water in the process solution produced by SMB chromatography was also 90% or more in all sections. According to the above results, the purification method of L-cysteine using SMB chromatography of the present application can be interpreted as a very effective method for purification and crystallization of a fermentation broth containing L-cysteine regardless of the L-cysteine concentration of the raw material solution. .

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims described below and their equivalents.

Claims (14)

(a) obtaining a separated solution after injecting a fermentation broth of pH 3.0 to 9.0 containing L-cysteine into a continuous chromatography apparatus using a strongly acidic cation exchange resin as a stationary phase;
(b) concentrating the separated solution; and
(c) recovering L-cysteine crystals from the concentrate,
The continuous chromatography of step (a) does not include the adsorption and elution process of L-cysteine,
The method for producing L-cysteine crystals, wherein the separated solution of step (a) has an L-cysteine content of 85% (w/w) or more in solids excluding water.
The method for producing L-cysteine crystals according to claim 1, further comprising adjusting the pH of the fermentation broth containing L-cysteine to be 3.5 to 7.5 before step (a).
The method for producing L-cysteine crystals according to claim 1, further comprising the step of concentrating the fermentation broth of pH 3.0 to 9.0 containing L-cysteine before step (a).
The method of claim 1, wherein the strongly acidic cation exchange resin of step (a) has a sulfuric acid functional group.
The method of claim 1, wherein the strongly acidic cation exchange resin in step (a) is a styrene-divinylbenzene copolymer having a sulfuric acid functional group.
The method of claim 1, wherein the continuous chromatography apparatus in step (a) is a simulated moving bed (SMB) chromatography apparatus.
The method of claim 1, wherein the yield of the continuous chromatography in step (a) is 50% (w/w) or more as the ratio of L-cysteine of the obtained separation solution to the injected fermentation broth. manufacturing method.
The method of claim 1, wherein in step (b), the separation solution is concentrated so that the concentration of L-cysteine is 200 to less than 800 g/L.
The method of claim 1, wherein in step (b), the concentration of the L-cysteine in the separation solution is 300 to 700 g/L.
The method of claim 1, further comprising cooling the concentrate before step (c).
The method of claim 10, wherein the concentrate is cooled to a temperature of 0 to 30°C.
The preparation of L-cysteine crystals according to claim 1, comprising the step of recovering the crystals in step (c) and adding the filtrate obtained to the fermentation broth of step (a) or the separation solution of step (b). method.
The method of claim 1, wherein the purity of the prepared L-cysteine crystal is 98% (w/w) or more.
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