OA19717A - Method for preparing natural L-cysteine crystals by continuous chromatography - Google Patents

Abstract

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

Description

[DESCRIPTION]

[Invention Title]

METHOD FOR PREPARING NATURAL L-CYSTEINE CRYSTALS BY

CONTINUOUS CHROMATOGRAPHY

[Technical Field]

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

[Background Art]

L-Cysteine is generally produced by decomposing animal-derived L-cystine, which uses duck feathers or human hair as a source material, or fermentation-derived Lcystine, which uses a microbial metabolism liquid as a source material, into L-cysteine by an electrochemical réduction reaction. In contrast, as methods for producing L-cysteine using microorganisms, there hâve been disclosed a process for producing natural Lcysteine by fermentation using a strain having a modified (9-acetyl transferase in a medium containing sulfide (US8802399B, US6946268B), and a process for producing natural L-cysteine by mixing O-phosphohomoserine produced by a microbial culture method with sulfide and inducing an enzyme catalytic reaction using G-phosphoserine sulfhydrylase (WO2013/089478, WO2012/053794).

Although it has been disclosed that L-cysteine produced by the microbial culture method can be separated by ion-exchange chromatography and other known methods, no information is given on the spécifie procedure, yield, purity, etc. (EP1645623A1, EP1298200B1, US20050221453A1, EP1234874A1, EP1571223A2, and EP1650296A).

CAMEROON

For example, there has been disclosed a process for purifying L-cysteine with a yield of 90% or more by lowering the pH of a fermentation broth containing L-cysteine to a pH of 5 or below and then bringing it into contact with an acidic or strongly acidic cation exchanger such that L-cysteine binds to the ion exchanger, and eluting the bound

L-cysteine with an aqueous hydrochloric acid solution (US8088949B).

In addition, there has been disclosed a process for purifying L-cysteine with a yield of 85% or more, by bringing an L-cysteine-containing fermentation broth having a pH of 6 to 9 into contact with a basic anion exchanger, such that L-cysteine binds to the anion exchanger, eluting the bound L-cysteine with an aqueous hydrochloric acid solution, then bringing the eluate into contact with an acidic cation exchanger at a pH of 4 or below, such that the L-cysteine binds to the cation exchanger, and eluting the bound Lcysteine with an aqueous hydrochloric acid solution (US9120729B).

However, the above-described processes hâve disadvantages in that they involve a Chemical reaction as they are carried out through repeated ion exchange steps, that they require a large amount of process water since a large amount of eluent is used as a subséquent step of the ion adsorption process, and that an additional purification step must be carried out.

Meanwhile, there has been disclosed a purification of L-cysteine by a Chemical method using a strongly acidic solvent (Chinese Patent Laid-Open Publication No. 105274554), but a strongly acidic solvent must be used in a large amount and a strongly basic solvent must also be used in a large amount for neutralization thereof, causing environmental problems.

Thus, there is a continuing need for a method for isolating L-cysteine with a higher yield and purity.

² 3'

I

Under these circumstances, the présent inventors hâve made extensive efforts to increase the purity and yield of L-cysteine crystals, and hâve completed a purification method including the advantages of having increased effective productivity and reduced water consumption as well as high yield and purity.

[Disclosure]

[Technical Problem]

One object of the présent disclosure is to provide a method for preparing Lcysteine crystals.

Another object of the présent disclosure is to provide L-cysteine crystals prepared by the method for preparing L-cysteine crystals.

[Technical Solution]

Hereinbelow, the présent disclosure will be described in detail.

Meanwhile, each of the explanations and exemplary embodiments disclosed herein can be applied to other explanations and exemplary embodiments. That is, ail combinations of various factors disclosed herein belong to the scope of the présent disclosure. Furthermore, the scope of the présent disclosure should not be limited by the spécifie disclosure provided hereinbelow.

In addition, those skilled in the art can recognize and identify numerous équivalents for the spécifie embodiments of the invention disclosed herein using no more than routine expérimentation, and ail such équivalents are believed to be within the scope of the invention.

Vi

In one aspect of the présent disclosure to overcome the objects above, there is provided a method for preparing L-cysteine crystals, comprising:

(a) obtaining a separated liquid after introducing a fermentation broth in a pH range of 3.0 to 9.0 containing L-cysteine into a continuous chromatography apparatus having a strongly acidic cation-exchange resin as a stationary phase;

(b) concentrating the separated liquid; and (c) recovering L-cysteine crystals from the concentrate.

As used herein, the term “L-cysteine” is one of the constituent amino acids and is the only sulfur-containing amino acid having a thiol group (R-SH) among L-amino acids. L-Cysteine may be those obtained by Chemical synthesis, or biological préparation through microbial fermentation, etc., but is not limited thereto. Specifically, in the présent disclosure, L-cysteine may be L-cysteine biologically produced through microbial fermentation, or may be natural L-cysteine obtained by inducing an enzyme catalytic reaction of O-phosphohomoserine, which is a precursor prepared through microbial fermentation, with a sulfide in the presence of phosphoserine sulfhydrylase. In terms of the préparation process, the natural L-cysteine may be L-cysteine obtained without going through a Chemical réaction, Chemical adsorption, or elution.

As used herein, the term “natural” indicates that something does not dépend on a Chemical reaction. According to the EU Flavorings Régulation 1334/2008, only substances obtained by a physical, enzymatic, or microbial process are defined as “natural” flavoring agents. From the above viewpoint, regardless of whether it is derived from an animal or microbial fermentation, L-cysteine produced by an electrochemical réduction reaction of L-cystine cannot be called entirely natural.

As used herein, the term “fermentation broth” refers to a culture medium obtained by culturing L-cysteine-producing microorganisms, a culture containing the microorganisms cultured together with the culture medium, or an enzyme conversion solution containing a precursor capable of producing L-cysteine and an enzyme. Specifically, the fermentation broth containing L-cysteine may be a culture medium or an enzyme conversion solution containing natural L-cysteine. More specifically, it may be an L-cysteine culture or culture medium biologically prepared by fermenting microorganisms having an L-cysteine-producing ability, or a natural L-cysteine enzyme conversion solution obtained by inducing an enzyme catalytic reaction of Ophosphohomoserine, which is a precursor prepared through microbial fermentation, with a sulfîde in the presence of phosphoserine sulfhydrylase. L-Cysteine crystals prepared by using the fermentation broth as a source liquid do not dépend on a Chemical reaction and thus can be referred to as natural L-cysteine.

In the présent disclosure, the fermentation broth can be used as a source liquid for a continuous chromatography process. That is, it may be introduced into the continuous chromatography apparatus of step (a).

The pH of the fermentation broth to be introduced into the continuous chromatography apparatus may vary depending on the préparation method, but may be in the range of 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, or 5.0 to 6.0. The fermentation broth itself may be used as a source liquid for the continuous chromatography, and a step of adjusting the fermentation broth containing L-cysteine to 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 may further be included, but is not limited thereto. For example, the pH may be adjusted by adding an acid such as sulfuric acid or 5

Λ'Λ hydrochloric acid, or a base such as sodium hydroxide (caustic soda), ammonia, lithium hydroxide, or potassium hydroxide, etc., but is not limited thereto. The pH-adjusting agent can be appropriately selected and used by those skilled in the art so long as Lcysteine crystals can be obtained without affecting the structure of L-cysteine.

As the pH of the fermentation broth of L-cysteine is lowered, there is a strong tendency for L-cysteine to cationize. As the stationary phase of the continuous chromatography used herein is a strongly acidic cation-exchange resin, it tends to adsorb cations, so that when L-cysteine is cationized, it may be partially adsorbed onto the stationary phase, thereby reducing the recovery rate of the continuous chromatography process. In addition, L-cysteine has a strong tendency to be oxidized and converted to Lcystine at higher pH, which may reduce the recovery rate of the continuous chromatography process. Thus, the fermentation broth containing L-cysteine may be 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.

However, as the recovery rate of the continuous chromatography process may be affected by various process parameters, such as the flow rate between resin towers of the continuous chromatography process, the température of the process, the composition of the mobile phase, the continuous chromatography sequence, etc., the recovery rate ofthe continuous chromatography process is not a factor that is limited only by the pH of the source liquid for the continuous chromatography.

In the présent disclosure, the method may further include a step of diluting or concentrating the fermentation broth containing L-cysteine prior to step (a). The step may be carried out before or after the step of adjusting pH described above.

AU

The concentration may be carried out in a conventional evaporator (for example, a forced circulation evaporator, a thin film evaporator, or a rotary evaporator, etc.).

The concentration of L-cysteine in the diluted or concentrated fermentation broth may be adjusted to 10 g/L to 180 g/L, specifically 10 g/L to 150 g/L, but it is not a factor that greatly affects the recovery rate of the continuous chromatography process and the quality of the separated liquid (specifically, the solid content of L-cysteine excluding moisture in the separated liquid) obtained through the continuous chromatography process. Thus, to adjust the concentration of the fermentation broth containing L-cysteine used as a source liquid for the continuous chromatography process is not an essential process for separating 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, so that low-quality L-cysteine crystals are produced which are inadéquate for recovery, resulting in the détérioration of the recovery rate of the continuous chromatography process. When the concentration of the source liquid for the continuous chromatography process is high, the amount of water used in the continuous chromatography process may be reduced relative to the amount of L-cysteine used for treatment. Such a feature cannot be found in an ion-exchange process in which the amount of water used is determined by the maximum adsorption amount of L-cysteine and an ion-exchange resin.

As used herein, the term “continuous chromatography” refers to a process by which a conventional batch chromatography process is carried out continuously. Specifically, a solid phase and a liquid phase can be continuously supplied to the chromatography apparatus, and the solid phase and the liquid phase move in opposite directions to each other to cause the countercurrent contact, thereby enabling the 7 •VH séparation of substances more efficiently. In the présent disclosure, the continuous chromatography may be used in the sense that it includes both true moving bed (TMB) chromatography and simulated moving bed chromatography (SMB). In addition, since the true moving bed chromatography and the simulated moving bed chromatography employ the same principles, they can be appropriately selected and used by those skilled in the art in considération of the productivity and other matters.

When the continuous chromatography process is employed in the présent disclosure, an adsorption/elution process is not required, and thus, it has the advantages of having a high productivity per hour compared to an ion-exchange process and reducing the amount of water used in the process. In addition, in order to obtain an L-cysteine powder product from a process liquid containing L-cysteine obtained by an ion-exchange process or common chromatography process with a high yield, a large amount of energy is required in a concentration crystallization process. In this regard, the energy cost may be reduced according to the method of the présent disclosure.

In an embodiment of the présent disclosure, an SMB chromatography apparatus may be used, and a schematic diagram of the SMB chromatography apparatus is shown in FIG. 2. The process parameters such as the number of resin towers, the volume of the resin towers, the filling capacity of resin, the flow rate of each section, the presence or absence of buffer tank installation, the movement time of the resin towers, etc. may vary, and are not limited to the fixed conditions specified above.

The stationary phase of the continuous chromatography apparatus may be an ionexchange resin, and specifically, it may be a strongly acidic cation-exchange resin. The functional group of the strongly acidic cation-exchange resin may be a sulfate group, but is not limited thereto. In addition, the parent compound of the strongly acidic cation8 exchange resin used herein is not limited as long as a strongly acidic functional group can be attached thereto. For example, a styrene-divinylbenzene copolymer may be used, but are not limited thereto. In a spécifie example, the strongly acidic cation-exchange resin may be a styrene-divinylbenzene sulfuric acid copolymer, but is not limited thereto.

When styrene-divinylbenzene copolymers having no functional group, exchange resins having no functional group such as méthacrylate polymers having no functional group; strongly basic anion-exchange resins such as trimethylamine styrenedivinylbenzene copolymers; weakly basic anion-exchange resins such as tertiary amine styrene-divinylbenzene copolymers; weakly basic cation-exchange resins such as carboxyl méthacrylate polymers, etc., are used in the présent disclosure as other types of stationary phases commonly used in the séparation and purification of amino acids in the art, it is difficult to purify L-cysteine in a solid content of 50% (w/w) or more excluding moisture in the separated liquid obtained through the continuous chromatography process. Meanwhile, when a strongly acidic cation-exchange resin such as a styrenedivinylbenzene sulfuric acid copolymer is used, it is possible to purify L-cysteine in a solid content of 80% (w/w) or more excluding moisture in the separated liquid obtained through the continuous chromatography process, specifically 90% (w/w) or more.

In the chromatography apparatus, water to which no Chemical compounds (such as an organic solvent such as methanol, isopropyl alcohol, acetonitrile, etc.) are added, a caustic soda diluted solution, a sulfuric acid diluted solution, a phosphoric acid diluted solution, a hydrochloric acid diluted solution, a potassium hydroxide diluted solution, or a mixture thereof may be used as a mobile phase, but is not limited thereto. In a spécifie example, water may be used as a mobile phase for the continuous chromatography. When a mobile phase containing a Chemical compound is used, the Chemical compound may remain in the final product, so that it may not be possible to sell the product as it may hâve exceeded the standard residue limit for the Chemical compound, or it may be impossible to distribute the product as a natural product. In addition, since no Chemical substances other than water are added to the process water, a production cost-saving effect can be expected. In the ion-exchange process, in order to elute the bound Lcysteine, a solvent such as hydrochloric acid or sulfuric acid is essentially used as an eluent, and accordingly, its use is inevitably accompanied by an increase in the product cost associated with the use and disposai of the Chemical substances.

When the fermentation broth containing L-cysteine is introduced into the continuous chromatography apparatus in step (a), a separated liquid containing L-cysteine can be obtained as L-cysteine is separated according to the continuous chromatography. As used herein, the separated liquid containing the separated L-cysteine may be briefly referred as a “separated liquid”, a “chromatography separated liquid”, or a “process liquid”.

The quality of the separated liquid may be evaluated by the content of L-cysteine in the solid excluding moisture in the separated liquid. The separated liquid of the présent disclosure may hâve a solid content of L-cysteine excluding moisture of 80% (w/w), specifically 85% (w/w), more specifically 90% (w/w) or more. L-Cysteine crystals having a purity of 90% or more can be prepared when the solid content of L-cysteine excluding moisture in the separated liquid is 80% or more. Specifically, L-cysteine crystals having a purity of 98% or more can be prepared when the solid content of Lcysteine excluding moisture in the separated liquid is 90% or more.

However, since the purity of the L-cysteine crystals may be affected by other process conditions such as concentration, crystallization, crystal séparation, etc., the

-Il purity ofthe L-cysteine crystals is not a factor that is limited only by the solid content of

L-cysteine excluding moisture in the process liquid produced by the continuons chromatography.

In the présent disclosure, step (a) may be referred to as a “continuons chromatography process”. The term “the recovery rate of the continuons chromatography process” refers to the recovery rate of L-cystine in the separated liquid obtained relative to the fermentation broth introduced in step (a), and is used to evaluate the efficiency of the process. The recovery rate of the continuous chromatography in the présent disclosure may be 50% (w/w), specifically 60% (w/w), more specifically 70% (w/w), and more specifically 80% (w/w) or more, but is not limited thereto.

Step (b) is a step for concentrating the separated liquid for crystallization. In step (b), a concentrate may be obtained by concentrating the separated liquid. The concentration may be carried out in a conventional evaporator (for example, a forced circulation evaporator, a thin film evaporator, or a rotary evaporator, etc.) by those skilled in the art via an appropriate sélection. The concentration of L-cysteine in the concentrate of step (b) may be in the range of 100 g/L to less than 800 g/L, specifically 200 g/L to less than 800 g/L, 300 g/L to less than 800 g/L, more specifically less than 300 g/L to 500 g/L, but is not limited thereto.

When the concentration of L-cysteine in the concentrate is low, crystallization does not occur during concentration due to lack of super saturai ion required for nucléation and crystal growth, and crystallization may be achieved during cooling, or crystallization may be carried out through an additional process. However, the recovery rate may be low, or the crystallization time may be prolonged. When the concentration of L-cysteine in the concentrate is 300 g/L or more, L-cysteine crystal nuclei may be formed during the 11 concentration process. Additionally, when the L-cysteine slurry concentrated to 300 g/L or more is cooled, the recovery rate of the crystallization process for L-cysteine may increase. When the concentration of L-cysteine in the concentrate is 800 g/L, solidification may occur due to the formation of a large amount of crystal particles, and accordingly, stirring of the crystal slurry and crystal séparation may not be possible. The concentration of L-cysteine in the separated liquid may be appropriately adjusted within the range of 100 g/L to less than 800 g/L, specifically 200 g/L to less than 800 g/L, more specifically 300 g/L to less than 800 g/L, or 300 g/L to 500 g/L. When the cooling crystallization is carried out with the separated liquid having the concentration within the range above, the purity of the L-cysteine crystals may be 98% or more. However, since the purity ofthe L-cysteine crystals may be affected by other process conditions such as concentration, crystallization, crystal séparation, etc., the purity ofthe L-cysteine crystals may not be a factor that is limited only by the concentration of L-cysteine in the concentrate obtained by concentrating the process liquid produced by SMB chromatography.

The crystallization of L-cysteine may occur during the concentration according to step (b). As used herein, the tenu “crystallization” refers to a phenomenon by which a liquid or a solid in amorphous state forms a crystal, and is accompanied by two phenomena called nucléation and crystal growth.

The concentrate may form and/or grow crystal nuclei through cooling and/or aging prior to recovery. Further, even when the L-cysteine crystals are not precipitated from the concentrate, crystals may be formed during cooling or aging ofthe concentrate.

The cooling step may specifically refer to cooling to a température of -10°C to 55°C over a period of 2 hours to 6 hours, specifically cooling to a température of 0°C to 12

45°C over a period of 2 hours to 6 hours, more specifically cooling to a température of

0°C to 30°C, and even more specifically cooling to a température of 0°C to 15°C over a period of 2 hours to 6 hours.

The aging step may refer to allowing to stand without changing the température. In the présent disclosure, it may refer to constantly maintaining the cooled température, or it may refer to constantly maintaining the température of the concentrate even when it is not cooled. Specifically, the aging may be achieved over a period of l hour to 3 hours.

Step (b) in the présent disclosure may be referred to as a “crystallization process”. The “recovery rate of the crystallization process” refers to the L-cysteine recovery rate of the L-cysteine crystals obtained relative to the L-cysteine in the separated liquid according to the continuous chromatography process, and is used to evaluate the efficiency of the crystallization process. The recovery rate of the continuous chromatography process recovery may be 50% (w/w), specifically 60% (w/w), more specifically 70% (w/w), even more specifically 80% (w/w) or more.

In step (c), the L-cysteine crystals precipitated from the concentrate may be recovered. Specifically, the L-cysteine crystals may be recovered from the slurry by subjecting the concentrate to a solid-liquid séparation. This may be carried out using a solid-liquid separator such as a reduced-pressure membrane filtration apparatus, a pressure membrane filtration apparatus, a centrifugal séparation apparatus, etc., but is not limited thereto. The slurry and/or precipitated cysteine crystals may be subjected to additional washing or drying.

In the présent disclosure, the filtrate obtained by recovering the L-cysteine crystals in step (c) is a mother liquid having residual L-cysteine, and may be wholly or partially added to the fermentation broth of step (a), or the separated liquid of step (b), in 13 /41 order to enhance the recovery rate during the final purification of L-cysteine, but is not limited thereto.

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

In another aspect of the présent disclosure to overcome the objects above, there is provided L-cysteine crystals prepared by the method for preparing L-cysteine crystals described above.

The L-cysteine crystals and the préparation method thereof are as described above.

[Advantageous Effects]

The method for preparing L-cysteine crystals of the présent disclosure is capable of separating and purifying the L-cysteine fermentation broth as it is in the natural State without a Chemical reaction or the use of artificial synthetic compounds, and obtaining Lcysteine crystals with a high recovery rate and/or purity at the same time. In addition, the préparation method of the présent disclosure shows efficient productivity, and in particular, water consumption can be remarkably reduced.

[Brief Description of Drawings]

FIG. I shows a représentative illustration of a process for preparing L-cysteine crystals by continuous chromatography from natural L-cysteine contained in a fermentation broth.

FIG. 2 shows the arrangement of resin towers and the flow rate for each section of SMB chromatography used in one embodiment of the présent disclosure.

[Mode for Invention]

Hereinafter, the présent disclosure will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and the scope of the invention is not intended to be limited by these Examples.

Test methods

Common analytical methods used in Examples of the présent disclosure are as follows:

(1) HPLC for quantitative analysis of L-cysteine

The conditions for HPLC analysis for analyzing the purity and concentration of L-cysteine in the présent disclosure are as follows:

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

Column: HP Cl 8 (150 mm x 3.9 mm; 5 pm)

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

Flow rate: 0.425 mL/min

Température: 30°C

Détection: UV at 220 nm

Volume of sample introduced: 2 pL (2) Method for measuring purity of L-cysteine crystals

In the présent disclosure, the quality of L-cysteine crystals is evaluated based on the purity of L-cysteine, and the procedure thereof is as follows:

(a) Placing L-cysteine crystals in a vacuum dryer containing silica gel for 24 hours under a vacuum of 20 mmHg or below to remove residual moisture and cooling the température of L-cysteine dride crystals to room température;

(b) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine crystals cooled to room température, placing it in a l L volumétrie flask, and diluting it with a 0.2 N HCl solution;

(c) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine crystals, placing it in a l L volumétrie flask, and diluting it with a 0.2 N HCl solution, after treating L-cysteine standard crystals (>99.0%) in the same manner as in step (a), subsequently, determining the purity of a standard product through the certificate ofthe standard reagent manufacturer, and then converting the concentration of L-cysteine in the sample (converted [concentration of L-cysteine] is 0.5000 g/L x [purity of standard product]); and (d) Analyzing the purity of the L-cysteine crystals used in step (a) by using the sample prepared in step (c) as an external standard and analyzing the sample prepared in step (b) by HPLC.

(3) Method for analyzing the content of L-cysteine based on solids in the fermentation broth or separated liquid according to chromatography process

In the présent disclosure, the quality of the fermentation broth or the separated liquid according to the chromatography process is evaluated based on the content of Lcysteine in the solids obtained by removing moisture from the solution, and the procedure thereof is as follows:

(a) Deodorizing a porcelain container with about 5 g of sea sand ( 10 to 20 mesh;

Daejung Chemicals) and removing the residual moisture by placing it in a forced circulation oven at !05°C for 3 hours, and then cooling it to room température by placing it in a vacuum dryer containing silica gel for l hour;

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

(c) Removing the residual moisture by placing the container in a forced circulation oven at 1O5°C for 3 hours, and then cooling it to room température by placing it in a vacuum dryer containing silica gel for 1 hour, subsequently, quantifying the amount of moisture removed using the weight différence, and converting the solid content by mass of the solution to be measured using the same ([solid content by mass] is ([mass of solution] - [mass of removed moisture]) / [mass of solution]);

(d) Measuring the density of the solution to be analyzed using a spécifie gravity analyzer;

(e) Measuring the concentration of L-cysteine in the solution to be analyzed by HPLC; and (f) Converting the content of L-cysteine in the solid excluding moisture in the solution to be analyzed using the solid content by mass of the solution, density, and the concentration of L-cysteine ([content of L-cysteine in the solid excluding moisture in the solution] is [concentration of L-cysteine] / [density of solution] / [solid content by mass of solution]).

Préparation Example (1) Préparation of fermentation broth containing L-cysteine

After obtaining an (9-phosphoserine fermentation broth by culturing a microorganism capable of producing O-phosphoserine in a fermentation medium, the fermentation broth was subjected to an enzyme conversion reaction with a sulfide using

O-phosphoserine sulfhydrase (OPS sulfhydrase) to obtain a fermentation broth containing

L-cysteine.

Specifically, KCCM 11103P (CA07-0022/pCL-prmf-5eM*(G336V)-5erC;

Korean Patent No. 10-1381048) strain, which is a modified E. coli W3110 strain in which serB is deleted and mutant serA* is introduced to hâve an OPS-producing ability, was cultured on an MMYE agar plate at 33°C for 24 hours and 1/10 of the cells on the plate were scraped from one plate and inoculated into a flask seed medium (10 g/L of glucose, 0.5 g/L of magnésium sulfate, 3 g/L of potassium dihydrogenphosphate, 10 g/L of yeast extract, 0.5 g/L sodium chloride, 1.5 g/L ammonium chloride, 12.8 g/L sodium pyrophosphate, 1 g/L glycine) in a baffle flask to carry out a seed culture at 200 rpm at 30°C for 6 hours. After the seed culture was completed, the seed culture medium with a volume corresponding to 16% of the volume of the main culture medium was inoculated into a 1 L small-size fermenter filled with 300 mL of the main culture medium, and the culture was carried out at 33°C at pH 7.0 to obtain an OPS fermentation broth. 50 mM OPS fermentation broth was reacted with Mycobacterium tuberculosis H37Rv-derived 50 mg/mL Msm-T enzyme under a condition of 100 mM Na₂S and 0.2 mM pyridoxal 5'phosphate (PLP) to obtain a fermentation broth containing L-cysteine (Korean Patent No. 10-1381048).

The pH of the L-cysteine fermentation broth was 9.3 and the concentration of Lcysteine was 26 g/L. The solid content of L-cysteine excluding moisture in the L18

RM cysteine fermentation broth was 26.7%. The pH ofthe fermentation broth was adjusted by lowering to a pH of 5.5 using 98% sulfuric acid. The fermentation broth was concentrated using a thin film evaporator to préparé an L-cysteine fermentation broth having an L-cysteine concentration of 120 g/L as a source liquid for SMB chromatography. The concentration conditions are as follows:

Internai pressure: 80 mmHg

Steam pressure: 2 bar

Maximum injection amount: 100 L

Forced circulation flow rate of process liquid: 10 L/min

Evaporation rate: about 25 L/hr (2) Obtaining separated liquid in which L-cysteine is separated using continuous chromatography apparatus

In order to obtain a separated liquid in 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, the apparatus was composed of a total of 15 resin towers. The volume of each tower was 1.5 L, and the resin was filled to 95% of the tower volume. The SMB source liquid was introduced into resin tower 8 at a flow rate of 15 mL/min. The mobile phase was introduced into resin tower 1 at a flow rate of 95 mL/min. The SMB production process liquid (separated liquid) was discharged from resin tower 3 at a flow rate of 50 mL/min. The SMB process waste liquid was discharged from resin tower 12 at a flow rate of 60 mL/min. The liquid discharged from resin tower 15 was mixed with the mobile phase at a flow rate of 65 mL/min and then the mixture

T was introduced into resin tower l at a total flow rate of 160 mL/min. AIL buffer tank was installed between resin towers 7 and 8 to enable automatic control by controlling the water level so that the source liquid for SMB chromatography could flow into the resin towers at a constant flow rate. The resin towers were moved in the direction of decreasing number every 8 minutes, but they were driven in a circulating manner such that resin tower l was moved to resin tower 15.

In the Préparation Example, each of TRILITE® MCK32L, PUROLITE® PCR642, or DIAION® UBK555 resins, which are styrene-divinylbenzene copolymer resins having a strong acid sulfate group as a functional group, was loaded into the resin tower ofthe chromatography apparatus, and O.l kL or more ofthe source liquid for SMB chromatography was introduced thereto. Then, the apparatus was operated to obtain an SMB production process liquid (separated liquid). The separated liquids had a pH of 6.1, 6.3, and 5.9, respectively, and the concentration of L-cysteine was 35.1 g/L, 34.8 g/L, and 33.9 g/L, respectively.

(3) Concentration

The separated liquid was concentrated by linearly connecting a thin film concentration tube and a forced circulation-type concentration tube until the concentration of L-cysteine reached 400 g/L. The L-cysteine crystals were precipitated during the concentration, and the température of the L-cysteine crystal slurry was 55°C immediately after the concentration. The concentration conditions are as follows:

Internai pressure: 80 mmHg

Steam pressure: 2 bar

Maximum injection amount: 100 L

Forced circulation flow rate of process liquid: 10 L/min

Evaporation rate: about 10 L/hr (4) Cooling and recovery of L-cysteine crystals

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

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

Washing liquid: triple-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 the séparation, the washing liquid was added 20% of the volume of the Lcysteine crystal slurry. After the séparation, the résultant was dried at 75°C for 2 hours or more using a fluidized bed dryer to lower the residual moisture to 12.0% or below, and fmally, L-cysteine crystals were prepared.

Accordingly, the yield of the SMB chromatography process, the solid content of L-cysteine (%) in the separated liquid obtained through the SMB chromatography process, and the purity of the L-cysteine crystals were measured. The recovery rate of the SMB chromatography process was calculated as the recovery rate of L-cysteine in the

separated liquid compared to the fermentation broth introduced into the SMB chromatography apparatus. The results are shown in Table l together with the results obtained in the Préparation Example. When a styrene-divinylbenzene copolymer resin having a sulfate group as a functional group was used as a stationary phase, ail of the experimental results showed that the yield of the SMB chromatography process was more than 90%, that the solid content of L-cysteine excluding moisture in the separated liquid obtained through the SMB chromatography process was 92.5% or more, and that the purity of the L-cysteine crystals was 98.6% or more.

Based on this, it can be confirmed that when the continuous chromatography is carried out by employing a stationary phase resin having a strongly acidic functional group, L-cysteine crystals can be obtained with a higher yield and purity.

Experimental Example 1 - Evaluation according to types of ion-exchange resins

In the préparation Example, L-cysteine crystals were prepared by only varying the stationary phase resins loaded into the SMB chromatography apparatus. Specifically, as the resins used as the stationary phase, those which can be industrially used without difficulty and which can be produced by mass production were selected. The resins were selected based on the functional groups, such that they contain a weakly acidic carboxyl group, a strong basic trimethylamine group, a weak basic tertiary amine group, and no functional group.

Accordingly, the yield of the SMB chromatography process, the solid content of L-cysteine (%) in the separated liquid obtained through the SMB chromatography process, and the purity of the L-cysteine crystals finally recovered were measured. The 22 recovery rate of the SMB chromatography process was calculated as the recovery rate of

L-cysteine in the separated liquid compared to the fermentation broth introduced into the

SMB chromatography apparatus. The results are shown in Table l together with the results obtained in the Préparation Example.

[Table l]

Type of . stationary phase	Component	Functio nal ; group	Yield of SMB chromato graphy (%)	Content of Lcysteine (%)	Purity of Lcysteine crystals (%)’
TRILITE® MCK32L	Styrenedivinylbenzene copolymer	Sulfate group	92.4	93.2	99.2
PUROLITE® PCR642	Styrenedivinylbenzene copolymer	Sulfate group	90.8	92.9	98.6
DIAION® UBK555	Styrenedivinylbenzene copolymer	Sulfate group	91.4	92.5	99.3
DIAION® SP850	Styrenedivinylbenzene copolymer	None	22.6	37.5	No Crystallization
MACRONE T®MN202	Styrenedivinylbenzene copolymer	None	25.4	44.5	No Crystallization
AMBERLIT E®XAl600	Styrenedivinylbenzene copolymer	None	25.8	31.8	No Crystallization
DIAION® HP2MGL	Méthacrylate polymer	None	16.2	39.1	No Crystallization
DIAION® WK10	Methacrylatedivinylbenzene copolymer	Carboxyl group	15.4	32.7	No Crystallization
TRILITE® AMP16	Styrenedivinylbenzene copolymer	Trimethy lamine group	13.5	38.9	No Crystallization
TRILITE® AW90	Styrenedivinylbenzene copolymer	Tertiary amine group	14.2	32.2	No Crystallization

When the styrene-divinylbenzene copolymer resin having a sulfate group as a functional group was used as a stationary phase, ail of the experimental results showed that the yield of the SMB chromatography process was more than 90%, that the content of L-cysteine was 92.5% or more, and that the purity of the L-cysteine crystals was 98.6% or more. In contrast, when the crystals were obtained by employing the resins having a weakly acidic carboxyl group, a strong basic trimethylamine group, a weak basic tertiary amine group, or no functional group, the yield of the SMB chromatography process was 13.5% to 25.8% and the solid content of L-cysteine excluding moisture in the separated liquid was 31.8% to 44.5%. That is, the yield and content were reduced by 50% or more compared to the yield and content obtained when a strongly acidic functional group was used as a stationary phase.

Based on this, it was confirmed that in the case where the resin having a strongly acidic functional group was used as a stationary phase when the fermentation broth of Lcysteine was separated and crystallized by the continuous chromatography, L-cysteine crystals could be obtained with a high yield, concentration, and purity.

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

In the Préparation Example (TRILITE® MCK32L used), L-cysteine crystals were prepared by only varying the pH of the fermentation broth containing L-cysteine. Specifically, after obtaining the fermentation broth containing L-cysteine in the same manner as in the Préparation Example, the pH of the fermentation broth was varied from 2.5 to 9.5 using 98% sulfuric acid or a 50% caustic soda solution.

/H

Accordingly, the yield of the SMB chromatography process, the solid content of L-cysteine (%) in the separated liquid obtained through the SMB chromatography process, and the purity ofthe L-cysteine crystals frnally recovered were measured. The recovery rate of the SMB chromatography process was calculated as the recovery rate of 5 L-cysteine in the separated liquid compared to the fermentation broth introduced into the SMB chromatography apparatus. The results are shown in Table 2.

[Table 2]

pH of fermentation broth	Recôvery rate of SMB chromatogrfiphy (%)	Content of Lcysteine (%)	Purity of Lcysteine crystals (%)
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 at a pH range of 3.0 to 9.0, 85% or more at a pH range of 4.5 to 7.0, and 90% at a pH range of

5.0 to 6.0. The solid content of L-cysteine excluding moisture in the separated liquid obtained through the SMB chromatography process was 85% or more at a pH range of 3.0 to 9.0 and 90% or more at a pH range of 3.5 to 7.5.

T

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

Even when the pH of the fermentation broth was less than 3.0 or 9.0 or more, it was possible to carry out the SMB chromatography using the L-cysteine fermentation broth, and L-cysteine crystals having a high concentration and purity could be obtained. However, it was confirmed that the process can be carried out in a more industrially effective way when the pH of the fermentation broth is from 3.0 to 9.0.

Experimental Example 3 - Evaluation according to concentration of concentrate

In the Préparation Example (TRILITE® MCK32L used), L-cysteine crystals were prepared by only varying the concentration of the concentrate obtained by concentrating the separated liquid from 100 g/L to 800 g/L.

Specifically, the concentration of L-cysteine in the separated liquid was 35.1 g/L, and a total of 8 experiments were conducted by concentrating the separated liquid and varying the concentration of L-cysteine from 100 g/L to 800 g/L.

Accordingly, the time at which nucléation occurred, the purity of the L-cysteine crystals finally recovered, and the recovery rate of the crystallization process were measured. The recovery rate of the crystallization process was calculated as the recovery rate of L-cysteine in the L-cysteine crystals obtained relative to L-cysteine in the separated liquid according to the SMB chromatography process. The results are shown in Table 3.

[Table 3]

Concentration (g/L)	Time at which nucléation occurred	Purity of L-cysteine crystals (%)	Recovery rate of crystallization process (%)
100	No crystal formation	No crystal formation	0
200	During cooling	99.7	16.7
300	During concentration	99.6	50.4
400	During concentration	99.2	58.6
500	During concentration	98.3	65.8
600	During concentration	95.1	70.3
700	During concentration	91.4	75.2
800	During concentration	No crystal séparation due to solidification	No crystal séparation due to solidification

The nucléation of L-cysteine occurred when the concentration was 200 g/L or more, but the nucléation occurred during concentration when the concentration was

300 g/L or more, and it was possible to carry out rapid crystallization.

The purity ofthe L-cysteine crystals was 91% or more at a concentration range of 200 g/L to 600 g/L, 95% or more at a concentration range of 200 g/L to 600 g/L, and 98% or more at a concentration range of 200 g/L to 500 g/L. The recovery rate of the crystallization process increased in proportion to the concentration. However, when the concentration was 700 g/L, L-cysteine crystals could be obtained with a high purity and a high recovery rate, whereas, when the concentration was 800 g/L, solidification of the Lcysteine crystal slurry occurred, and accordingly, stirring and séparation of the crystals were impossible. Thus, it is expected that L-cysteine crystals can be obtained with a high purity and a high recovery rate at a concentration less than 800 g/L.

Based on these results, it was confïrmed that when the concentration of the separated liquid for the SMB chromatography was from 200 g/L or more to less than

800 g/L, the L-cysteine crystals were easily obtained. In particular, it was confïrmed that when the concentration was in the range of 300 g/L to 700 g/L, the crystals could be obtained with a faster crystallization process, at a high purity and a high recovery rate.

Experimental Example 4 - Evaluation according to change of cooling condition at low concentration

The concentrate, from which no L-cysteine crystals were formed even when cooled to 15°C, when the concentration of the separated liquid for the SMB chromatography was 100 g/L in Example 3, was cooled to -10°C at a constant cooling rate for 2 hours and 30 minutes in a jacket tank together with stirring, and then stirred at the same température as the température at the completion of cooling for 12 hours. As a resuit, L-cysteine crystals were formed. The crystals were subjected to a solid-liquid séparation using a reduced-pressure membrane filtration apparatus, added with 100 mL of a washing liquid, and dried in an oven dryer at 35°C for 12 hours to reduce the residual moisture to 12.0% or less. Finally, L-cysteine crystals were prepared. The purity ofthe L-cysteine crystals was 99.7%, and the recovery rate of the crystallization process was found to be 4.2%.

Based on this, it was confïrmed that even when the concentration of the separated liquid for the SMB chromatography was less than 200 g/L, the L-cysteine crystals could be obtained by controlling the cooling température. However, the recovery rate of the crystallization process was too low to be applied industrially. In addition, there were disadvantages in that the cooling crystallization process must be carried out even under a harsh température condition below zéro and that the crystallization time is long.

Experimental Example 5 - Evaluation according to cooling température

In the Préparation Example, L-cysteine crystals were prepared by only varying the cooling température of the L-cysteine crystal slurry. Specifically, a total of 5 crystallization experiments were carried out including stirring the L-cysteine crystal slurry at 55°C for 2 hours without cooling, and cooling the L-cysteine crystal slurry to a various température range from 0°C to 45°C with a cooling rate of 10°C/h while stirring. The initial volume ofthe L-cysteine slurry used in each experiment was l L.

Accordingly, the purity of the L-cysteine crystals and the recovery rate of the crystallization process are shown in Table 4. The recovery rate of the crystallization process was calculated as the recovery rate of L-cysteine in the L-cysteine crystals obtained relative to the L-cysteine in the separated liquid according to the SMB chromatography process. The results are shown in Table 4.

[Table 4]

Cooling température (°C) ‘	Purity of L-cysteine crystals (%)	Recovery rate of crystallization process (%)
0	98.9	68.2
15	99.2	58.6
30	99.4	45.1
45	99.5	38.3
55 (No crystallization)	99.5	34.2

The purity of the L-cysteine crystals was 98% or more in ail cases. The recovery rate of the crystallization process increased in inverse proportion to the cooling

température. That is, the recovery rate of the crystallization process increased as the cooling température was lowered. In addition, the process recovery rate was found to be

45% or more at a température of 30°C or below, and it was expected that the process recovery rate would be 50% or more at a température below 25°C.

Example 6 - Evaluation according to concentration of fermentation broth containing L-cysteine used as source material for SMB chromatography

In the Préparation Example, L-cysteine crystals were prepared by only varying the concentration of the fermentation broth (pH 5.5) containing L-cysteine used as a source material for SMB chromatography. Specifically, a total of 6 experiments were conducted including using the L-cysteine fermentation broth having a concentration of 26 g/L obtained in the Préparation Example as a source liquid for SMB chromatography, using a fermentation broth having an L-cysteine concentration of 10 g/L as a source liquid for SMB chromatography by dilution with water, and using a fermentation broth having an L-cysteine concentration from 60 g/L to 150 g/L as a source liquid for SMB chromatography by concentration using a thin-film evaporator. When the concentration of L-cysteine was increased to 180 g/L, the SMB chromatography process was not carried out as the L-cysteine crystals were precipitated. Since the precipitated L-cysteine crystals were precipitated as fine crystals, it was difficult to separate the crystals, and also the purity was 50% or below, which was very low.

The volume of the source liquid used in each experiment was 0.1 kL or more. The yield of the SMB chromatography process and the solid content of L-cysteine excluding moisture in the process liquid produced by SMB chromatography are shown in Table 5.

[Table 5]

Concentration of Lcysteine in source liquid for SMB chromatography (g/L)	Yield of SMB chromatography process (%)	Solid content of L-cysteine (%)
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 ail sections, and the solid content of L-cysteine excluding moisture in the process liquid produced by

SMB chromatography was 90% or more in ail sections. According to the above results, it can be interpreted that the method for purifying L-cysteine by SMB chromatography of the présent disclosure is very effective for purifying and crystallizing the fermentation broth containing L-cysteine regardless of the concentration of L-cysteine in the source liquid.

While the présent disclosure has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the présent disclosure pertains that the présent disclosure may be embodied in other spécifie forms without departing from the technical spirit or essential characteristics of the présent disclosure. Therefore, the embodiments described above are considered to be illustrative in ail respects and not restrictive. Furthermore, the scope of the présent disclosure is defined by the appended claims rather than the detailed description, and it should be understood that ail modifications or variations derived from the meanings and scope of the présent disclosure and équivalents thereof are included in the scope of the appended claims.

Claims (10)

1. [CLAIMS]

   [Claim l]

   A method for preparing L-cysteine crystals, comprising:

   (a) obtaining a separated liquid after introducing a fermentation broth in a pH range of 3.0 to 9.0 containing L-cysteine into a continuous chromatography apparatus having a strongly acidic cation-exchange resin as a stationary phase;

   (b) concentrating the separated liquid; and (c) recovering L-cysteine crystals from the concentrate.
2. [Claim 2]

   The method of claim l, further comprising adjusting the fermentation broth containing L-cysteine to a pH of 3.5 to 7.5 prior to step (a).
3. [Claim 3]

   The method of claim l or 2, further comprising concentrating the fermentation broth in a pH range of 3.0 to 9.0 containing L-cysteine prior to step (a).
4. [Claim 4]

   The method of any one of claims l to 3, wherein the strongly acidic cationexchange resin in step (a) has a sulfuric acid functional group.
5. [Claim 5]

   The method of any one of claims l to 4, wherein the strongly acidic cationexchange resin in step (a) is a styrene-divinylbenzene copolymer having a sulfuric acid functional group.
6. [Claim 6]

   The method of any one of claims l to 5, wherein the continuons chromatography apparatus in step (a) is a simulated moving bed (SMB) chromatography apparatus.
7. [Claim 7]

   The method of any one of claims l to 6, wherein the separated liquid in step (a) has a solid content of L-cysteine excluding moisture of 85% (w/w) or more.
8. [Claim 8]

   The method of any one of claims l to 7, wherein the yield of continuous chromatography in step (a), as a ratio of L-cysteine in the separated liquid obtained relative to the fermentation broth introduced, is 50% (w/w).
9. [Claim 9]

   The method of any one of claims l to 8, wherein step (b) is carried out such that the concentration of L-cysteine in the separated liquid is from 200 g/L to less than 800 g/L.
10. [Claim 10]

    The method of any one of claims l to 9, wherein step (b) is carried out such that the concentration of L-cysteine in the separated liquid is from 300 g/L to less than 700 g/L.

    [Claim H]

    The method of any one of claims 1 to 10, further comprising cooling the concentrate prior to step (c).

    [Claim 12]

    The method of claim 11, wherein the concentrate is cooled to a température of

    0°C to 30°C.

    [Claim 13]

    The method of any one of claims 1 to 12, comprising adding a filtrate obtained by recovering the crystals in step (c) to the fermentation broth of step (a) or the separated liquid of step (b).

    [Claim 14]

    The method of any one of claims 1 to 13, wherein the purity of the prepared Lcysteine crystals is 98% (w/w) or more.

    [Claim 15]

    L-Cysteine crystals prepared according to the préparation method of any one of claims 1 to 14.