TECHNICAL FIELD

The present 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 L-cystine, which uses a microbial metabolism liquid as a source material, into L-cysteine by an electrochemical reduction reaction. In contrast, as methods for producing L-cysteine using microorganisms, there have been disclosed a process for producing natural L-cysteine by fermentation using a strain having a modified O-acetyl transferase in a medium containing sulfide (U.S. Pat. Nos. 8,802,399B, 6,946,268B), 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 O-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 specific procedure, yield, purity, etc. (EP1645623A1, EP1298200B1, US20050221453A1, EP1234874A1, EP1571223A2, and EP1650296A).

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 (U.S. Pat. No. 8,088,949B).

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 L-cysteine with an aqueous hydrochloric acid solution (U.S. Pat. No. 9,120,729B).

However, the above-described processes have 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 subsequent 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.

Under these circumstances, the present inventors have made extensive efforts to increase the purity and yield of L-cysteine crystals, and have 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 present disclosure is to provide a method for preparing L-cysteine crystals.

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

Technical Solution

Hereinbelow, the present 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, all combinations of various factors disclosed herein belong to the scope of the present disclosure. Furthermore, the scope of the present disclosure should not be limited by the specific disclosure provided hereinbelow.

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

In one aspect of the present 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 preparation through microbial fermentation, etc., but is not limited thereto. Specifically, in the present 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 preparation process, the natural L-cysteine may be L-cysteine obtained without going through a chemical reaction, chemical adsorption, or elution.

As used herein, the term “natural” indicates that something does not depend on a chemical reaction. According to the EU Flavorings Regulation 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 reduction 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 O-phosphohomoserine, which is a precursor prepared through microbial fermentation, with a sulfide in the presence of phosphoserine sulfhydrylase. L-Cysteine crystals prepared by using the fermentation broth as a source liquid do not depend on a chemical reaction and thus can be referred to as natural L-cysteine.

In the present 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 preparation 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 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 L-cysteine 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 L-cystine 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 temperature of the process, the composition of the mobile phase, the continuous chromatography sequence, etc., the recovery rate of the continuous chromatography process is not a factor that is limited only by the pH of the source liquid for the continuous chromatography.

In the present 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.

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 inadequate for recovery, resulting in the deterioration 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 separation of substances more efficiently. In the present 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 consideration of the productivity and other matters.

When the continuous chromatography process is employed in the present 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 present disclosure.

In an embodiment of the present disclosure, an SMB chromatography apparatus may be used, and a schematic diagram of the SMB chromatography apparatus is shown inFIG. 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 ion-exchange 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 cation-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 specific 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 methacrylate polymers having no functional group; strongly basic anion-exchange resins such as trimethylamine styrene-divinylbenzene copolymers; weakly basic anion-exchange resins such as tertiary amine styrene-divinylbenzene copolymers; weakly basic cation-exchange resins such as carboxyl methacrylate polymers, etc., are used in the present disclosure as other types of stationary phases commonly used in the separation 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 styrene-divinylbenzene 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 specific 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 have 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 L-cysteine, 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 disposal 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 present disclosure may have 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 L-cysteine 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 separation, etc., the purity of the 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 continuous chromatography.

In the present disclosure, step (a) may be referred to as a “continuous chromatography process”. The term “the recovery rate of the continuous 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 present 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 selection. 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 supersaturation required for nucleation 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 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 separation 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 of the L-cysteine crystals may be affected by other process conditions such as concentration, crystallization, crystal separation, etc., the purity of the 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 term “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 nucleation 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 of the concentrate.

The cooling step may specifically refer to cooling to a temperature of −10° C. to 55° C. over a period of 2 hours to 6 hours, specifically cooling to a temperature of 0° C. to 45° C. over a period of 2 hours to 6 hours, more specifically cooling to a temperature of 0° C. to 30° C., and even more specifically cooling to a temperature 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 temperature. In the present disclosure, it may refer to constantly maintaining the cooled temperature, or it may refer to constantly maintaining the temperature of the concentrate even when it is not cooled. Specifically, the aging may be achieved over a period of 1 hour to 3 hours.

Step (b) in the present 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 separation. 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 separation apparatus, etc., but is not limited thereto. The slurry and/or precipitated cysteine crystals may be subjected to additional washing or drying.

In the present 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 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 preparation method of the present disclosure may be 95% (w/w) or more, specifically 98% (w/w), more specifically 99% (w/w).

In another aspect of the present 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 preparation method thereof are as described above.

Advantageous Effects

The method for preparing L-cysteine crystals of the present 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 L-cysteine crystals with a high recovery rate and/or purity at the same time. In addition, the preparation method of the present disclosure shows efficient productivity, and in particular, water consumption can be remarkably reduced.

