Method for preparing natural L-cysteine hydrochloride hydrate crystals by continuous chromatography

Abstract

The present disclosure relates to a method for preparing L-cysteine hydrochloride hydrate crystals, and L-cysteine hydrochloride hydrate crystals prepared by the method. Through the method for preparing L-cysteine hydrochloride hydrate crystals of the present disclosure, L-cysteine hydrochloride hydrate 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.

Claims

1. A method for preparing L-cysteine hydrochloride hydrate 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, wherein the strongly acidic cation exchange resin comprises a sulfonic acid functional group, and the continuous chromatography excludes adsorption or elution of L-cysteine; (b) adding hydrochloric acid to the separated liquid such that an equivalence ratio ([HCl]/[L-cysteine]) of hydrochloric acid to L-cysteine is from 0.9 to 3.0; (c) concentrating the separated liquid to which hydrochloric acid is added to form a concentrate; and (d) recovering L-cysteine hydrochloride hydrate crystals from the concentrate.

2. The method of claim 1, further comprising adjusting the fermentation broth containing L-cysteine to a pH of 3.5 to 7.5 prior to step (a).

3. The method of claim 1, further comprising concentrating the fermentation broth in a pH range of 3.0 to 9.0 containing L-cysteine prior to step (a).

4. The method of claim 1, wherein the strongly acidic cation exchange resin is a styrene-divinylbenzene copolymer.

5. The method of claim 1, wherein the continuous chromatography apparatus is a simulated moving bed (SMB) chromatography apparatus.

6. The method of claim 1, wherein the separated liquid in step (a) has a solid content of L-cysteine excluding moisture of 85% (w/w) or more.

7. The method of claim 1, wherein a recovery rate of the continuous chromatography process, as a ratio of L-cysteine in the separated liquid obtained relative to the fermentation broth introduced, is 50% (w/w).

8. The method of claim 1, wherein the equivalence ratio ([HCl]/[L-cysteine]) of hydrochloric acid to L-cysteine in step (b) is from 1.5 to 2.5.

9. The method of claim 1, wherein step (c) is carried out such that a concentration of L-cysteine in the separated liquid, to which hydrochloric acid is added, is from 200 g/L to less than 900 g/L.

10. The method of claim 1, wherein step (c) is carried out such that a concentration of L-cysteine in the separated liquid, to which hydrochloric acid is added, is from 500 g/L to less than 900 g/L.

11. The method of claim 1, further comprising cooling the concentrate prior to step (d).

12. The method of claim 11, wherein the concentrate is cooled to a temperature from 0° C. to 30° C.

13. The method of claim 1, further comprising adding a filtrate obtained by recovering the crystals in step (d) to the fermentation broth, the separated liquid of step (b), or the separated liquid to which hydrochloric acid is added.

14. The method of claim 1, wherein a purity of the prepared L-cysteine hydrochloride hydrate crystals is 98% (w/w) or more.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a representative illustration of a process for preparing L-cysteine hydrochloride hydrate crystals by continuous chromatography from natural L-cysteine contained in a fermentation broth.

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

MODE FOR INVENTION

(3) Hereinafter, the present 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.

(4) Test Methods

(5) Common analytical methods used in Examples of the present disclosure are as follows:

(6) (1) HPLC for Quantitative Analysis of L-Cysteine Hydrochloride Monohydrate

(7) The conditions for HPLC analysis for analyzing the purity and concentration of L-cysteine hydrochloride monohydrate in the present disclosure are as follows:

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

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

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

(11) Flow rate: 0.425 mL/min

(12) Temperature: 30° C.

(13) Detection: UV at 220 nm

(14) Volume of sample introduced: 2 μL

(15) (2) Method for Measuring Purity of L-Cysteine Hydrochloride Monohydrate Crystals

(16) In the present disclosure, the quality of L-cysteine hydrochloride monohydrate crystals is evaluated based on the purity of L-cysteine hydrochloride monohydrate, and the procedure thereof is as follows:

(17) (a) Preparing L-cysteine hydrochloride anhydrous crystals by placing L-cysteine hydrochloride monohydrate 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 temperature of L-cysteine hydrochloride anhydrous crystals to room temperature;

(18) (b) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine hydrochloride anhydrous crystals cooled to room temperature, placing it in a 1 L volumetric flask, and diluting it with triple-distilled water;

(19) (c) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine hydrochloride anhydrous crystals, placing it in a 1 L volumetric flask, and diluting it with triple-distilled water, after treating L-cysteine hydrochloride monohydrate standard crystals (Sigma, ≥99.0%) in the same manner as in step (a), subsequently, determining the purity of a standard product through the certificate of the standard reagent manufacturer, and then converting the concentration of L-cysteine hydrochloride anhydrous in the sample (converted [concentration of L-cysteine hydrochloride anhydrous] is 0.5000 g/L×[purity of standard product]); and

(20) (d) Analyzing the purity of the L-cysteine hydrochloride anhydrous 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 (the purity of the analyzed L-cysteine hydrochloride anhydrous crystals is the same as the purity of the L-cysteine hydrochloride monohydrate crystals).

(21) (3) Method for Analyzing the Content of L-Cysteine Based on Solids in the Fermentation Broth or Separated Liquid According to Chromatography Process

(22) In the present disclosure, the quality of the fermentation broth or the separated liquid according to the chromatography process is evaluated based on the content of L-cysteine in the solids obtained by removing moisture from the solution, and the procedure thereof is as follows:

(23) (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 105° C. for 3 hours, and then cooling it to room temperature by placing it in a vacuum dryer containing silica gel for 1 hour;

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

(25) (c) Removing the residual moisture by placing the container in a forced circulation oven at 105° C. for 3 hours, and then cooling it to room temperature by placing it in a vacuum dryer containing silica gel for 1 hour, subsequently, quantifying the amount of moisture removed using the weight difference, 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]);

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

(27) (e) Measuring the concentration of L-cysteine in the solution to be analyzed by HPLC; and

(28) (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]).

PREPARATION EXAMPLE

(29) (1) Preparation of Fermentation Broth Containing L-Cysteine

(30) After obtaining an O-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.

(31) Specifically, KCCM 11103P (CA07-0022/pCL-prmf-serA*(G336V)-serC; 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 have 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 magnesium 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 Na.sub.2S and 0.2 mM pyridoxal 5′-phosphate (PLP) to obtain a fermentation broth containing L-cysteine (Korean Patent No. 10-1381048).

(32) The pH of the L-cysteine fermentation broth was 9.3 and the concentration of L-cysteine was 26 g/L. The solid content of L-cysteine excluding moisture in the L-cysteine fermentation broth was 26.7%. The pH of the 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 prepare 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:

(33) Internal pressure: 80 mmHg

(34) Steam pressure: 2 bar

(35) Maximum injection amount: 100 L

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

(37) Evaporation rate: about 25 L/hr

(38) (2) Obtaining Separated Liquid in which L-Cysteine is Separated Using Continuous Chromatography Apparatus

(39) 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.

(40) 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 was introduced into resin tower 1 at a total flow rate of 160 mL/min. A 1 L 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 1 was moved to resin tower 15.

(41) In the Preparation Example, each of TRILITE® MCK32L, PUROLITE® PCR642, or DIAION® UBK555 resins, which are styrene-divinylbenzene copolymer resins having a strong acid sulfonic group as a functional group, was loaded into the resin tower of the chromatography apparatus, and 0.1 kL or more of the 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.

(42) (3) Hydrochloric Acid Addition Reaction and Concentration

(43) 36% hydrochloric acid was added to the above separated liquid so that the [HCl]/[L-cysteine] equivalence ratio was 2, and the 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 700 g/L. The L-cysteine hydrochloride monohydrate crystals were precipitated during the concentration, and the temperature of the L-cysteine hydrochloride monohydrate crystal slurry was 55° C. immediately after the concentration. The concentration conditions are as follows:

(44) Internal pressure: 80 mmHg

(45) Steam pressure: 2 bar

(46) Maximum injection amount: 100 L

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

(48) Evaporation rate: about 10 L/hr

(49) (4) Cooling and Recovery of L-Cysteine Hydrochloride Crystals

(50) The L-cysteine hydrochloride monohydrate 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 temperature as the temperature at the completion of cooling. Thereafter, the L-cysteine hydrochloride monohydrate crystals were subjected to a solid-liquid separation from the L-cysteine hydrochloride monohydrate crystal slurry using a basket centrifugal separator. The separation conditions of the basket separator are as follows:

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

(52) Washing liquid: triple-distilled water

(53) Filter type: Polyamide multifilament fiber filter fabric

(54) Filter air permeability: 250 L/m.sup.2/s (at 2 mbar)

(55) Bowl rotation speed: 3,000 rpm

(56) Bowl rotation time: 20 min

(57) During the separation, the washing liquid was added 10% of the volume of the L-cysteine hydrochloride monohydrate crystal slurry. After the separation, the resultant was dried at 35° C. for 2 hours or more using a fluidized bed dryer to lower the residual moisture to 12.0% or below, and finally, L-cysteine hydrochloride monohydrate crystals were prepared.

(58) 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 hydrochloride monohydrate 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 1 together with the results obtained in the Preparation Example. When a styrene-divinylbenzene copolymer resin having a sulfonic group as a functional group was used as a stationary phase, all 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 hydrochloride monohydrate crystals was 99.5% or more.

(59) 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 hydrochloride monohydrate crystals can be obtained with a higher yield and purity.

Experimental Example 1—Evaluation According to Types of Ion-Exchange Resins

(60) In the preparation Example, L-cysteine hydrochloride hydrate crystals were prepared by only varying the stationary phase resins loaded into the SMB chromatography apparatus. 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.

(61) 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 hydrochloride monohydrate crystals finally recovered 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 1 together with the results obtained in the Preparation Example.

(62) TABLE-US-00001 TABLE 1 Purity of Type of Yield of SMB Content of L-cysteine hydrochloride stationary Functional chromatography L-cysteine monohydrate crystals phase Component group (%) (%) (%) TRILITE ® Styrene-divinylbenzene Sulfonic group 92.4 93.2 99.7 MCK32L copolymer PUROLITE ® Styrene-divinylbenzene Sulfonic group 90.8 92.9 99.5 PCR642 copolymer DIAION ® Styrene-divinylbenzene Sulfonic group 91.4 92.5 99.6 UBK555 copolymer DIAION ® Styrene-divinylbenzene None 22.6 37.5 No SP85O copolymer Crystallization MACRONET ® Styrene-divinylbenzene None 25.4 44.5 No MN202 copolymer Crystallization AMBERLITE ® Styrene-divinylbenzene None 25.8 31.8 No XA1600 copolymer Crystallization DIAION ® Methacrylate None 16.2 39.1 No HP2MGL polymer Crystallization DIAION ® Methacrylate-divinylbenzene Carboxyl group 15.4 32.7 No WK10 copolymer Crystallization TRILITE ® Styrene-divinylbenzene Trimethylamine 13.5 38.9 No AMP16 copolymer group Crystallization TRILITE ® Styrene-divinylbenzene Tertiaryamine 14.2 32.2 No AW90 copolymer group Crystallization

(63) When the styrene-divinylbenzene copolymer resin having a sulfonic group as a functional group was used as a stationary phase, all 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 hydrochloride monohydrate crystals was 99.5% 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 content of L-cysteine 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.

(64) 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 L-cysteine was separated and crystallized by the continuous chromatography, L-cysteine hydrochloride monohydrate crystals could be obtained with a high yield, concentration, and purity.

Experimental Example 2—Evaluation of Fermentation Broth Containing L-Cysteine According to pH

(65) In the Preparation Example (TRILITE® MCK32L used), L-cysteine hydrochloride hydrate 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 Preparation 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.

(66) 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 hydrochloride monohydrate crystals finally recovered 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 2.

(67) TABLE-US-00002 TABLE 2 Purity of pH of Recovery L-cysteine hydro- fermen- rate of SMB Content of chloride mono- tation chromatography L-cystiene hydrate crystals broth (%) (%) (%) 2.5 30.1 81.3 98.2 3.0 59.4 85.4 98.9 3.5 68.2 90.1 99.6 4.0 76.5 91.2 99.5 4.5 88.2 93.7 99.6 5.0 90.2 94.5 99.7 5.5 92.4 93.2 99.7 6.0 91.8 92.1 99.5 6.5 89.4 92.7 99.4 7.0 86.1 92.2 99.5 7.5 69.2 90.3 99.3 8.0 60.3 88.4 99.0 8.5 58.2 87.9 99.1 9.0 50.4 85.7 98.7 9.5 22.7 58.2 75.6

(68) 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.

(69) In addition, the purity of the L-cysteine hydrochloride monohydrate crystals was found to be 98% or more at a pH range of 2.5 to 9.0 and 99% or more at a pH range of 3.5 to 8.5. That is, it was confirmed that when the method of the present disclosure was employed, the L-cysteine hydrochloride monohydrate crystals could be obtained with a very high purity without greatly affecting the pH range.

Experimental Example 3—Evaluation According to the Amount of Hydrochloric Acid Added

(70) In the Preparation Example (TRILITE® MCK32L used), L-cysteine hydrochloride hydrate crystals were prepared by only varying the [HCl]/[L-cysteine] equivalence ratio of hydrochloric acid added in the range of 0.80, 0.85, 0.90, 1.0, 1.5, 2.0, 2.5, 3.0, and 3.5.

(71) Accordingly, the yield of the SMB chromatography and the purity of the L-cysteine hydrochloride monohydrate crystals finally recovered were measured. The recovery rate of the crystallization process was calculated as the recovery rate of L-cysteine contained in the L-cysteine hydrochloride monohydrate crystals finally recovered relative to L-cysteine in the separated liquid according to the SMB chromatography process. The results are shown in Table 3.

(72) TABLE-US-00003 TABLE 3 Purity of Recovery [HCl]/ L-cysteine hydro- rate of [L-cysteine] chloride mono- crystalliza- equivalence Type of hydrate crystals tion process ratio crystals (%) (%) 0.80 L-Cysteine — — 0.85 L-Cysteine — — 0.90 L-Cysteine 99.5 43.7 hydrochloride monohydrate 1.0 L-Cysteine 99.7 46.1 hydrochloride monohydrate 1.5 L-Cysteine 99.5 60.2 hydrochloride monohydrate 2.0 L-Cysteine 99.7 79.1 hydrochloride monohydrate 2.5 L-Cysteine 99.6 64.9 hydrochloride monohydrate 3.0 L-Cysteine 99.6 44.9 hydrochloride monohydrate 3.5 L-Cysteine 99.7 24.8 hydrochloride monohydrate

(73) All crystals were precipitated during the concentration, but when the [HCl]/[L-cysteine] equivalence ratio was 0.85 or less, the L-cysteine crystals were precipitated, and when the equivalence ratio was 0.9 or more, the L-cysteine hydrochloride monohydrate crystals were precipitated. That is, it can be interpreted that when the equivalence ratio was greater than 0.85, the L-cysteine hydrochloride monohydrate crystals were precipitated.

(74) The purity of L-cysteine hydrochloride monohydrate crystals was found to be 98% or more when the L-cysteine hydrochloride monohydrate was formed regardless of the [HCl]/[L-cysteine] equivalence ratio. The recovery rate of the crystallization process for the L-cysteine hydrochloride monohydrate was found to be 40% or more at the [HCl]/[L-cysteine] equivalence ratio of 0.9 to 3.0, and it is expected to be more than 50% at the equivalence ratio greater than 1.0 and less than 3.0. In addition, the purity was found to be 60% or more at the equivalence ratio of 1.5 to 2.5, and it is predicted that there may be an optimum condition for reaching the highest recovery rate at the equivalence ratio of 1.5 to 2.5.

Experimental Example 4—Evaluation According to Concentration of Concentrate

(75) In the Preparation Example (TRILITE® MCK32L used), L-cysteine hydrochloride hydrate crystals were prepared by only varying the concentration of the concentrate obtained by concentrating the separated liquid to which hydrochloric acid was added from 100 g/L to 900 g/L.

(76) Accordingly, the time at which nucleation occurred, the purity of the L-cysteine hydrochloride monohydrate 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 contained in the L-cysteine hydrochloride monohydrate crystals finally recovered relative to L-cysteine in the separated liquid according to the SMB chromatography process. The results are shown in Table 4.

(77) TABLE-US-00004 TABLE 4 Purity of Recovery L-cysteine hydro- rate of Concen- Time at which chloride mono- crystalliza- tration nucleation hydrate crystals tion process (g/L) occurred (%) (%) 100 No crystal No crystal 0  formation formation 200 During 99.8 28.3 cooling 300 During 99.7 50.7 concentration 400 During 99.5 62.1 concentration 500 During 99.7 70.6 concentration 600 During 99.6 73.4 concentration 700 During 99.7 79.1 concentration 800 During 99.1 83.2 concentration 900 During No crystal No crystal concentration separation due separation due to solidification to solidification

(78) The nucleation of the L-cysteine hydrochloride monohydrate occurred when the concentration was 200 g/L or more, but the nucleation occurred during concentration when the concentration was 300 g/L or more, and it was possible to carry out rapid crystallization.

(79) The purity of the L-cysteine hydrochloride monohydrate crystals was 99% or more in all cases where crystals were formed. That is, it was confirmed that when the crystals were obtained using the method of the present disclosure, the L-cysteine hydrochloride monohydrate crystals could be obtained with a very high purity regardless of the concentration of the separated liquid. The recovery rate of the crystallization process increased in proportion to the concentration. However, when the concentration was 800 g/L, L-cysteine crystals could be obtained with a high purity and a high recovery rate, whereas, when the concentration was 900 g/L, solidification of the L-cysteine hydrochloride monohydrate crystal slurry occurred, and accordingly, stirring and separation 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 900 g/L.

(80) Based on these results, it was confirmed that when the concentration of the separated liquid for the SMB chromatography was from 200 g/L to less than 900 g/L, the L-cysteine crystals were easily obtained. In particular, it was confirmed that when the concentration was in the range of 300 g/L to 800 g/L, the crystals could be obtained with a faster crystallization process, at a high purity and a high recovery rate.

Experimental Example 5—Evaluation According to Change of Cooling Condition at Low Concentration

(81) The concentrate, from which no L-cysteine hydrochloride monohydrate 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 4, 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 temperature as the temperature at the completion of cooling for 12 hours. As a result, L-cysteine hydrochloride monohydrate crystals were formed. The crystals were subjected to a solid-liquid separation 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 hydrochloride monohydrate crystals were prepared. The purity of the L-cysteine hydrochloride monohydrate crystals was 99.8%, and the recovery rate of the crystallization process was found to be 9.7%.

(82) Based on this, it was confirmed that even when the concentration of the separated liquid for the SMB chromatography was less than 200 g/L, the L-cysteine hydrochloride monohydrate crystals could be obtained by controlling the cooling temperature. 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 temperature condition below zero and that the crystallization time is long.

Experimental Example 6—Evaluation According to Cooling Temperature

(83) In the Preparation Example, L-cysteine hydrochloride monohydrate crystals were prepared by only varying the cooling temperature of the L-cysteine hydrochloride monohydrate crystal slurry. Specifically, a total of 5 crystallization experiments were carried out including stirring the L-cysteine hydrochloride monohydrate crystal slurry at 55° C. for 2 hours without cooling, and cooling the L-cysteine hydrochloride monohydrate crystal slurry to a various temperature range from 0° C. to 45° C. with a cooling rate of 10° C./h while stirring. The initial volume of the L-cysteine hydrochloride monohydrate slurry used in each experiment was 1 L.

(84) Accordingly, the purity of the L-cysteine hydrochloride monohydrate crystals and the recovery rate of the crystallization process are shown in Table 5. The recovery rate of the crystallization process was calculated as the recovery rate of L-cysteine contained in the L-cysteine hydrochloride monohydrate crystals finally recovered relative to the L-cysteine in the separated liquid according to the SMB chromatography process. The results are shown in Table 5.

(85) TABLE-US-00005 TABLE 5 Purity of Recovery L-cysteine hydro- rate of Cooling chloride mono- crystalliza- temperature hydrate crystals tion process (° C.) (%) (%) 0 99.5 83.5 15 99.7 79.1 30 99.7 74.2 45 99.8 68.9 55 (No 99.8 64.3 crystallization)

(86) The purity of the L-cysteine hydrochloride monohydrate crystals was 99.5% or more in all cases. In addition, the recovery rate of the crystallization process was 64% or more even when cooling did not occur, and the recovery rate of the crystallization process increased as the cooling temperature decreased. The process recovery rate was found to be 70% or more at a temperature below 45° C., and it was expected that the process recovery rate would be 74% or more at a temperature 30° C. or below.

Example 7—Evaluation According to Concentration of Fermentation Broth Containing L-Cysteine Used as Source Material for SMB Chromatography

(87) In the Preparation Example, L-cysteine hydrochloride hydrate 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 Preparation 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.

(88) 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 6.

(89) TABLE-US-00006 TABLE 6 Concentration of L-cysteine in Yield of SMB Solid content of source liquid for chromatography L-cysteine in SMB chromatography process separated liquid (g/L) (%) (%) 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

(90) The yield of the SMB chromatography process was 90% or more in all sections, and the solid content of L-cysteine excluding moisture in the process liquid produced by SMB chromatography was 90% or more in all sections. According to the above results, it can be interpreted that the method for purifying L-cysteine by SMB chromatography of the present 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.

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