Method for preparing natural L-cysteine crystals by continuous chromatography

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.

Claims

1. 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, wherein the continuous chromatography excludes adsorption or elution of L-cysteine; (b) concentrating the separated liquid; and (c) recovering L-cysteine 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 in step (a) has a sulfonic acid functional group.

5. The method of claim 1, wherein the strongly acidic cation-exchange resin in step (a) is a styrene-divinylbenzene copolymer having a sulfonic acid functional group.

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

7. 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.

8. The method of claim 1, 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. The method of claim 1, 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. The method of claim 1, 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.

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

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

13. The method of claim 1, 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).

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

15. L-Cysteine crystals prepared according to the preparation method of claim 1.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1 shows a representative illustration of a process for preparing L-cysteine 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 Common analytical methods used in Examples of the present disclosure are as follows:

(5) (1) HPLC for Quantitative Analysis of L-Cysteine

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

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

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

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

(10) Flow rate: 0.425 mL/min

(11) Temperature: 30° C.

(12) Detection: UV at 220 nm

(13) Volume of sample introduced: 2 μL

(14) (2) Method for Measuring Purity of L-Cysteine Crystals

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

(16) (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 temperature of L-cysteine dried crystals to room temperature;

(17) (b) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine crystals cooled to room temperature, placing it in a 1 L volumetric flask, and diluting it with a 0.2 N HCl solution;

(18) (c) Preparing a sample of 0.5000 g/L by quantitatively measuring 0.5000 g of the L-cysteine crystals, placing it in a 1 L volumetric flask, and diluting it with a 0.2N 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 of the standard reagent manufacturer, and then converting the concentration of L-cysteine in the sample (converted [concentration of L-cysteine] is 0.5000 g/L×[purity of standard product]); and

(19) (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.

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

(21) 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:

(22) (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;

(23) (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;

(24) (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]);

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

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

(27) (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

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

(29) 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.

(30) 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).

(31) 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:

(32) Internal pressure: 80 mmHg

(33) Steam pressure: 2 bar

(34) Maximum injection amount: 100 L

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

(36) Evaporation rate: about 25 L/hr

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

(38) 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.

(39) 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.

(40) 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.

(41) (3) Concentration

(42) 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 temperature of the L-cysteine crystal slurry was 55° C. immediately after the concentration. The concentration conditions are as follows:

(43) Internal pressure: 80 mmHg

(44) Steam pressure: 2 bar

(45) Maximum injection amount: 100 L

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

(47) Evaporation rate: about 10 L/hr

(48) (4) Cooling and Recovery of L-Cysteine Crystals

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

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

(51) Washing liquid: triple-distilled water

(52) Filter type: Polyamide multifilament fiber filter fabric

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

(54) Bowl rotation speed: 3,000 rpm

(55) Bowl rotation time: 20 min

(56) During the separation, the washing liquid was added 20% of the volume of the L-cysteine crystal slurry. After the separation, the resultant 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 finally, L-cysteine crystals were prepared.

(57) 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 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 crystals was 98.6% or more.

(58) 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

(59) In the preparation 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.

(60) 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 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.

(61) TABLE-US-00001 TABLE 1 Purity of Yield of SMB Content of L-cysteine Type of stationary Functional chromatography L-cysteine crystals phase Component group (%) (%) (%) TRILITE ® Styrene- Sulfonic 92.4 93.2 99.2 MCK32L divinylbenzene group copolymer PUROLITE ® Styrene- Sulfonic 90.8 92.9 98.6 PCR642 divinylbenzene group copolymer DIAION ® Styrene- Sulfonic 91.4 92.5 99.3 UBK555 divinylbenzene group copolymer DIAION ® Styrene- None 22.6 37.5 No SP850 divinylbenzene Crystallization copolymer MACRONET ® Styrene- None 25.4 44.5 No MN202 divinylbenzene Crystallization copolymer AMBERLITE ® Styrene- None 25.8 31.8 No XA1600 divinylbenzene Crystallization copolymer DIAION ® Methacrylate None 16.2 39.1 No HP2MGL polymer Crystallization DIAION ® Methacrylate- Carboxyl 15.4 32.7 No WK10 divinylbenzene group Crystallization copolymer TRILITE ® Styrene- Trimethylamine 13.5 38.9 No AMP16 divinylbenzene group Crystallization copolymer TRILITE ® Styrene- Tertiary 14.2 32.2 No AW90 divinylbenzene amine Crystallization copolymer group

(62) 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 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.

(63) 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 crystals could be obtained with a high yield, concentration, and purity.

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

(64) In the Preparation 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 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.

(65) 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 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.

(66) TABLE-US-00002 TABLE 2 pH of Recovery Purity of fermentation rate of SMB Content of L-cysteine broth chromatography (%) L-cysteine (%) 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

(67) 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.

(68) 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.

(69) 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

(70) In the Preparation 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.

(71) 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.

(72) Accordingly, the time at which nucleation 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.

(73) TABLE-US-00003 TABLE 3 Recovery rate of Concentration Time at which Purity of L-cysteine crystallization (g/L) nucleation occurred crystals (%) 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 separation No crystal separation due to solidification due to solidification

(74) The nucleation of L-cysteine 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.

(75) The purity of the 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 L-cysteine crystal slurry occurred, and accordingly, stirring and separation of the crystals were impossible. Thus, itis 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.

(76) Based on these results, it was confirmed 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 confirmed 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

(77) 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 temperature as the temperature at the completion of cooling for 12 hours. As a result, L-cysteine 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 crystals were prepared. The purity of the L-cysteine crystals was 99.7%, and the recovery rate of the crystallization process was found to be 4.2%.

(78) 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 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 5—Evaluation According to Cooling Temperature

(79) In the Preparation Example, L-cysteine crystals were prepared by only varying the cooling temperature 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 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 slurry used in each experiment was 1 L.

(80) 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.

(81) TABLE-US-00004 TABLE 4 Purity of Recovery rate of Cooling L-cysteine crystallization temperature (° C.) crystals (%) 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

(82) The purity of the L-cysteine crystals was 98% or more in all cases. The recovery rate of the crystallization process increased in inverse proportion to the cooling temperature. That is, the recovery rate of the crystallization process increased as the cooling temperature was lowered. In addition, the process recovery rate was found to be 45% or more at a temperature of 30° C. or below, and it was expected that the process recovery rate would be 50% or more at a temperature below 25° C.

Example 6—Evaluation According to Concentration of Fermentation Broth Containing L-Cysteine Used as Source Material for SMB Chromatography

(83) In the Preparation 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 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. 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.

(84) 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.

(85) TABLE-US-00005 TABLE 5 Concentration of Yield of SMB L-cysteine in source liquid chromatography Solid content for SMB chromatography (g/L) process (%) 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

(86) 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.

(87) 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.