METHOD FOR PRODUCING D-PSICOSE FROM D-PSICOSE BORATE COMPLEX USING CHROMATOGRAPHY AND COMPOSITION CONTAINING D-PSICOSE

Abstract

The present application relates to a method for producing D-psicose, the method comprising the steps of: putting a composition containing a D-psicose borate complex into a chromatography comprising divalent cations; and separating the composition containing the D-psicose borate complex into a D-psicose-containing fraction (i) and a borate-containing fraction (ii).

Claims

1. A method for producing D-psicose, the method comprising: a step of putting a composition comprising a D-psicose borate complex into a chromatography comprising divalent cations; and a step of separating the composition comprising the D-psicose borate complex into a D-psicose-containing fraction (i) and a borate-containing fraction (ii).

2. The method of claim 1, further comprising, before the step of putting, a step of obtaining a composition comprising the D-psicose borate complex by bringing a D-fructose and a borate into contact with a D-psicose 3-epimerization enzyme, a strain expressing the enzyme or a culture of the strain.

3. The method of claim 2, wherein the D-psicose 3-epimerization enzyme is a wild-type psicose epimerization enzyme derived from Agrobacterium tumefaciens or Kaistia granuli, or a variant thereof.

4. The method of claim 1, wherein the composition comprising the D-psicose borate complex has a borate content of less than 25% (w/w) on a dry solid basis.

5. The method of claim 1, wherein the D-psicose-containing fraction (i) has a borate content of less than 0.5 ppm (w/w) on a dry solid basis.

6. The method of claim 1, wherein the D-psicose-containing fraction (i) has a D-psicose content of 85% (w/w) or more on a dry solid basis.

7. The method of claim 1, wherein the chromatography is a simulated moving bed chromatography, and the divalent cations are included in a columnar form filled with a cation exchange resin.

8. The method of claim 1, wherein the divalent cations are one or more of calcium ions, barium ions, and strontium ions.

9. The method of claim 7, wherein the simulated moving bed chromatography comprises four or more columns.

10. The method of claim 7, wherein the simulated moving bed chromatography performs elution with water of 50-70 C.

11. The method of claim 1, wherein a content of the borate-containing fraction (ii) is 95 or higher parts by weight with respect to 100 parts by weight of a borate content in the composition comprising the D-psicose borate complex.

12. The method of claim 1, wherein the composition comprising the D-psicose borate complex further comprises a fructose.

13. The method of claim 12, wherein the borate-containing fraction (ii) has a borate content of 95 or higher parts by weight with respect to 100 parts by weight of a borate content in the composition comprising the D-psicose borate complex, and has a fructose content of 95 or higher parts by weight with respect to 100 parts by weight of a fructose content in the composition comprising the D-psicose borate complex.

14. A composition comprising D-psicose, wherein a content of the D-psicose is equal to or higher than 99% (w/w) and lower than 100% (w/w) on a dry solid basis, and a content of a borate is more than 0 and lower than 0.5 ppm (w/w).

15. The composition of claim 14, wherein the composition comprising the D-psicose comprises 2% (w/w) or less of D-fructose on a dry solid basis.

16. The composition of claim 14, wherein the composition comprising the D-psicose is a fraction comprising D-psicose separated with a chromatography.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 is a HPLC chromatogram for the results of converting D-fructose to D-psicose, respectively obtained when a borate is added (experimental example 1) and not added (comparative example 1) to a D-psicose epimerization enzyme reaction of example 1 of the present application.

[0043] FIG. 2 is a HPLC chromatogram of a D-psicose fraction fractionated by the simulated moving bed (SMB) chromatography of the present application.

[0044] FIG. 3 is a HPLC chromatogram of a borate fraction fractionated by the simulated moving bed (SMB) chromatography of the present application.

[0045] FIG. 4 is a D-psicose production flowchart of the present application.

MODE FOR CARRYING OUT THE INVENTION

[0046] Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, the examples are to explain the present application by way of example only, and thus the scope of the present application is not limited by the examples. Details not included herein are readily recognized and appreciated to those skilled in the art at the time of filing the present application, and the description therefore will thus be omitted.

EXAMPLE 1

Production of D-Psicose using Borate and Enzymatic Conversion Method

[0047] An enzymatic conversion reaction was carried out using Corynebacterium glutamicum (KCCM11403P), which is a recombinant strain known to express a variant of a psicose epimerase derived from Agrobacterium tumefaciens (Korean Patent No. 10-1455759). 0.1 M fructose was dissolved in water and 0.05 M borate and 10 mM MnCl.sub.2 were added thereto (experimental example 1) or only 10 mM MnCl.sub.2 was added thereto (comparative example 1) to prepare a raw material for enzymatic conversion reaction. Then, the strain (20% w/w) was added to the raw material and the reaction was carried out for 3 hours under the condition of pH of 8.0 and the temperature of 55 C.

[0048] Afterwards, the conversion rate was calculated by comparing the peak area values of the substrate (D-fructose) of the psicose epimerization enzyme and the product (psicose) through HPLC analysis, and the HPLC analysis was performed under the conditions that the sample is injected to a column (BP-100 Ca.sup.2+ carbohydrate column) set at 80 C. and distilled water as a mobile phase flows at a rate of 0.6 ml/min through the column.

[0049] As a result, the conversion rate to D-psicose was 25% for comparative example 1, but 56% for experimental example 1. Thus, it could be confirmed that the conversion rate of experimental example 1 was increased by 224% compared to that of comparative example 1 (FIG. 1).

EXAMPLE 2

Borate Separation

[0050] 2-1. Confirmation of Borate Separation using SMB Chromatography

[0051] An SMB (Simulated Moving Bed) chromatography was used to separate borate-removed D-psicose from a composition comprising D-psicose borate complex (borate, D-fructose and D-psicose, hereinafter referred to as feed). The SMB chromatography used an advanced simulated moving-bed system (Organo, Japan) instrument, and the instrument includes successively connected six columns (each having a height of 1.2 m, and a diameter of 4.3 cm), a feed pump, a recirculation pump, an eluent pump, a heat exchanger and a valve for controlling flow rate. The conditions for operating the SMB chromatography are as the Table 1 below.

TABLE-US-00001 TABLE 1 Mixture of fructose, psicose Feed and borate Feed concentration 60% (w/w) Cation exchange resin Amberlite (Amberlite (adsorbent) filled in columns CR-1310; Ca-type) Size and number of columns 43 mm 1200 mm * 6 columns Desorbent (eluent) O Flow speed 2.49 m/H Eluent temperature 60 C.

[0052] Additionally barium ions (Ba.sup.2+) and strontium ions (Sr.sup.2+), as divalent cations, were filled and used instead of calcium ions, and an experiment using sodium ions (Na.sup.+), as monovalent cations, was additionally performed as a control group. As a result, it was confirmed that the amounts of the borate remaining in a psicose fraction in the case of using calcium ions, strontium ions and barium ions were 0.2 ppm, 0.3 ppm and 0.4 ppm respectively, which are lower than the boron content standard in drinking water recommended by WHO, which is 0.5 ppm, and the rate of removal was 99.999% (DS%, w/w). On the other hand, it was confirmed that the psicose fraction in the case of using sodium ions was 62100 ppm, which is a significantly higher value than 0.5 ppm. Additionally, the purities of a psicose in psicose fractions were 99.350% (DS%, w/w) (FIG. 3), 99.150% (DS%, w/w) and 90.850% (DS%, w/w), respectively. Additionally compared to those of the feed, the recovery rate of the borate were found to be approximately 100% for the borate fraction in the case of using calcium ions and strontium ions and the recovery rate of the fructose was 99.264%, which was a high recovery rate (borate content in borate fraction/(psicose fraction+borate fraction)*100). Thus a fructose and a borate, which are the raw materials used for producing a psicose, can be separated while obtaining a high-purity psicose using the SMB chromatography (see Table 2 and FIG. 3), and therefore it could be confirmed that the fructose and the borate remaining after production of the psicose could be reused (FIG. 4).

TABLE-US-00002 TABLE 2 Amberlite (Amberlite Psicose Borate CR-1310; Ca-type) Feed fraction fraction Borate (DS %, w/w) 12.050 0.00002 12.050 D-psicose (DS %, w/w) 49.255 99.350 0.280 D-fructose (DS %, w/w) 38.695 0.650 87.670 Borate (DS %, w/w) 12.050 0.00003 12.050 D-psicose (DS %, w/w) 49.255 99.150 0.350 D-fructose (DS %, w/w) 38.695 0.850 87.600 Borate (DS %, w/w) 12.050 0.00004 12.050 D-psicose (DS %, w/w) 49.255 90.850 8.750 D-fructose (DS %, w/w) 38.695 9.150 79.200 Borate (DS %, w/w) 12.050 6.210 8.870 D-psicose (DS %, w/w) 49.255 55.850 37.280 D-fructose (DS %, w/w) 38.695 37.940 53.850 *DS: Dry solid

[0053] 2-2. Confirmation of Content of Borate Separable by Smb Chromatography

[0054] The purity of psicose in a psicose fraction was 99% (DS%, w/w) or higher and 0.5 ppm or less of boron concentration in drinking water recommended by WHO was satisfied, whereby it could be confirmed that SMB chromatography is applicable for such a borate content in feed. The contents of borate, psicose and D-fructose in the feed are as the Table 3 below.

[0055] As a result, it could be confirmed that when the quantity of added borate was 21.05% (DS%, w/w) or less, the rate of borate removal was all 99.999%, the quantity of borate remaining in the psicose fraction was 0.5 ppm (w/w) or less, and the purity of psicose was 99% (DS%, w/w) or more (Table 3).

TABLE-US-00003 TABLE 3 Experimental Experimental Experimental Experimental Experimental Experimental example 2 example 3 example 4 example 5 example 6 example 7 Borate content in 0.00 6.25 11.76 16.67 21.05 25.00 feed (DS %, w/w) Content of Borate 0 0.000015 0.000021 0.000032 0.000044 0.000137 psicose fraction (DS %, w/w) D- 0.78 0.75 0.68 0.56 0.54 1.23 fructose Psicose 99.22 99.25 99.32 99.44 99.46 98.77 *DS: Dry solid

[0056] The present application has been described above in detail with reference to the specific features, and it will be apparent to those skilled in the art that this detailed description is only for a preferred embodiment and does not limit the scope of the present application. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.