Method for extracting alpha-ketoglutarate and pyruvate simultaneously from microbial fermentation broth or enzymatic conversion solution

Abstract

The present disclosure provides a method for extracting alpha-ketoglutarate and pyruvate simultaneously from microbial fermentation broth or enzyme transformation solution, which is related to the technical field of biological separation and extraction. The method comprises the following steps: centrifuging the microbial fermentation broth or enzymatic conversion solution containing -KG and PA to remove the cells and other visible solids; removing the macromolecular impurities by ultrafiltration; evaporating and concentrating under reduced pressure conditions; extracting with the water-insoluble extraction after acidification; separating crude crystals of -KG and crude liquid of PA by evaporation crystallization method (if concentration of PA is great higher than that of -KG, crystallization separation should be conducted after distilling partial pure pyruvate); washing the crude crystal of -KG with water-insoluble organic solvent as ethyl acetate or butyl acetate, drying and crushing to obtain qualified -KG; distilling to gain qualified PA product applying high vacuum distillation (or molecular distillation).

Claims

1. A method for extracting alpha-ketoglutarate (-KG) and pyruvate (PA) simultaneously, wherein said method comprises: pretreating microbial fermentation broth or enzymatic conversion solution by separating microorganisms and other visible solids from the microbial fermentation broth or enzymatic conversion solution containing -KG and pyruvate by a centrifugal process, and removing macromolecular impurities though a filtration process, and concentrating -KG or pyruvate to a concentration of 120 g/L, and obtaining a pretreatment solution after crossing the basic ion exchange resin to remove high-valent cations; separating crude -KG and crude pyruvate by acidifying the pretreatment solution to obtain a acidified solution, and extracting -KG and pyruvate to obtain an extraction solution from the acidified solution using a water-insoluble extractive agent; wherein if C.sub.-KG: C.sub.PA2 in the extraction solution, then further concentrating the extraction solution to obtain a higher concentration of -KG by evaporation, and crystallizing at low temperature and separating the crude -KG crystals and crude pyruvate liquid by centrifuging after crystallization; wherein if C.sub.-KG: C.sub.PA2 in the extraction solution, removing the extractive agent and the dissolved water therein by distillation, and harvesting part of a pure pyruvate with further distillation, and then adding a small amount of water to residue of distillation, and separating crude -KG crystals and remaining crude pyruvate liquid by centrifuging after crystallization at low temperature; purifying PA by obtaining pure pyruvate through distilling crude pyruvate liquid under decompression conditions.

2. The method of claim 1, wherein pretreating microbial fermentation broth or enzymatic conversion solution comprises one or more of centrifugation, ultrafiltration, microfiltration and frame filtration steps.

3. The method of claim 1, wherein acidifying the pretreatment solution comprises adjusting pH of the pretreatment solution to be 1.5.

4. The method of claim 1, wherein said the water-insoluble extractive agent is ethyl acetate or butyl acetate.

5. The method of claim 1, comprising extracting -KG and pyruvate at 540 C. for 1060 min.

6. The method of claim 1, comprising crystallizing at low temperature at 15 C.

7. The method of claim 1, comprising obtaining pure -KG after a series of steps by leaching with water-immiscible organic solvent, drying at a drying temperature of 5080 C. and crushing; wherein a volume of water-immiscible organic solvent is less than -fold that of the crude -KG crystals.

8. The method of claim 1, comprising obtaining pure pyruvate by distilling crude pyruvate liquor at 0.070.1 Mpa and 3060 C.

9. The method of claim 1, further comprising: A: separating the microorganisms and other visible solids from the microbial fermentation broth or enzymatic conversion solution containing -KG and PA to obtain a supernatant; B: using a ultrafiltration membrane module to remove macromolecular substances, cell fragments, pigments, lipids, proteins and polysaccharides, from the supernatant, and washing residual filtrate with a small amount of water; wherein the ultrafiltration membrane module is selected from a group comprising a roll membrane, a tubular membrane or a plate membrane with molecular weight cut-off of 5003,000; C: concentrating -KG or pyruvate to a concentration of 120 g/L by decompression distillation at 0.070.095 Mpa and 5080 C.; D: passing a concentrated solution obtained from step C through a basic ion exchange resin to remove high-valent cations; E: adjusting a liquid obtained from step D to pH1.5 by adding sulfuric acid while stirring; F: extracting -KG and pyruvate by adding water-insoluble organic solvent to an acidizing solution obtained from step E at 540 C. for 1060 min, with the volume of the extractant to be 2-fold that of the acidified solution, and the time of extraction to be 2; wherein if C.sub.-KG: C.sub.PA of a resulting extractant is 2, separating -KG and pyruvate by following steps G-1 to K-1; wherein if C.sub.-KG: C.sub.PA of the resulting extractant is 2, separating -KG and pyruvate by following steps G-2 to L-2; G-1: concentrating -KG to a concentrated of 200 g/L by distilling the water-immiscible extractant ethyl acetate or butyl acetate under conditions of 0.070.095 Mpa and 3560 C.; H-1: cooling a concentrated liquid from step G-1 rapidly and crystalling at temperature15 C. for 15 days, and separating crude crystals of -KG and crude liquid of pyruvate by centrifugation; I-1: washing crude crystals of -KG obtained from the step H-1 with a water-immiscible organic solvent ethyl acetate or butyl acetate, with a volume of the water-insoluble organic solvent to be -fold of that of crude products; and harvesting the pure -KG after a series of steps by leaching with water-insoluble organic solvent, drying under 5080 C. condition and crushing into powder; J-1: harvesting the crude pyruvate by distilling off extractant and water therein from the crude liquid of pyruvate gained from the step H-1, under conditions of 0.070.095 Mpa and 3560 C.; K-1: obtaining the pure pyruvate by distilling the crude pyruvate from above step applying high vacuum distillation or molecular distillation under conditions of 0.0980.1 Mpa and 2560 C.

10. The method of claim 1, comprising A: separating the microorganisms and other visible solids from the microbial fermentation broth or enzymatic conversion solution containing -KG and pyruvate to obtain a supernatant; B: removing the macromolecular substances, cell fragments, pigments, lipids, proteins and polysaccharides from the supernatant, and washing the residual filtrate with a small amount of water, wherein a ultrafiltration membrane module is selected from a group comprising a roll membrane, a tubular membrane and a plate membrane with molecular weight cut-off of 5003,000; C: concentrating -KG or pyruvate concentration to a concentration of 120 g/L by concentrating under decompression conditions at 0.070.095 Mpa at 5080 C.; D: passing a concentrated solution obtained from step C through the basic ion exchange resin to remove high-valent cations; E: adjusting a liquid obtained from step D is a pH1.5 by adding sulfuric acid while stirring; F: extracting -KG and pyruvate by adding water-insoluble organic solvent to an acidizing solution obtain from step E at 540 C. for 1060 min, with the volume of the extractant to be 2-fold that of the acidified solution, and the time of extraction to be 2; resulting an extractant with the C.sub.-KG: C.sub.PA to be 2 in the; G-2: wherein if C.sub.-KG: C.sub.PA is 2 in the extractant obtained in step F, distilling off the extractant and the water therein under a conditions of 0.070.095 Mpa and 3560 C.; H-2: obtaining a partial pure pyruvate by distilling the crude mixture of organic acid by applying high vacuum distillation or molecular distillation under conditions of 0.0980.1 Mpa and 2560 C.; I-2: separating the crude -KG crystals and crude pyruvate liquid after adding a small amount of water to dissolve a residual solid completely from above distillation step and immediately crystallizing at low temperature of 15 C. for 15 days; J-2: obtaining the crude crystals of -KG from the above step I-2 by washing with a water-immiscible organic solvent ethyl acetate or butyl acetate, and wherein the volume of the water-insoluble organic solvent is -fold that of crude products; and harvesting pure -KG after a series of steps by leaching with water-insoluble organic solvent, drying under 5080 C. condition and crushing into powder; K-2: harvesting crude pyruvate by distilling off extractant and water therein from the crude liquid of pyruvate gained from the step I-2, under conditions of 0.070.095 Mpa and 3560 C.; L-2: obtaining remaining pure pyruvate by distilling the crude pyruvate from the step K-2 with high vacuum distillation or molecular distillation under conditions of 0.0980.1 Mpa and 2560 C.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0038] FIG. 1. Flow chart of extraction process in one embodiment of present disclosure which use ethyl acetate as extractant.

EXAMPLES

Determination of -KG and PA

[0039] The concentrations of -KG and PA are determined by high-performance liquid chromatography (HPLC) equipped with a Bio-Rad Aminex HPX-87H column, eluted with 5 mM H.sub.2SO.sub.4 at a flow rate of 0.5 mL/min and 40 C. -KG and PA were detected by an Agilent 1260 (Agilent, Palo Alto, Calif., USA) UV absorbance detector at 210 nm.

Calculation of Purity

[0040] [00001]$W=\frac{f_2.Math.m_1.Math.p}{f_1.Math.m_2}$

f.sub.1peak area of standard sample solution determined by HPLC;
f.sub.2peak area of experimental sample solution determined by HPLC;
m.sub.1quality of standard sample (g);
m.sub.2quality of experimental sample (g);
ppurity of standard sample (%).

Calculation of Yield

[0041] [00002]$N=\frac{M.Math.W}{C.Math.V}$

Mtotal quality of the obtained pure sample (g);
Wpurity of the obtained sample (%);
Cconcentration of the experimental sample in fermentation broth (g/L);
Vvolume of the fermentation broth after removal of the cells (L).

[0042] The following examples are the method for simultaneous extraction of -KG and PA from microbial fermentation broth or enzymatic conversion solution containing -KG and PA. The fermentation broth was obtained by using Yarrowia lipolytica WSH-Z06 CCTCC M20714 or Candida glabrata CCTCC M202019 as fermented strain respectively.

[0043] Other similar microbial fermentation broth or enzymatic conversion solution containing -KG and PA could be carried out according to the embodiment of the present invention, and the high purity of -KG and PA also could be obtained through simple modification.

[0044] The present disclosure was described in detail referencing to the accompanying drawings.

Example 1

[0045] Extraction of -KG and PA from the fermentation broth obtained by using Y. lipolytica WSH-Z06 as fermented strain was carried out as the following steps:

[0046] (1) Centrifugation filtration: the fermentation broth containing -KG and PA was centrifuged to remove cells and other visible solids, with the centrifuge conditions of room temperature and 3,000 r/min.

[0047] (2) Membrane filtration: the macromolecular substances, such as cell fragments, pigments, lipids, proteins and polysaccharides, were removed from the above obtained supernatant with ultrafiltration membrane module, and then the residual filtrate was washed with a small amount of water to increase the yield of extraction. The applied ultrafiltration membrane module was a roll membrane with a molecular weight cut-off of 5003,000, and the ultrafiltration process was conducted under the conditions of room temperature and operating pressure of 410 Bar.

[0048] (3) Concentration: the concentration of -KG was concentrated to 120 g/L by distilling the obtained ultrafiltrate under reduced pressure condition of 0.08 Mpa at 65 C.

[0049] (4) Removal of impurities by ion exchange resin: The concentrated solution flowed through 732 cation exchange resin, which was pre-acidified to pH 2.0 and washed to neutral (sodium resin was converted to hydrogen type resin). The purpose of this operation is to remove high-valent cations from the concentrated solution and acidify the solution at the same time.

[0050] (5) Acidification: the pH of concentrated liquor gained after ion exchange column treatment was adjusted to 1.5 by adding sulfuric acid with stirring under normal temperature condition.

[0051] (6) Extraction: 3-fold volume of ethyl acetate that of acidified solution was used to extracted organic acids at 15 C. for 20 min, and then the aqueous and organic phases were separated. This extraction experiments were repeated 3 times. The C.sub.-KG: C.sub.PA of the resulted solution was 2.

[0052] (7) Distillation: The extractant ethyl acetate containing organic acid obtained from the extraction process was distilled under reduced pressure of 0.08 Mpa at 50 C. until the concentration of -KG increased to 200 g/L.

[0053] (8) Separation of -KG crude crystals and PA crude liquor: the crude -KG crystals and crude PA liquid are separated after distilling off extractant ethyl acetate to thick liquid of organic acid mixture and immediately crystallizing at 10 C. for 1 day. The crystal is cultured for one day and then separated from mother liquor by centrifugation.

[0054] (9) Obtainment of pure -KG: The -KG crude crystal was washed with ethyl acetate, of which the volume is -fold than that of crude crystal. After removing the ethyl acetate by centrifugation and drying at 65 C. in a constant temperature drying oven, the pure -KG was collected. The yield of this procedure was 79.8% and the purity of -KG was 99.3%.

[0055] (10) Harvest of crude PA: the PA crude liquor was distilled to remove ethyl acetate under reduced pressure of 0.09 Mpa at 40 C. and then temperature was raised to 55 C. to distill off the water therein.

[0056] (11) Obtainment of pure PA: The pure PA, light yellow transparent liquid, was obtained with high vacuum distillation at 0.099 Mpa and 50 C. For liquid product of PA, the purity gradually decreases as the color of the liquid changes from a light yellowish transparent to a yellowish transparent to a red transparent, then to a reddish viscous. The yield of this procedure was 80.6% and the purity of PA was 99.5%.

Example 2

[0057] The embodiment is the same as Example 1 except that the C.sub.-KG: C.sub.PA of the resulted solution in step (6) was 3.5 and step (4) was not performed.

[0058] The content of -KG and PA was determined. The purity of -KG was 97.8% and its yield was 80.4%. The purity of PA was 99.5% and its yield was 72.7%.

Example 3

[0059] Taking the fermentation broth obtained from Y. lipolytica WSH-Z06 as fermented strain (C.sub.-KG: C.sub.PA=3.5), The embodiment is the same as Example 1 except that the C.sub.-KG: C.sub.PA of the resulted solution in step (6) was 3.5 and the step (2) is not carried out. The centrifuged liquid obtained in step 1 was distilled under the conditions of step (3) directly.

[0060] The content of -KG and PA was determined. The purity of -KG was 96.2% and its yield was 81.7%. The purity of PA was 99.3% and its yield was 65.2%.

Example 4

[0061] The embodiment is the same as Example 1 except that the C.sub.-KG: C.sub.PA of the resulted solution in step (6) was 3.5 and the pH of concentrated solution was adjusted to 3 in the step (5).

[0062] The content of -KG and PA was determined. The purity of -KG was 99.2% and its yield was 45.8%. The purity of PA was 99.3% and its yield was 40.9%.

Example 5

[0063] The embodiment is the same as Example 1 except that the C.sub.-KG: C.sub.PA of the fermentation culture was 3.5 and step (6) is not carried out.

[0064] The content of -KG and PA was determined. The purity of -KG was 99.4% and its yield was 52.8%. The purity of PA was 99.6% and its yield was 56.3%.

Example 6

[0065] Taking the fermentation broth obtained from Y. lipolytica WSH-Z06 as fermented strain (C.sub.-KG: C.sub.PA=3.5), The embodiment is the same as Example 1 except that the C.sub.-KG: C.sub.PA of the resulted solution in step (6) was 3.5 and the extraction (step 6) and the purification (step 9) were carried out using butyl acetate instead of ethyl acetate.

[0066] The content of -KG and PA was determined. The purity of -KG was 99.4% and its yield was 82.1%. The purity of PA was 99.6% and its yield was 79.8%.

Example 7

[0067] Extraction of -KG and PA from the fermentation broth obtained by using C. glabrata as fermented strain was carried out as the following steps:

[0068] (1) Centrifugation filtration: the fermentation broth containing -KG and PA was centrifuged to remove cells and other visible solids, with the centrifuge conditions of room temperature and 3,000 r/min.

[0069] (2) Membrane filtration: the macromolecular substances, such as cell fragments, pigments, lipids, proteins and polysaccharides, were removed from the above obtained supernatant with ultrafiltration membrane module, and then the residual filtrate was washed with a small amount of water to enhance the yield of extraction. The applied ultrafiltration membrane module was a roll membrane with molecular weight cut-off of 5003,000, and the ultrafiltration process was conducted under the conditions of room temperature and operating pressure of 410 Bar.

[0070] (3) Concentration: the concentration of PA was concentrated to 200 g/L by distilling the obtained ultrafiltrate under reduced pressure condition of 0.08 Mpa at 65 C.

[0071] (4) Removal of impurities by ion exchange resin: The concentrated solution flowed through 732 cation exchange resin, which was pre-acidified to pH 2.0 and washed to neutral (sodium resin was converted to hydrogen type resin). The purpose of this operation is to remove high-valent cations from the concentrated solution and acidify the solution at the same time.

[0072] (5) Acidification: the pH of concentrated liquor gained after ion exchange column treatment was adjusted to 1.0 by adding sulfuric acid with stirring under normal temperature condition.

[0073] (6) Extraction: Ethyl acetate was used to extracted organic acids at 35 C. for 15 min and the volume of ethyl acetate was 5-fold than that of acidified solution. The aqueous and organic phases were separated after extraction. This extraction experiments were repeated 2 times. The C.sub.-KG: C.sub.PA of the resulted solution was 0.5.

[0074] (7) Distillation off ethyl acetate and water: the extractant was firstly distilled off under the conditions of 0.08 Mpa at 50 C. to remove ethyl acetate and then distilled at 0.09 Mpa and 53 C. remove water.

[0075] (8) Obtainment of partial pure PA: partial pure PA is obtained by distilling the organic acid mixture under 0.099 Mpa and 30 C.

[0076] (9) Separation of -KG crude crystals and PA crude liquor: a small amount of water was added to dissolve the residual solid completely, and then the mixture immediately crystallized at 10 C. for 2 days. After crystallization, the crude -KG crystals and crude PA liquid are separated.

[0077] (10) Obtainment of pure -KG: The -KG crude crystal was washed with ethyl acetate, of which the volume is -fold than that of crude crystal. Then the pure -KG was obtained after removing the ethyl acetate by centrifugation, drying at 65 C. and crushing into powder.

[0078] The yield of this procedure was 80.9% and the purity of -KG was 99.4%.

[0079] (11) Harvest of crude PA: the PA crude liquor was firstly distilled at 0.09 Mpa and 40 C. to remove ethyl acetate and then distilled at 55 C. to remove water.

[0080] (12) Obtainment of remaining pure PA: The pure PA, light yellow transparent liquid, was obtained by distilling crude PA employing molecular distiller under conditions of 0.099 Mpa and 30 C. The yield of this procedure was 82.8% and the purity of PA was 99.6%.

[0081] While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.