Preparation of (R)-3-hydroxybutyric acid or its salts by one-step fermentation

Abstract

The subject invention relates to a process of preparing (R)-3-hydroxybutyric acid or a salt thereof by one-step fermentation with a nonpathogenic microorganism. The fermentation of (R)-3-hydroxybutyric acid was performed by supplying with certain carbon and nitrogen sources. These microorganisms include a Glutamic acid Bacterium HR057 strain or one type of genetically engineered Corynebacterium Glutamicum.

Claims

1. A process for producing (R)-3-hydroxybutyric acid, wherein the process comprises culturing Corynebacterium glutamicum strain deposited under the accession number CGMCC No. 13111 at the China General Microbiological Culture Collection Center in a fermentation broth, wherein the fermentation broth comprises carbon and nitrogen sources, and the carbon and nitrogen sources are directly converted into (R)-3-hydroxybutyric acid by a one-step fermentation, wherein the (R)-3-hydroxybutyric acid is recovered from the fermentation broth after it is excreted during fermentation.

2. The process of claim 1, wherein the carbon source is selected from the group consisting of glucose, sucrose, maltose, molasses, starch and glycerol.

3. The process of claim 1, wherein the nitrogen source is selected from the group consisting of an organic nitrogen source and an inorganic nitrogen source.

4. The process of claim 3, wherein the organic nitrogen source is selected from the group consisting of corn steep liquor, bran hydrolysate, soybean cake hydrolysate, yeast extract, yeast powder, peptone, and urea.

5. The process of claim 3, wherein the inorganic nitrogen source is selected from the group consisting of ammonium sulfate, ammonium nitrate, and aqueous ammonia.

6. The process of claim 1, wherein the (R)-3-hydroxybutyric acid is prepared in the form of (R)-3-hydroxybutyrate sodium salt, (R)-3-hydroxybutyrate potassium salt, (R)-3-hydroxybutyrate magnesium salt, or (R)-3-hydroxybutyrate calcium salt.

Description

EXAMPLE 1

Pre-Culture and Fermentation

(1) A glycerol stock CGMCC No. 13111 stored at −80° C. was thawed and inoculated to a 5000 mL flask containing 500 mL seed medium (75 g/L of glucose, 25-30 g/L of corn steep liquor, 20 g/L of (NH.sub.4).sub.2SO.sub.4, 1.5 g/L of KH.sub.2PO.sub.4, 0.5 g/L of MgSO.sub.4.7H.sub.2O, 1.0 g/L of urea, 30 mg/L of histidine, 25 g/L of molasses, 100 μg/L of biotin, pH 7.0), and cultured at 30° C. for 18 hours. The seed culture cultivation was completed when OD=0.4-0.5.

(2) 500 mL seed culture was inoculated into a 7-liter fermenter filled with 5 liters medium. The composition of fermentation medium was the same as the seed medium described above, and the pH was controlled at 6.4˜6.7 after sterilization. Feed medium contains 500 g/L ammonium sulfate and 650 g/L glucose. The fermentation temperature was set at 30° C., the tank pressure was kept at 0.05 Mpa, and the initial ventilation ratio was 1 vvm. Stirring speed was 600 rpm. The pH of the fermentation was about pH 6.5.

(3) The pH was controlled at 6.7 and the temperature was raised to 35° C. at the later phase of fermentation. The dissolved oxygen constant (dO2) was controlled at 15˜25% by adjusting ventilation and stirring speed. To control residual sugar level, sugar was fed slowly while the concentration of the original sugar dropped to about 3.0% and the residual sugar was controlled at 1.5%˜2.0%. (R)-3-hydroxybutyric acid was accumulated to 11.8 g/L after 72 hours.

EXAMPLE 2

Isolation of Fermentation Broth and Extraction of (R)-3-hydroxybutyric Acid

(4) 5.2 liters of the fermentation broth obtained in Example 1 was centrifuged at 4500 rpm and the cells were discarded. The supernatant was filtered with 1% diatomaceous earth. Clear filtrate was recovered after stirring for 30 minutes mixed with 1% activated carbon.

(5) The filtrate was concentrated through nanofiltration membrane, and the resulting filtrate was passed through a 732 cation exchange resin to get a 1000 g/L concentrated filtrate. The concentrated collection was oily and collected while hot to give 56.8 g of (R)-3-hydroxybutyric acid with a yield of 92.5%. The purity of (R)-3-hydroxybutyric acid was determined by high performance liquid chromatography. The chromatographic column was Shim-pack Vp-ODSC18 column (150 L×4.6). The mobile phase consisted of acetonitrile: water (v/v)=15: 85, UV detection wavelength was 210 nm, injection volume was 20 μL, flow rate was 1 mL/min, column temperature was 10° C. The purity of (R)-3-hydroxybutyric acid was 98% and the specific optical value was [α] D20=−25° (C=6%, H.sub.2O).

EXAMPLE 3

Preparation of Sodium (R)-3-hydroxybutyrate

(6) A neutralization reaction was performed by mixing 5 g of (R)-3-hydroxybutyric acid obtained in Example 2 with an equivalent amount of a 2.0 N sodium hydroxide solution at 25° C. or lower. 4.9 g of sodium (R)-3-hydroxybutyrate powder was finally obtained with an 81% yield by rotating concentration, standing for 2 hours, and being collected by suction filtration and dried at 60° C. The salt has a melting point of 152° C. and a specific optical value [α] D20 of −14.1° (C=10%, H2O).

EXAMPLE 4

Racemization of (R)-3-hydroxybutyric Acid

(7) 10 g of (R)-3-hydroxybutyric acid was slowly added to 100 mL of a 2.0 N sodium hydroxide solution, and the mixture was heated to 60° C. for 4 hours. The optical value of product was 0° at room temperature. A neutralization reaction was carried out by adding hydrochloric acid to adjust the pH of the mixture to 7.0. 10.3 g of sodium 3-hydroxybutyrate powder was obtained with a yield of 85% by rotary evaporation until a solid was observed, allowing to stand for 2 hours, and then filtration. The results showed that racemic 3-hydroxybutyrate was obtained.

(8) In summary, the present invention disclosed that the engineered Corynebacterium glutaricum by genetic modification could ferment (R)-3-hydroxybutyric acid in a one-step process at a high yield, which was proved to be safe and non-toxic food grade. The engineered strain and manufacturing process that were disclosed in this invention has broad industrial application prospects.