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Production method of (R)-3-hydroxybutyric acid

A technology of hydroxybutyric acid and hydroxybutyrate, which is applied in the field of bioengineering, can solve the problems of long reaction time, high consumption of organic solvents, low yield, etc., and achieve the effect of ensuring food safety and broad industrial application prospects

Active Publication Date: 2017-08-22
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method for chemically synthesizing (R)-3-hydroxybutyric acid requires high temperature, high-pressure reaction conditions and expensive chiral metal catalysts, etc.; and enzymatic degradation of poly-3-hydroxybutyric acid to produce (R)-3-hydroxybutyric acid requires A large amount of organic solvent is consumed, the reaction time is long, and high-purity poly-3-hydroxybutyrate is required as a starting material, and racemization is very easy to occur, but this method is still at the laboratory level. If it enters the industrial scale Production still has problems such as high cost and low yield

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 : Preculture and fermentation

[0046] Thaw the bacterial strain CGMCC No.13957 preservation liquid that is contained in the glycerol tube, inoculate in seed culture medium (glucose 75g / L, corn steep liquor 25-30g / L, (NH 4 ) 2 SO 4 20g / L, KH 2 PO 4 1.5g / L, MgSO 4 ·7H 2 O 0.5g / L, urea 1.0g / L, histidine 30mg / L, molasses 25g / L, biotin 100μg / L, pH 7.0), 5000mL Erlenmeyer bottle liquid volume 500mL, 30 ℃ for 18 hours, absorbance value When ΔOD=0.4-0.5, shake the bed to obtain seed culture solution. After testing, no bacteria.

[0047] Inoculate 500 mL of seed culture solution into a 7-liter fermenter with a liquid volume of 5 liters. The composition of the medium is the same as that of the seed medium, pH=6.4-6.7 (after sterilization). Feed medium: ammonium sulfate 500g / L, glucose 650g / L. The culture temperature is 30°C, the tank pressure is 0.05Mpa, and the initial aeration ratio is 1vvm. Stirring 600rpm. During the fermentation, the pH is controll...

Embodiment 2

[0048] Example 2 : Separation of fermentation broth and extraction and purification of (R)-3-hydroxybutyric acid

[0049] 5.2 liters of fermented broth obtained in Example 1 was centrifuged at 4500 rpm, and the thallus was discarded. Add 1% celite to the supernatant and filter. Then add 1% activated carbon, stir and decolorize for 30 minutes and then filter to obtain a clear filtrate.

[0050] The filtrate is subjected to nanofiltration treatment with a nanofiltration membrane, the obtained filtrate is passed through 732 cation exchange resin, and the permeate is collected. Concentrate to 1000g / L, in the form of oil, pour it out while hot to obtain 55.9g (R)-3-hydroxybutyric acid, yield 91%. The purity of (R)-3-hydroxybutyric acid was determined by high performance liquid chromatography. The chromatographic column is a Shim-pack Vp-ODSC18 column (150L×4.6), the mobile phase is acetonitrile:water (v / v)=15:85, the UV detection wavelength is 210nm, the injection volume is 20...

Embodiment 3

[0051] Example 3 : Preparation of (R)-3-hydroxybutyrate sodium

[0052] Slowly add 5 g of (R)-3-hydroxybutyric acid obtained in Example 2 into an equivalent amount of 2N sodium hydroxide solution to carry out neutralization reaction, and the reaction temperature is controlled below 25°C. Concentrate by rotary evaporation until solid precipitates, let stand for 2 hours, collect the solid by suction filtration, and blow dry at 60°C to obtain 4.8 g of (R)-3-hydroxybutyrate sodium powder with a yield of 79%. Melting point is 152℃, specific rotation: [α] D 20 =-14.2° (C=10%, H 2 O).

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Abstract

The invention discloses a method for producing (R)-3-hydroxybutyric acid through one-step fermentation of a microbe, and the method adopts a non-pathogenic microbe and directly converts a carbon source and a nitrogen source into (R)-3-hydroxybutyric acid through fermentation. The produced (R)-3-hydroxybutyric acid can be further prepared into salts such as a sodium salt, a potassium salt, a magnesium salt, a calcium salt and the like to be used as medicinal active components or nutritional supplements, so that the (R)-3-hydroxybutyric acid has extensive industrial application prospects. The microbe used in the method comprises corynebacterium glutamicum constructed through genetic engineering, and the corynebacterium glutamicum is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 13957.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for producing (R)-3-hydroxybutyric acid and its salts through microbial fermentation. Background technique [0002] (R)-3-Hydroxybutyrate ((R)-3-Hydroxybutyrate, 3HB) is an optically active chiral compound with a CAS number of 625-72-9. (R)-3-Hydroxybutyric acid is a compound produced by the metabolism of long-chain fatty acids in the liver in mammals. It exists as the main ketone body in plasma and peripheral tissues and can be used as an energy source in most tissues of the body. . In addition to its nutritional function, (R)-3-hydroxybutyric acid also has the effect of treating many diseases, including: 1. It can treat many diseases that benefit from increased levels of ketone bodies (such as neurological disorders including epilepsy and myoclonus) disorders and neurodegenerative diseases including Alzheimer's disease and dementia); 2. Reduce free radical d...

Claims

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Application Information

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IPC IPC(8): C12P7/42C12N1/20C12R1/15
CPCC12N1/20C12P7/42C12N1/205C12R2001/15
Inventor 范文超
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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