Preparation method of R-3-aminobutyric acid

A kind of aminobutyric acid, R-3- technology, applied in the field of biocatalysis, to achieve the effect of high product purity and high selectivity

Active Publication Date: 2018-08-07
CHANGXING PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Compared with chemical methods, biocatalysis has the advantages of mild reaction conditions, high selectivity, and high product purity. Utilizing bio-enzymatic cata

Method used

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  • Preparation method of R-3-aminobutyric acid
  • Preparation method of R-3-aminobutyric acid
  • Preparation method of R-3-aminobutyric acid

Examples

Experimental program
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Example Embodiment

[0031] Example 1

[0032] 1. Construction of aspartase clone strain

[0033] After the cryopreserved Bacillus is resuscitated, cultured and subcultured, a pair of primers are designed according to the Bacillus aspartase gene (such as GenBank: AB028242.1) sequence published on NCBI using the bacterial liquid as the DNA template:

[0034] Forward primer (ASP-P-BamH I): 5’-CGCGGATCCATGAATACCGATGTTCGT-3’;

[0035] Reverse primer (ASP-R-Xho I): 5’-CCGCTCGAGTATTTTCTTCCAGCAATTCCCGG-3’;

[0036] Then PCR amplification was carried out. The reaction conditions were 94°C pre-denaturation for 5 minutes, then 30 cycles (94°C for 30s, 59°C for 30s, 72°C for 1.5min), and 72°C for another 10 minutes.

[0037] After the PCR is completed, gel verification and product recovery are performed. Then the target fragment and pET-28a(+) plasmid were subjected to a double digestion system consisting of BamH I and Xho I for digestion. After the reaction, the digestion product was recovered, and then the digested...

Example Embodiment

[0044] Example 2 Biocatalytic reaction of 1000L system

[0045] In the 1000L catalytic system, add 250kg of crotonic acid, 2.4kg of magnesium sulfate, 58kg of ammonium sulfate, then adjust the pH to 9.0 with ammonia water, stir at 40°C, add 10kg of aspartase bacteria sludge obtained in Example 1 above, start reaction. After the reaction for 12 hours, the concentration of R-3-aminobutyric acid in the solution can reach 294 g / L, and the conversion rate can reach more than 98%.

[0046] Please refer further Figure 3 to Figure 5 As shown, image 3 In order to adjust the pH of the liquid phase after adding the substrate, Figure 4 It is a liquid phase profile of the reaction 4h, and the reaction speed is very fast. Figure 5 It is the liquid phase spectrum of the reaction for 12 hours. It can be seen from this figure that the substrate has been basically reacted, and the residue of the substrate is very low.

[0047] After the reaction, the reaction solution is treated with a microfilt...

Example Embodiment

[0049] Example 3 Biocatalytic reaction of 1000L system

[0050] In the 1000L catalytic system, add 270kg of crotonic acid, 2.4kg of magnesium sulfate, 72.5kg of ammonium acetate, then adjust the pH to 9.2 with ammonia water, stir at 38°C, add 11kg of aspartase bacteria mud, and start the reaction. After the reaction for 12 hours, the concentration of R-3-aminobutyric acid in the solution can reach 319 g / L, and the conversion rate can reach more than 98%.

[0051] After the reaction, the reaction solution is treated with a microfiltration membrane to retain the aspartase bacteria mud and most of the enzyme protein, and the supernatant is passed through a nanofiltration membrane to remove SO 4 2- And most of the pigments. Microfiltration uses a microfiltration membrane with a relative molecular mass greater than 3000-5000 Daltons for separation, and nanofiltration with a sodium filter membrane with a relative molecular mass greater than 120-200 Daltons for separation. The obtained n...

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Abstract

The invention discloses a preparation method of R-3-aminobutyric acid. The method comprises the following steps: taking crotonic acid and ammonium salt as substrates; adding salt containing magnesiumions and regulating the pH (Potential of Hydrogen) by utilizing ammonia water; then adding recombinant aspartase as a biological enzyme catalyst; reacting at proper temperature and under an alkaline condition; after reacting, separating, purifying and crystallizing to obtain the R-3-aminobutyric acid. According to the method disclosed by the invention, ammonium ions of a reaction system are controlled, and reverse reaction is controlled and generation of byproducts is reduced; impurities including bacterial sludge, zymoprotein, sulfate ions, pigments and the like are intercepted by utilizing microfiltration and nanofiltration; high-quality products with high chiral purity and high impurity purity are obtained through continuous concentration and crystallization.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis, and in particular relates to a method for preparing R-3-aminobutyric acid by catalyzing crotonic acid with aspartase. Background technique [0002] R-3-aminobutyric acid (R-3-aminobutyric acid), CAS: 3775-73-3, is mainly used as the precursor of pharmaceutical intermediate R-3-aminobutyric acid. R-3-amino-1-butanol, CAS: 61477-40-5, is a key intermediate used in the treatment of AIDS drug Dolutegravir. Dolutegravir is a human immunodeficiency virus type I (HIV-1) integrase inhibitor drug, and its chemical synthesis requires many raw materials, and R-3-aminobutyric acid is one of its important raw materials. The molecular structural formula of R-3-aminobutyric acid is shown in formula (I): [0003] [0004] R-3-aminobutyric acid can be obtained through a one-step reduction reaction to obtain R-3-aminobutanol, the key intermediate of dolutegravir. The chiral purity of R-3-aminobutanol de...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 杨卫华张飞龙钱敏帆肖延铭谈聪
Owner CHANGXING PHARMA
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