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

AI Technical Summary

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 catalysis technology is a feasible solution, but there is currently no information on the preparation of R-3-aminobutyric acid by bio-enzymatic methods. related reports

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

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Experimental program
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Effect test

Embodiment 1

[0032] 1. Construction of Aspartase Cloning Strain

[0033] After the frozen bacillus was revived and cultured, a pair of primers were designed according to the sequence of the bacillus aspartase gene published on NCBI (such as GenBank: AB028242.1) using the bacterial solution as a DNA template:

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

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

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

[0037]After the completion of PCR, gel verification and product recovery were carried out. Subsequently, the target fragment and the pET-28a(+) plasmid were digested by a double enzyme digestion system composed of BamH I and Xho I. After the reaction, the digested product was recovered, and then the digested target ...

Embodiment 2

[0044] Example 2 1000L system biocatalytic reaction

[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 sludge obtained in Example 1 above, and start reaction. After 12 hours of reaction, 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 For adding the substrate and adjusting the pH, the liquid phase chromatogram, Figure 4 The liquid phase chromatogram of reaction 4h shows that the reaction speed is very fast. Figure 5 It is the liquid phase spectrum when reacting 12h, can find out from this figure that the substrate has been reacted substantially, and the residue of substrate is very low.

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

Embodiment 3

[0049] Example 3 1000L system biocatalytic reaction

[0050] In a 1000L catalytic system, add 270kg of crotonic acid, 2.4kg of magnesium sulfate, 72.5kg of ammonium acetate, adjust the pH to 9.2 with ammonia water, stir at 38°C, add 11kg of aspartase sludge, and start the reaction. After 12 hours of reaction, 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 is over, the reaction solution is treated with a microfiltration membrane to retain aspartase sludge and most of the enzyme protein, and the supernatant is passed through a nanofiltration membrane to remove SO 4 2- and most pigments. For microfiltration, microfiltration membranes with a relative molecular weight greater than 3000-5000 Daltons are selected for separation, and nanofiltration is used for separation with sodium filtration membranes with a relative molecular weight greater than 120-200 Daltons. The obtain...

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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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