Synthesis and purification method of (R)-3-aminobutyric acid

A technology of aminobutyric acid and amino acids, applied in the field of bioengineering, can solve problems such as clogging of biotransformation reaction liquid, affecting efficiency, membrane fouling, etc., and achieve high-efficiency expression

Active Publication Date: 2021-12-14
THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the prior art, there are generally two problems in the preparation of (R)-3-aminobutyric acid by enzymatic method: 1) The preparation of (R)-3-aminobutyric acid by enzymatic method usually requires the addition of ammonium salt to improve the conversion rate , the addition of ammonium salt will increase the complexity of the preparation and purification of (R)-3-aminobutyric acid in the later stage; 2) the purification of (R)-3-aminobutyric acid is mostly performed by membrane treatment methods such as microfiltration, ultrafiltration, and nanofiltration To pretreat the catalytic reaction system, but the membrane treatment biotransformation reaction solution is very easy to cause clogging and membrane fouling, which affects the efficiency

Method used

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  • Synthesis and purification method of (R)-3-aminobutyric acid
  • Synthesis and purification method of (R)-3-aminobutyric acid
  • Synthesis and purification method of (R)-3-aminobutyric acid

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Experimental program
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Effect test

Embodiment 1

[0067] Embodiment 1 Aminotransferase gene and the acquisition of genetically engineered cells

[0068] Through gene prediction and database comparison, a gene encoding aminotransferase was obtained from the metagenome of marine samples, its nucleotide sequence is shown in SEQ ID NO: 1, and its protein amino acid sequence is shown in SEQ ID NO: 2 Show. The gene was optimized according to the codon preference of Escherichia coli, and the codon-optimized gene was obtained, the nucleotide sequence of which was shown in SEQ ID NO:3. Entrust the synthesis of the nucleotide sequence shown in SEQ ID NO: 3 and clone the aminotransferase gene into the pET30 expression vector NdeI and XhoI to obtain a plasmid expressing the aminotransferase, which is named pET30-AT plasmid. The plasmid is transformed into BL21(DE3) cells to obtain aminotransferase gene-positive genetically engineered bacteria BL21(DE3)[pET30-AT].

Embodiment 2

[0069] The high-density fermentation culture of embodiment 2 aminotransferase engineering bacteria

[0070] 1) The cultivation of the seed liquid of aminotransferase engineering bacteria

[0071] Get the genetically engineered bacterium BL21 (DE3) [pET30-AT] that embodiment 1 prepares, it is inoculated on the petri dish flat plate to activate bacterial classification, culture time 14h; Pick single bacterium colony from flat plate and inoculate to LB medium (peptone 10g / L, yeast powder 5g / L, NaCl 10g / L), cultivated in a constant temperature shaker at 37°C and 180rpm for 16h to obtain a fermented seed culture solution.

[0072] 2) High-density fermentation culture of aminotransferase engineering bacteria

[0073] A 5L fermenter was used for high-density cell fermentation of the aminotransferase, and the filling volume was 2.5L. The seed liquid that step 1) is cultivated is inserted in the fermentation medium (peptone 30g / L, yeast powder 30g / L, glycerol 50g / L, Na2HPO4 16.4g / L, ...

Embodiment 3

[0074] Whole-cell catalytic synthesis of embodiment 3 (R)-3-aminobutyric acid

[0075] After the high-density fermentation in Example 2 was finished, the thalline was collected by centrifugation as a whole-cell catalyst. Preparation of biotransformation reaction liquid: add the thallus obtained after fermentation in Example 2 to water, the addition amount is 50g / L according to the wet weight of the thallus, add 200g / L crotonic acid, adjust the pH to 9.0 with ammonia water, and set the volume to 1000ml . The biotransformation reaction was carried out at 45°C. Sampling was taken at intervals in the middle process to detect the reaction between the substrate and the product. After 9 hours, the substrate was completely converted, and the concentration of (R)-3-aminobutyric acid in the supernatant was 227 g / L, and the conversion rate was 95%. The monitoring diagram of the conversion process is shown in the Figure 4 shown.

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Abstract

The invention relates to a synthesis and purification method of (R)-3-aminobutyric acid. The invention firstly provides a novel aminotransferase, the amino acid sequence of the novel aminotransferase is shown as SEQ ID NO: 2, and the novel aminotransferase can effectively catalyze crotonic acid to synthesize (R)-3-aminobutyric acid under the condition that no ammonium salt is added; the invention also provides a high-density fermentation method which can enable the engineering bacteria expressing the aminotransferase to realize high-density growth in a short time and realize high-efficiency expression of the aminotransferase. The invention further provides a separation and purification method of (R)-3-aminobutyric acid, and (R)-3-aminobutyric acid with the purity higher than 99% can be simply and efficiently obtained without complex membrane treatment equipment of different levels.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing and purifying (R)-3-aminobutyric acid. Background technique [0002] (R)-3-aminobutyric acid (R-3-aminobutyric acid), CAS: 3775-73-3, is a human immunodeficiency virus type I (HIV-1) integrase inhibitor drug Dolutegravir ) is a key intermediate in the synthesis. At present, the preparation methods of (R)-3-aminobutyric acid mainly include chemical synthesis and enzymatic catalysis. Chemical synthesis includes, for example, using ethyl acetoacetate as a raw material, condensing with acetamide, asymmetric hydrogenation, and hydrolysis to obtain (R)-3-aminobutyric acid. In the past few years, as the market demand for (R)-3-aminobutyric acid is increasing, due to the requirements of reducing costs and reducing pollution, the use of enzymatic methods to catalyze the production of (R)-3-aminobutyric acid has been reported Gradually increase. [0003...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/63C12N15/70C12P13/04C07C227/40C07C229/08C12R1/19
CPCC12N9/1096C12Y206/01C12N15/635C12N15/70C12P13/04C07C227/40C07B2200/07C07C229/08
Inventor 王昭凯胡凡杨隆河
Owner THIRD INST OF OCEANOGRAPHY MINIST OF NATURAL RESOURCES
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