A kind of method for preparing l-2-aminobutyric acid with double enzymes in series

A kind of aminobutyric acid and double-enzyme technology, applied in the field of bioengineering, can solve the problems of unsuitability for industrial production, high substrate price of amino donor and cofactor regeneration system, and only 50% of theoretical yield, and achieve low price and low cost. The effect of simple production cost and process

Active Publication Date: 2022-03-04
JIANGNAN UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

There are two main types of biosynthesis, one is the enzymatic resolution method, the mixed-rotation DL-2-aminobutyric acid is catalyzed by the corresponding enzyme to generate L-2-aminobutyric acid, but the theoretical yield is only 50%; the other is Enzyme synthesis method, the substrate is threonine, threonine generates 2-ketobutyrate under the action of threonine dehydrogenase, and 2-ketobutyrate is reduced to L-2 under the action of transaminase or dehydrogenase -Aminobutyric acid, this method has the disadvantages of needing a large amount of amino donors, a cofactor regeneration system and a high substrate price, and is not suitable for industrial production

Method used

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  • A kind of method for preparing l-2-aminobutyric acid with double enzymes in series
  • A kind of method for preparing l-2-aminobutyric acid with double enzymes in series

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Embodiment 1

[0023] Embodiment 1 Construction of recombinant Escherichia coli BL21 / pET-28-GlmES

[0024]The fusion L-glutamate mutase is connected by (glycine-glutamine) repeated decapeptides from the glmE subunit from Clostridium tetanomorphum and the mutS subunit from Clostridium cochlearium, and the nucleotide sequence is as SEQ ID NO 5, the gene was synthesized by Suzhou Jinweizhi Company and linked to pET-28a. The recombinant plasmid pET-28a-glmES was transformed into Escherichia coli BL21 strain to obtain recombinant Escherichia coli BL21 / pET-28a-glmES.

Embodiment 2

[0025] Example 2 Construction of recombinant Escherichia coli expressing L-aspartic acid-β-decarboxylase

[0026] The gene encoding the L-aspartic acid-β-decarboxylase mutant shown in the nucleotide sequence as SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO.8 or SEQ ID NO.9 is connected to Plasmid pET-28a, obtain recombinant plasmid pET-28a-K18A / V287I or pET-28a-K18A / V287L or pET-28a-K18S / V287I or pET-28a-K18S / V287L, and transfer the recombinant plasmid into Escherichia coli BL21, Recombinant Escherichia coli BL21 / pET-28a-K18A / V287I or BL21 / pET-28a-K18A / V287L or BL21 / pET-28a-K18S / V287I or BL21 / pET-28a-K18S / V287L were obtained by screening.

Embodiment 3

[0027] Expression of embodiment 3 L-glutamic acid mutase

[0028] Recombinant Escherichia coli BL21 / pET-28a-glmES was inoculated in 5 mL of LB medium with a kanamycin concentration of 50 μg / mL, and cultured overnight at 37° C. with shaking at 200 rpm. The above overnight culture was inoculated into 2YT medium containing 50 μg / mL of kanamycin at an inoculum size (V / V) of 1%, and cultured with shaking at 37°C and 200 rpm until the bacterial liquid OD 600 To 0.6-0.8, add IPTG to a final concentration of 0.2mmol / L, induce culture at 30°C for about 20h to obtain bacteria. After collecting the bacteria by centrifugation at 6000rpm, they were ultrasonically crushed, purified by His Trap HP affinity column, and detected the target protein by SDS-PAGE.

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Abstract

The invention discloses a method for preparing L-2-aminobutyric acid in tandem with two enzymes, belonging to the field of bioengineering. The present invention obtains L-glutamic acid mutase and L-aspartic acid-β-decarboxylase by respectively cultivating recombinant Escherichia coli expressing L-glutamic acid mutase and L-aspartic acid-β-decarboxylase decarboxylase. The two enzymes are added to the reaction system with a certain mass ratio, and L-glutamic acid is used as a substrate to carry out an enzyme reaction to prepare L-2-aminobutyric acid. When the amount of L-aspartic acid-β-decarboxylase 2mg / mL, reacted for 24h, converted into 8.5mmol / L L-2 aminobutyric acid, and the molar conversion rate was 85.00%. Compared with the chemical production method, this method has a safe production process and no environmental pollution. Compared with the multi-enzyme synthesis system using threonine as the substrate, the substrate is cheaper and the process is simpler.

Description

technical field [0001] The invention relates to a method for preparing L-2-aminobutyric acid in series with double enzymes, belonging to the technical field of bioengineering. Background technique [0002] L-2-aminobutyric acid (L-ABA) is a non-protein amino acid and an important pharmaceutical intermediate, which is widely used in the fields of medicine, pesticides and food. For example, it can be used to synthesize the antiepileptic drug levetiracetam And anti-tuberculosis drugs ethambutol, buvaracetam and so on. Currently, the synthesis methods of L-2-aminobutyric acid mainly include chemical synthesis and biosynthesis. The chemical asymmetric synthesis of L-2-aminobutyric acid needs to use highly toxic chemical reagents such as cyanide and bromine as synthetic raw materials, and then use a chemical chiral resolving agent to achieve the resolution of racemic intermediate products. The reaction process has disadvantages such as highly toxic compounds, serious environment...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04C12N9/90C12N9/88C12N15/60C12N15/61C12N15/70C12R1/19
CPCC12P13/04C12N9/90C12N9/88C12N15/70C12Y504/03009C12Y401/01012
Inventor 周哲敏刘中美刘宇锋周丽崔文璟郭军玲
Owner JIANGNAN UNIV
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