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Method for synthesizing L-2-aminobutyric acid by enzymatic method

A technology of aminobutyric acid and enzymatic synthesis, which can be used in fermentation and other directions, and can solve problems such as low conversion rate

Active Publication Date: 2017-08-04
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to provide a new enzymatic method for synthesizing L-2-aminobutyric acid, in order to solve the problem of low conversion rate in the prior art

Method used

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  • Method for synthesizing L-2-aminobutyric acid by enzymatic method
  • Method for synthesizing L-2-aminobutyric acid by enzymatic method
  • Method for synthesizing L-2-aminobutyric acid by enzymatic method

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Embodiment 1

[0051] Embodiment 1 A method for enzymatically synthesizing 2-aminobutyric acid, the specific steps are as follows:

[0052] 1. Heterologous expression of alanine dehydrogenase alaD and formate dehydrogenase FDH:

[0053] 1. Heterologous expression and enzyme activity determination of alanine dehydrogenase alaD:

[0054] (1) Obtain alaD gene fragment

[0055] Primers were designed according to the alanine dehydrogenase (alaD) gene sequence (Genbank accession number: EF154460, SEQ ID NO.1) and the front and back sequences, and the better upstream primers of alanine dehydrogenase (alaD) obtained after screening The upstream primer is 5'-aaGGATCCatgaagatcggcattccaaaag-3' (the uppercase is the BamHI restriction site, SEQ ID NO.2); the downstream primer is 5'-ttGAATTCtcatccctgcagcaacgaatgaac-3' (the uppercase is the EcoRI restriction site, SEQ ID NO.3). The alaD gene fragment can be efficiently obtained by amplifying with the above-mentioned upstream and downstream primers.

[...

Embodiment 2

[0086] Embodiment 2 A kind of method of enzymatically synthesizing 2-aminobutyric acid

[0087] Except that the following steps are different, all the other steps are the same as in Example 1.

[0088] A genetically engineered bacterium co-expressing alaD and FDH was constructed, and a bacterium co-expressing FDH and alaD was obtained through fermentation. The construction of the plasmids pET32a-alaD and pET28a-FDH is the same as in Example 1, and the plasmids pET32a-alaD and pET28a-FDH are transferred into the same strain E.coli BL21(DE3) to obtain the strain BL21-alaD / FDH co-expressing alaD and FDH , recombinant protein induction of co-expression strains see Figure 5 (Swimming lane 3 arrow shows the co-expressed induced protein, induction 2 is the empty vector negative control, induction 1 is the protein molecular weight standard).

[0089] (1) The fermentation of the co-expression bacteria was the same as in Example 1.

[0090] (2) Add the following pharmaceutical prepa...

Embodiment 3

[0091] Embodiment 3 A kind of method of enzymatically synthesizing 2-aminobutyric acid

[0092] In addition to the following steps "Cultivate the strain BL21-alaD in the fermentation medium, add 0.1% lactose to induce expression at 30°C for 16 hours, collect the cells by centrifugation to obtain the expression cells of alaD, and store the expressed cells at -20°C ; Culture the bacterial strain BL21-FDH in the fermentation medium, add 0.1% lactose to induce expression at 30°C for 16 hours, collect the cells by centrifugation to obtain the FDH expressing cells, and store the expressing cells at -20°C for induction temperature. Be that 30 ℃ is changed into 25 ℃, all the other are with embodiment 1.

[0093] Then obtain the expression cells of alaD and FDH by the method in the first step, add the following medicine preparation catalyst system successively in the 250ml Erlenmeyer flask: 0.5mol of 2-ketobutyric acid, 0.5mol of ammonium formate, wet cells of formate dehydrogenase 20...

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Abstract

The invention discloses a method for synthesizing L-2-aminobutyric acid by an enzymatic method. According to the method, 2-ketobutyric acid is catalyzed by the aid of alanine dehydrogenase and formate dehydrogenase to produce the L-2-aminobutyric acid. The method particularly includes the steps: uniformly mixing 0.5-1.5mol of 2-ketobutyric acid, 0.5-1.5mol of ammonium formate, o-0.5g / L of NAD (nicotinamide adenine dinucleotide) and 20g / L of formate dehydrogenase and alanine dehydrogenase co-expression wet cells (or 10-30g / L of formate dehydrogenase wet cells, 10-30g / L of alanine dehydrogenase wet cells) in a rector; adjusting a pH (potential of hydrogen) value to be 7.0-9.0; performing catalytic reaction for 16-20h at the temperature ranging from 30 DEG C to 37 DEG C to obtain the L-2-aminobutyric acid. The method is rapid in reaction, reverse oxidation deamination activity is low, and product loss is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of chemical synthesis, and in particular relates to a method for enzymatically synthesizing L-2-aminobutyric acid. Background technique [0002] L-2-aminobutyric acid is a key intermediate for the production of the new antiepileptic drug levetiracetam, and is also a key chiral precursor for the synthesis of the antibacterial and anti-tuberculosis drug ethambutol, and is an important chiral precursor for many chiral drugs. sex intermediate. In recent years, the synthesis technology of L-2-aminobutyric acid has become a research focus of genetic engineering pharmaceuticals. Many studies use FDH to construct NADH regeneration system and co-express it with leucine dehydrogenase. By constructing a dual-enzyme coupled catalytic system, 2-ketobutyric acid is used as a substrate to generate L-2-aminobutyric acid, which solves the problem of The problem of expensive NADH saves the cost to a great extent. [0003] ...

Claims

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Application Information

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IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 张娟冯志彬陈国忠
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
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