A method for enzymatically synthesizing l-2-aminobutyric acid

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: 2020-06-30
SHANDONG YANGCHENG BIOLOGY TECH CO LTD
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for enzymatically synthesizing l-2-aminobutyric acid
  • A method for enzymatically synthesizing l-2-aminobutyric acid
  • A method for enzymatically synthesizing l-2-aminobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

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...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

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

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12P13/04
CPCC12P13/04
Inventor 张娟冯志彬陈国忠
Owner SHANDONG YANGCHENG BIOLOGY TECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap