Method for the preparation of 2,4-dihydroxybutyrate

A technology of hydroxybutyrate and carbonyl, which is applied in the field of preparation of 2,4-dihydroxybutyrate, and can solve problems such as undetermined enzyme reactions

Active Publication Date: 2015-03-25
AVEATIS ANIMAL NUTRITION SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Only few studies have reported the production of DHB in patients deficient in succinate semialdehyde dehydrogenase (Shinka et al., 2002), but the enzymatic reactions involved in the production of DHB have not been identified

Method used

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  • Method for the preparation of 2,4-dihydroxybutyrate
  • Method for the preparation of 2,4-dihydroxybutyrate
  • Method for the preparation of 2,4-dihydroxybutyrate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1: Demonstration of OHB reductase activity

[0077] Construction of plasmids containing wild-type genes encoding lactate dehydrogenase or malate dehydrogenase:

[0078] Will encode (L)-malate dehydrogenase in Escherichia coli, Ec-mdh (SEQ ID No.1), (D)-lactate dehydrogenase in Escherichia coli, Ec-ldhA (SEQ ID No.3), lactic acid Streptococcus (L)-lactate dehydrogenase, Ll-ldhA (SEQ ID No.5), Bacillus subtilis (L)-lactate dehydrogenase, Bs-ldh (SEQ ID No.7), thermophilic fat The (L)-lactate dehydrogenase of Bacillus, Gs-ldh (SEQ ID No.9), two hypotypes of (L)-lactose dehydrogenase of rabbit, Oc-ldhA (SEQ ID No.11 and The gene of SEQ ID No.13) was extended by PCR using the high-fidelity polymerase Phusion TM (Fermantas) and the primers listed in Table 1, the genomes of Escherichia coli MG1655, Lactococcus IL1403 and Bacillus subtilis chain 168 were used as templates. The genes Oc-ldhA and Gs-ldh were codon optimized for expression in E. coli and synthesized by ...

Embodiment 2

[0093] Example 2: Construction of lactose dehydrogenase with improved OHB reductase activity

[0094] Site-directed mutagenesis of the lactis ldhA gene was performed using the pET28-Ll-ldhA plasmid as a template. PCR using the oligonucleotide pairs listed in Table 3 (Phusion 1U, HF buffer 20% (v / v), dNTPs 0.2mM, forward and reverse primers 0.04uM, template plasmid 30-50ng, water) to introduce site-directed mutagenesis to alter the amino acid sequence. In addition to functional mutations that facilitate identification of mutant clones, the genes mutated by PCR contained new restriction sites listed in Table 3 (introduced using silent mutations). The template DNA was removed by digesting the PCR product with Dpnl at 37° C. for 1 h, and the PCR product was transformed into competent E. coli DH5α (NEB) cells. Mutated plasmids were identified by restriction site analysis and confirmed to carry the desired mutation by DNA sequencing.

[0095] table 3 : an oligonucleotide used t...

Embodiment 3

[0101] Example 3: Construction of malate dehydrogenase with improved OHB reductase activity

[0102] Site-directed mutagenesis of the mdh gene from E. coli was performed as described in Example 2 using the primers listed in Table 5. Plasmid pET28-Ec-mdh was used as template.

[0103] table 5 Oligonucleotides used to mutate malate dehydrogenase mdh from Escherichia coli (nnk denotes a degenerate codon where k denotes thymine or cytosine)

[0104]

[0105] The mutated enzyme was expressed, purified and tested for OHB and pyruvate reductase activity as described in Example 1. exist image 3 Activity measurements for OHB and pyruvate are summarized in . The results showed that replacement of Arg81 by alanine, cysteine, glycine, histidine, isoleucine, leucine, methionine, asparagine, glutamine, serine, threonine or valine achieved Significant OHB reductase activity, and concomitant decrease in pyruvine reductase activity.

[0106] Combining R81A mutations in Ec-Mdh with o...

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Abstract

The present invention concerns a method for the preparation of 2,4-dihydroxybutyric acid from homoserine comprising a first step of conversion of the primary amino group of homoserine to a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and a second step of reduction of the obtained 2-oxo-4-hydroxybutyrate (OHB) to 2,4-dihydroxybutyrate

Description

technical field [0001] The present invention relates to a new method for preparing 2,4-dihydroxybutyrate (2,4-DHB) from homoserine, which comprises two steps: [0002] - the first step, converting the primary amino group of homoserine into a carbonyl group to obtain 2-oxo-4-hydroxybutyrate, and [0003] - In a second step, the 2-oxo-4-hydroxybutyrate (OHB) obtained is reduced to obtain 2,4-DHB. [0004] The carboxylic acids described in the present invention are named according to the salt form (such as 2,4-dihydroxybutyrate, 2,4-dihydroxyburyrate) or according to the acid form (such as 2,4-dihydroxybutyric acid, 2,4-dihydroxybutyric acid ) names have the same meaning. Background technique [0005] 2,4-Dihydroxybutyric acid (equivalent to 2,4-DHB or DHB) is a compound of considerable economic value. By adjusting the appropriate pH, DHB can be converted to α-hydroxy-γ-butyrolactone in aqueous medium. α-Hydroxy-γ-butyrolactone is the main precursor for the preparation of m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P7/42C12P7/16C12N1/00
CPCC12N1/14C12N1/16C12N1/20C12P7/42C12Y101/01027C12Y101/01028C12Y101/01037C12Y206/01001C12Y206/01042C12Y206/01057C12N9/0006C12N9/1096C12Y101/01082C12Y101/01272C12Y101/01299C12Y206/01C12N15/09C12N15/70
Inventor 托马斯·瓦尔特伊莲娜·科迪尔克莱芒蒂娜·德雷赛尔让-玛丽·弗朗索瓦罗伯特·胡艾特
Owner AVEATIS ANIMAL NUTRITION SA
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