Ketoacid reductase, gene, engineering bacteria and its application in the synthesis of chiral aromatic 2-hydroxy acids

A technology of reductase and engineering bacteria, applied in genetic engineering, oxidoreductase, application and other directions, can solve the problems of low substrate loading and yield, limit catalytic efficiency, etc., and achieve low cost, simple process and high catalytic efficiency. Effect

Active Publication Date: 2021-07-27
ZHEJIANG UNIV OF TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] When applying a redox cascade deracemization strategy to prepare optically pure chiral 2-hydroxyacids, it was found that the activity of ketoacid reductases limited the overall catalytic efficiency, resulting in relatively low substrate loading and yields

Method used

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  • Ketoacid reductase, gene, engineering bacteria and its application in the synthesis of chiral aromatic 2-hydroxy acids
  • Ketoacid reductase, gene, engineering bacteria and its application in the synthesis of chiral aromatic 2-hydroxy acids
  • Ketoacid reductase, gene, engineering bacteria and its application in the synthesis of chiral aromatic 2-hydroxy acids

Examples

Experimental program
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Effect test

Embodiment 1

[0052] Embodiment 1: screening efficient ketoacid reductase LlKAR

[0053]Using the amino acid sequence of the known ketoacid reductase LeKAR (shown in SEQ ID NO.2, the corresponding nucleotide sequence is shown in SEQID NO.1) as a template, five potential ketoacid reductions were obtained by NCBI-Blastp online alignment Enzyme sequences are respectively LlKAR (shown in amino acid sequence SEQ ID NO.4, nucleotide sequence shown in SEQ ID NO.3), LpKAR (shown in amino acid sequence SEQ ID NO.6, nucleotide sequence shown in SEQ ID shown in NO.5), LmKAR (shown in amino acid sequence SEQ ID NO.8, nucleotide sequence shown in SEQ ID NO.7), KoKAR (shown in amino acid sequence SEQ ID NO.10, nucleotide sequence is Shown in SEQ ID NO.9) and SnKAR (shown in amino acid sequence SEQ ID NO.12, nucleotide sequence shown in SEQ ID NO.11), the amino acid sequence homology is respectively 84%, 78%, 74%, 49% and 49%. Subsequently, the corresponding nucleotide sequences were synthesized in vitr...

Embodiment 2

[0060] Embodiment 2: Construction of recombinant bacteria E.coli BL21(DE3) / pET28b-HADH / pCDFDuet-LlKAR-GDH

[0061] Glucose dehydrogenase (GDH) gene (nucleotide sequence shown in SEQ ID NO.15, amino acid sequence shown in SEQ ID NO.16) derived from Exiguobacterium sibiricum (WP_012369122.1) was synthesized corresponding nucleosides in vitro The acid sequence was inserted into the expression plasmid pET28b to obtain the recombinant plasmid pET28b-GDH, which was transformed into E.coli BL21(DE3), spread on the LB plate containing 50 μg / mL kanamycin, and screened the recombinant bacteria E. coli BL21(DE3) / pET28b-GDH.

[0062] With the seamless cloning kit ( II, Vazyme Biotech Co., Ltd), the nucleotide sequence of GDH and the preferred L1KAR were successively connected to the expression vector pCDFDuet-1 to obtain the recombinant plasmid pCDFDuet-L1KAR-GDH, which was transformed into E.coli BL21(DE3), Spread on LB plates containing 50 μg / mL streptomycin to screen recombinant bac...

Embodiment 3

[0066] Example 3: Inducing the co-expression of recombinant bacteria E.coli BL21(DE3) / pCDFDuet-LlKAR-GDH dual enzymes

[0067] Inoculate recombinant Escherichia coli E. coli BL21(DE3) / pCDFDuet-LlKAR-GDH into LB liquid medium containing 50 μg / mL streptomycin, shake and culture at 37°C and 150 rpm for 8-10 hours to obtain seed liquid; Put the seed solution into the LB liquid medium containing 50 μg / mL streptomycin according to the inoculum amount of 2% (volume concentration), and cultivate it with shaking at 37°C and 150rpm until the OD 600When it reaches 0.6, add IPTG to a final concentration of 0.1mM, shake and culture at 28°C and 150rpm for 10-12h, collect the wet cells by centrifugation and wash twice with saline to obtain E.coli BL21(DE3) / pCDFDuet-LlKAR - Resting cells of GDH. Using uninduced E.coli BL21(DE3) / pCDFDuet-LlKAR-GDH as a control, SDS-PAGE protein electrophoresis analysis was carried out, and the results were as follows Figure 4 As shown, it can be seen that a...

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Abstract

The invention discloses a ketoacid reductase, gene, engineering bacteria and its application in the synthesis of chiral aromatic 2-hydroxy acids. The amino acid sequence of the ketoacid reductase is SEQ ID NO.4, SEQ ID NO.8 or Shown in one of SEQ ID NO.10; the present invention provides a high-efficiency ketoacid reductase derived from Leuconostoc lactis, which can catalyze a broad-spectrum aromatic 2-ketoacid, and use acetophenone as a substrate Substrate loading was increased from 100 mM to 400 mM. A single-bacteria double-plasmid three-enzyme tandem redox cascade system established by ketoacid reductase, 2-hydroxyacid dehydrogenase and glucose dehydrogenase can catalyze the efficient deracemization of most racemic aromatic 2-hydroxyacids It is a chiral aromatic (R)-2-hydroxy acid, and the yield and e.e. value are both greater than 99%. Finally, it is applied to deracemize 300mM o-chloromandelic acid to prepare optically pure (R)-o-chloromandelic acid, and the yield is as high as 83.8g / (L·d).

Description

[0001] (1) Technical field [0002] The invention relates to a ketoacid reductase gene derived from Leuconostoc lactis, an encoding enzyme, a carrier, an engineering bacterium and an application in synthesizing chiral aromatic 2-hydroxy acid. [0003] (2) Background technology [0004] Chiral 2-hydroxy acids are a class of compounds with hydroxyl (-OH) substitutions at the C1 position of the carboxyl (-COOH) side. They are widely distributed and active in nature. They are important chiral building blocks for the production of pharmaceuticals and fine chemicals. Chemical, pharmaceutical and other fields have important application value. Among them, the more representative ones are a class of derivatives containing a benzene ring in the structure, such as (R)-mandelic acid is a key intermediate for the production of semi-synthetic penicillins, cephalosporins, antineoplastic drugs and weight loss drugs, and is also an important (R)-o-chloromandelic acid is a key chiral building b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/04C12N15/53C12N1/21C12P7/42C12R1/19
CPCC12N9/0006C12P7/42C12Y101/01169C12P41/001C12Y101/01047
Inventor 薛亚平郑裕国王闯柳志强
Owner ZHEJIANG UNIV OF TECH
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