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NAD<+>-independent aldehyde oxidase catalyzing production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde, and its application

An aldehyde oxidase, hydroxypropionic acid technology, applied in the directions of oxidoreductase, enzymes, biochemical equipment and methods, etc., can solve problems such as excessive consumption, coenzyme metabolism disorder, etc.

Inactive Publication Date: 2014-07-30
BEIJING UNIV OF CHEM TECH
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Problems solved by technology

[0003] Overexpressed aldehyde dehydrogenase leads to intracellular NAD in the biological production of 3-hydroxypropionate + Excessive consumption, resulting in the disorder of coenzyme metabolism in the body

Method used

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  • NAD&lt;+&gt;-independent aldehyde oxidase catalyzing production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde, and its application
  • NAD&lt;+&gt;-independent aldehyde oxidase catalyzing production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde, and its application

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Embodiment Construction

[0012] The following specific methods can enable those skilled in the art to understand the present invention more comprehensively, but do not limit the present invention in any way.

[0013] The concrete steps of the inventive method comprise:

[0014] 1. Cloning of Aldehyde Oxidase Enzyme from Klebsiella pneumoniae

[0015] Bacterial strains and plasmids: Escherichia coli (E.coli) top10 was purchased from Beijing Bomed Company, Klebsiella pneumoniae (Klebsiella pneumoniae) DSM2026 was purchased from German DSM, and the expression vector pET-pk was preserved in our laboratory.

[0016] Medium: LB medium (g / L) peptone 10, yeast extract 5, NaCl10, pH 7.0. Add 1.5% agar to the solid medium. The resistant medium needs to be added with 50 μg / mL kanamycin sulfate.

[0017] Klebsiella pneumoniae fermentation medium (g / L): K 2 HPO 4 ·3H 2 O, 3.4; KH 2 PO 4 , 1.3; (NH 4 ) 2 SO 4 , 4; MgSO 4 ·7H 2 O, 0.5; CaCO 3 , 0.1; yeast powder, 3; glycerin, 40; trace elements, 1.25mL...

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Abstract

The invention discloses an NAD<+>-independent aldehyde oxidase catalyzing production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde, and its application, belongs to the biochemical production field, and relates to the NAD<+>-independent aldehyde oxidase enabling Klebsiella pneumonia to maintain the intracellular coenzyme balance in the catalysis process of the production of 3-hydroxypropionic acid from 3-hydroxypropionaldehyde. The sequence of the aldehyde oxidase is represented by SEQ ID NO:1 in a sequence table. Mass screening results show that the aldehyde oxidase in Pseudomonas sp. is independent of NAD<+>, and can catalyze the oxidation of aldehydes to corresponding carboxylic acids. The overexpression of the aldehyde oxidase in the Klebsiella pneumonia is carried out, and the in-vivo and in-vitro 3-hydroxypropionaldehyde catalysis ability is determined. The above enzyme has good 3-hydroxypropionic acid production ability.

Description

technical field [0001] The invention belongs to the field of biochemical production and relates to a NAD-independent + The aldehyde oxidase of Klebsiella pneumoniae can maintain the balance of intracellular coenzymes in the process of catalyzing 3-hydroxypropionaldehyde to produce 3-hydroxypropionate. Background technique [0002] 3-Hydroxypropionic acid is an important platform compound. Due to the difficulty of chemical synthesis, biosynthesis has received more attention in recent years. Klebsiella pneumoniae is an ideal host for biological production of 3-hydroxypropionic acid because of its rapid growth and high glycerol tolerance. Glycerol is catalyzed by glycerol dehydratase in Klebsiella pneumoniae to generate the intermediate product 3-hydroxypropionaldehyde, which is then absorbed by NAD + Dependent aldehyde dehydrogenase catalyzes the production of 3-hydroxypropionate. [0003] Overexpressed aldehyde dehydrogenase leads to intracellular NAD in the biological pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/02C12P7/42
CPCC12N9/0008C12P7/42C12Y102/03001
Inventor 田平芳李映
Owner BEIJING UNIV OF CHEM TECH
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