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Reduction method of NAD analog

A technology of analogs and reducing agents, which is applied in the biological field to achieve the effects of high reaction efficiency, high product selectivity and low price

Active Publication Date: 2019-06-04
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the regeneration cycle of NAD analogs is of great significance to the fields of biocatalysis and synthetic biology, there are few literatures on the efficient reduction of NAD analogs by modifying the structure of the enzyme, and there is no literature on how to modify formate dehydrogenase to efficiently reduce NAD analogs

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  • Reduction method of NAD analog

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0031] Example 1: Using formic acid as a reducing agent, formate dehydrogenase catalyzes the reduction of NAD analogs

[0032] NAD analogues NCD, NFCD, NBrCD, NClCD, NMeCD, NGD, NTD and NUD, and formate dehydrogenase cboFDH, cboFDH-G171Y, cboFDH-I170T / A229S, pseFDH and pseFDH-H223S / L257R, one by one NAD analogues -Formate dehydrogenase combination, react as follows: dissolve 1mM NAD analogue, 4mM formic acid and 40μg formate dehydrogenase in 1mL HEPES buffer solution with a concentration of 50mM and pH 7.5, and react at 30°C 20min, take 20μL analysis.

[0033] According to the analysis method of Comparative Example 1, it was found that all the samples used had characteristic absorption peaks at 340nm, but the absorption peak intensities obtained by different combinations were significantly different, indicating that formate dehydrogenase can catalyze the reduction of NAD analogs with formate. Due to the molar extinction coefficient ε of the reduced state product of the NAD an...

Embodiment 2

[0037] Example 2: Preparation of reduced NAD analogs

[0038] The reaction system in Example 1 is scaled up and can be used to prepare reduced NAD analogs. Take NUDH as an example to illustrate the preparation process. 20 mM NUD, 25 mM sodium formate and 5 mg of formate dehydrogenase cboFDH-G171Y were dissolved in 10 mL of sodium phosphate buffer solution with a concentration of 50 mM and a pH of 5.7, mixed well, and reacted at 30°C for 80 min. Freeze-dry directly after the reaction, concentrate to a total volume of about 4 mL, separate with a formic acid-type anion-exchange resin column, track and collect the product at an ultraviolet wavelength of 340 nm, and freeze-dry to obtain 11.6 mg of a white powder with a yield of about 90%.

[0039] The above-mentioned white powder sample was subjected to high-resolution mass spectrometry analysis to measure the precise molecular weight (M+H) + It is 643.1026, and the theoretical molecular weight of NUDH (C 20 h 29 N 4 o 16 P ...

Embodiment 3

[0041] Example 3: Using sodium formate as a reducing agent, formate dehydrogenase catalyzes the reduction of NAD analogs

[0042] Dissolve 0.1 mM NBrCD, 0.4 mM sodium formate and 4 μg of formate dehydrogenase cboFDH-G171Y in 1 mL of 50 mM, pH 8.0 PIPES buffer, mix well, react at 40°C for 3 min, and take 20 μL for analysis.

[0043] According to the method analysis of Comparative Example 1, it was found that the sample had a characteristic absorption peak at 340nm. The concentration of generated NBrCDH reached 78 μM, that is, the yield reached 78%.

[0044] The results of Example 1 and Example 3 show that in the reaction of formate dehydrogenase catalyzing the reduction of NAD analogues, using sodium formate or formic acid as a reducing agent can reduce NAD analogues.

[0045] According to the method of Example 3, the same amount of NBrCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R was used to produce NAD analogs, and the concentration of NBrCDH produced reached ...

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Abstract

The invention discloses a reduction method of a NAD analog and an application of the reduction method. According to the method, a reducing agent is a formic acid compound, a catalyst is hydrogenlyasewhich can use the formic acid compound, and when the hydrogenlyase is used for oxidating the formic acid compound, the NAD analog is converted into a reducing state. The method can be used for producing the reducing state NAD analog or a deuterated reducing state analog, and can also be used for providing reducing power for an enzyme reaction consuming the reducing state NAD analog, and the reducing state NAD analog can also be used as coenzyme to be used for a reduction reaction of enzyme catalysis of malic enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like. Wide application of the NAD analog is facilitated. According to the reducing method of the NAD analog, under the mild condition, the regenerated reducing state NAD analog can be used for preparing malic acid or lactic acid, and can also be used as oxidation-reduction power for adjusting and controlling the metabolism intensity of the malic acid or the lactic acidin microorganisms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for reducing NAD analogues. Specifically, formic acid compounds are used as reducing agents to convert NAD analogues into their reduced states under the catalysis of enzymes, and can be used as NAD analogues by other enzymes. Coenzymes are used in reduction reactions. Background technique [0002] Nicotinamide adenine dinucleotide (NAD) and its reduced state NADH are important coenzymes in the life process, participating in redox metabolism and a series of other important biochemical processes in living organisms. These coenzymes can be used in the production of chiral chemicals and in the preparation of isotopic labels. Since many oxidoreductases use NADH or NADPH as a coenzyme, any operation that changes the concentration of NAD and its redox state will have a global impact on the cell, and it is difficult to control specific oxidoreductases in biological systems at the coe...

Claims

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Application Information

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IPC IPC(8): C12P19/60
Inventor 赵宗保郭潇佳刘武军
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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