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Method for reducing NAD analogue by using formic acid

A technology of analogs and formic acid compounds, applied in the biological field, to achieve high activity, beneficial regeneration, and high product selectivity

Active Publication Date: 2020-10-23
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • 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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  • Method for reducing NAD analogue by using formic acid
  • Method for reducing NAD analogue by using formic acid
  • Method for reducing NAD analogue by using formic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0032] NAD analogs were combined with formate dehydrogenase mutant E4 (V198I / C256I / P260S / E261P / S381N), mutant A2 (C256I / E261P / S381I), mutant 3C4 (A199C / E261P / S381N), mutant 2A4 ( A199C / E261P / S381I), mutant 4C4 (V198I / C256I / P260S / E261P / S381N / H224C), mutant 4C1 (T197A / C256I / H260S / E261P / S381N), mutant 4B4 (T197C / C256I / H260S / E261 / S381N), mutant 4A2 (T197V / C256I / H260S / E261P / S381N) and 3A3 (V198I / C256I / P260S / E261P / S381N / S383F), carry out NAD analogue-formate dehydrogenase combination one by one, as follows Reaction: Dissolve 1 mM NAD analogue, 4 mM formic acid and 40 μg of formate dehydrogenase in 1 mL of HEPES buffer with a concentration of 50 mM and pH 7.5, mix well, react at 30°C for 20 min, and take 20 μL for analysis.

[0033] According to the analysis method of Comparative Example 1, it was found that all the samples used had characteristic absorption peaks...

Embodiment 2

[0038] Example 2: Preparation of reduced NAD analogs

[0039] The reaction system in Example 1 is enlarged and can be used to prepare reduced NCD. Dissolve 20 mM NCD, 25 mM sodium formate and 5 mg formate dehydrogenase A2 (C256I / E261P / S381I) in 10 mL of sodium phosphate buffer solution with a concentration of 50 mM and a pH of 5.7, mix well, and react 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%.

[0040] The above-mentioned white powder sample was subjected to high-resolution mass spectrometry analysis to measure the precise molecular weight (M+H) + is 640.1118, and the theoretical molecular weight of NCDH (C 20 h 27 HN 5 o 15 P 2 - , 640.1125) consistent, indicating that the reduced product NCDH was obta...

Embodiment 3

[0042] Example 3: Using sodium formate as a reducing agent, formate dehydrogenase catalyzes the reduction of NAD analogs

[0043] Dissolve 0.1 mM NCD, 0.4 mM sodium formate and 4 μg formate dehydrogenase 3C4 (A199C / E261P / S381N) in 1 mL of PIPES buffer with a concentration of 50 mM and pH 8.0, mix well, react at 40°C for 3 min, and take 20 μL for analysis.

[0044] Analysis by the method of Comparative Example 1 found that the sample had a characteristic absorption peak at 340nm. The concentration of generated NCDH reached 78 μM, that is, the yield reached 78%.

[0045] The results of Example 1 and Example 3 show that in the reaction of NCD catalyzed by formate dehydrogenase, using sodium formate or formic acid as a reducing agent can reduce NCD.

[0046] According to the method of Example 3, the same amount of NCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R was used to produce NAD analogs, and the concentration of NCDH generated reached 73 μM, that is, the yield...

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Abstract

The invention discloses a method for reducing an NAD analogue by using formic acid and application of the method. In the method, a reducing agent is a formic acid compound, a catalyst is formate dehydrogenase capable of utilizing the formic acid compound, and while the formate dehydrogenase oxidizes the formic acid compound, the NAD analogue is converted into a reduction state. The method can be used for producing a reduction-state NAD analogue or a deuterated reduction-state analogue. The reducing power can also be provided for the enzymatic reaction consuming the reduction-state NAD analogue; the reduction-state NAD analogue can be used as a coenzyme to be applied to enzyme-catalyzed reduction reactions of malic enzyme ME-L310R / Q401C, D-lactic dehydrogenase DLDH-V152R, saccharomyces cerevisiae alcohol dehydrogenase and the like, and wide application of the NAD analogue is facilitated. The reduction method of the NAD analogue can be used for regenerating the reduced NAD analogue for preparing malic acid or lactic acid under a mild condition; and can also be used as a redox force to regulate the metabolic intensity of malic acid or lactic acid in microorganisms.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an enzyme-catalyzed reduction method and application of coenzyme nicotinamide adenine dinucleotide (NAD) analogues. Specifically, formic acid compounds are used as reducing agents to reduce NAD analogues under enzyme catalysis. The substance is converted into its reduced state, and can be used as a coenzyme by other enzymes in the reduction reaction. 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 dif...

Claims

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Application Information

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IPC IPC(8): C12P19/36
CPCC12P19/36
Inventor 赵宗保郭潇佳王雪颖
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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