Method for reducing NAD analogues by utilizing methanol

An analogue, methanol technology, applied in the direction of fermentation, etc., can solve the problem of transforming methanol dehydrogenase that has not been reported in the literature, and achieve the effect of selective delivery, abundant reserves, and low price

Active Publication Date: 2020-04-07
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

[0006] 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 methanol dehydrogenase to efficiently reduce NAD analogs

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  • Method for reducing NAD analogues by utilizing methanol
  • Method for reducing NAD analogues by utilizing methanol
  • Method for reducing NAD analogues by utilizing methanol

Examples

Experimental program
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Effect test

Embodiment 1

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

[0032] NAD and its analogs NCD, NFCD, NBrCD, NClCD, NMeCD, NGD, NTD or NUD, and methanol dehydrogenase BsMDH, BsMDH-Y171G, BsMDH-Y171G / A238N, BsMDH-N240A / A243K, BsMDH-K242Y / A243M , BsMDH-I196Q, BsMDH-D195E / A238V, BmMDH, BmMDH-V210S, BmMDH-N123S, BmMDH-V210T / M219K, BmMDH-D212E / M219R, BmMDH-Q217E, BmMDH-D153T / V210S were de-NAD analog-methanol one by one Hydrogenase combination, react according to the following method: dissolve 1mM NAD or similar, 4mM methanol and 80μg methanol dehydrogenase in 1mL HEPES buffer with a concentration of 50mM and pH 7.5, mix well, and react at 30°C for 20min, 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 at 340 nm, but the intensity of absorption peaks obtained by different combinations was significantly differen...

Embodiment 2

[0038] Example 2: Preparation of reduced NAD analogs

[0039] 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. 20mM NUD, 25mM methanol and 5mg methanol dehydrogenase BsMDH-Y171G were dissolved in 10mL sodium phosphate buffer solution with a concentration of 50mM and a pH of 5.7, mixed well, and reacted at 30°C for 80min. 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 5.6 mg of a white powder with a yield of about 44%.

[0040] 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 2 + , 643.1054) co...

Embodiment 3

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

[0043] Dissolve 0.1mM NBrCD, 0.4mM methanol and 80μg methanol dehydrogenase BsMDH-Y171G / A238N in 1mL PIPES buffer with a concentration of 50mM and pH 8.0, mix well, react at 40°C for 3min, and take 20μL 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 NBrCDH reached 60 μM, that is, the yield reached 60%.

[0045] The results of Example 1 and Example 3 show that methanol dehydrogenase can reduce NAD analogues by using methanol as a reducing agent.

[0046] According to the method of Example 3, the same amount of NBrCD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R was used to produce NAD analogues, and the concentration of generated NBrCDH reached 43 μM, that is, the yield reached 43%. It shows that both methanol dehydrogenase Bs...

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Abstract

The invention discloses a method for reducing NAD analogues by utilizing methanol and applications. In the method, a reducing agent is the methanol, a catalyst is the methanol dehydrogenase capable ofutilizing the methanol, and NAD analogues can be converted into the reduced states of the NAD analogues while the methanol dehydrogenase oxidizes the methanol; the method can be used for producing the reduced state NAD analogues or the reduced state analogues of deuteration and can provide reduced state coenzymes for the enzymatic reaction consuming the reduced state NAD analogues; the reduced state NAD analogues can be used as coenzymes to be applied to the reduction reaction catalyzed by enzymes such as malic enzyme ME-L310R/Q401C, D-lactic dehydrogenase DLDH-V152R and saccharomyces cerevisiae alcohol dehydrogenase, so that the wide application of the NAD analogues can be realized; the reduction method of the NAD analogues can be performed under a mild condition; and the regenerated reduced state NAD analogues can be used for preparing malic acid or lactic acid and can be used as redox power to control and regulate the metabolic intensity of the 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, methanol is used as a reducing agent to decompose NAD analogues under enzyme catalysis. Converted to its reduced state, and can be used by other enzymes as a coenzyme for 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 oxid...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/36
CPCC12P19/36
Inventor 赵宗保郭潇佳刘武军
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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