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Method for reducing NAD analogue by using 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

Pending Publication Date: 2020-10-27
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

[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 analogue by using methanol
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  • Method for reducing NAD analogue by using methanol

Examples

Experimental program
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Embodiment 1

[0033] Example 1: Using methanol as a reducing agent, methanol dehydrogenase catalyzes the reduction of NAD analogs

[0034] NAD and its analog NCD, NTD or NUD, and methanol dehydrogenase BsMDH, BsMDH-V237T, BsMDH-V237T / N240E, BsMDH-V237T / N240E / K241A, BsMDH-Y171R / V237T / N240E / K241A, BsMDH-Y171R / I196V / V237T / N240E / K241A, carry out NAD analogue-methanol dehydrogenase combination one by one, and react as follows: dissolve 1mM NAD or analogue, 8mM methanol and 80μg methanol dehydrogenase in 1mL concentration 50mM, pH 7.5 HEPES buffer, mix well, react at 30°C for 20min, take 20μL for analysis.

[0035] 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 different, indicating that methanol dehydrogenase can catalyze the reduction of NAD analogues by methanol. Due to the molar extinction coefficient ε o...

Embodiment 2

[0040] Example 2: Preparation of reduced NAD analogs

[0041] 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. Dissolve 20 mM NUD, 20 mM methanol and 5 mg methanol dehydrogenase BsMDH-V237T 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 5.6 mg of white powder with a yield of about 44%.

[0042] 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) consi...

Embodiment 3

[0044] Example 3: Using methanol as a reducing agent, methanol dehydrogenase catalyzes the reduction of NAD analogs

[0045] Dissolve 0.1 mM NUD, 0.8 mM methanol and 80 μg methanol dehydrogenase BsMDH-V237T / N240E 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.

[0046] 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%.

[0047] The results of Example 1 and Example 3 show that in the reaction of methanol dehydrogenase catalyzed reduction of NAD analogues, methanol can be used as a reducing agent to reduce NAD analogues.

[0048] According to the method of Example 3, NAD analogs were produced with the same amount of NUD, sodium phosphite and phosphite dehydrogenase rsPDH-I151R, and the concentration of NUDH generated reached 43 μM, that is, the yield reached 40%. It shows th...

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Abstract

The invention discloses a method for reducing an NAD analogue by using methanol and application of the method. In the method, the methanol is taken as a reducing agent, methanol dehydrogenase capableof utilizing the methanol is taken as a catalyst, and the NAD analogue is transformed into a reduction state while the methanol is oxidized by the methanol dehydrogenase. The method can be used for producing a reduction-state NAD analogue or a deuterated reduction-state analogue, and can also provide reduction-state coenzyme for an enzymatic reaction consuming the reduction-state NAD analogue; thereduction-state NAD analogue can be used as a coenzyme to be applied to reduction reactions catalyzed by enzymes such as malic enzyme ME-L310R / Q401C, D-lactic dehydrogenase DLDH-V152R, and saccharomyces cerevisiae alcohol dehydrogenase, and wide application of the NAD analogue is facilitated. According to the NAD analogue reduction method, the reduction-state NAD analogue can be regenerated undera mild condition for preparing malic acid or lactic acid; and the reduction-state NAD analogue can also serve as an oxidation-reduction 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, 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 a series of redox metabolism and 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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IPC IPC(8): C12P19/36
CPCC12P19/36
Inventor 赵宗保王俊婷
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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