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NADH analog dependent cytochrome P450 reductase and application thereof

A technology of cytochromes and analogs, applied in the direction of oxidoreductases, enzymes, enzymes, etc., can solve the problems of weak host substrate supply capacity, restrictions on industrial applications, and low overall efficiency

Active Publication Date: 2020-06-02
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, other P450-like enzyme biotransformation systems still have problems such as low overall efficiency, dependence on reduced coenzyme, and weak host substrate supply ability, which seriously restrict their industrial application.

Method used

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  • NADH analog dependent cytochrome P450 reductase and application thereof
  • NADH analog dependent cytochrome P450 reductase and application thereof
  • NADH analog dependent cytochrome P450 reductase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Enzymatic reduction of NAD analogs NCD, NTD and NUD to reduced NADH analogs NCDH, NTDH and NUDH.

[0037] The NAD analogs NCD, NTD and NUD were reduced with malic enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R, phosphorous acid dehydrogenase PDH-I151R or formate dehydrogenase FDH-G171Y, respectively, according to The following method was used for the reaction: 1 mM of NAD analogs, 4 mM of substrate (one of malic acid, D-lactic acid, phosphorous acid or formic acid) and 10 U of enzyme (malic enzyme ME-L310R / Q401C, D- One of lactate dehydrogenase DLDH-V152R, phosphite dehydrogenase PDH-I151R or formate dehydrogenase FDH-G171Y) was dissolved in 1 mL of HEPES buffer with a concentration of 50 mM and pH 7.5, and reacted at 30°C for 20 min. Take 20 μL for analysis.

[0038] Analysis found that all samples had characteristic absorption peaks at 340nm, indicating that malic enzyme ME-L310R / Q401C, D-lactate dehydrogenase DLDH-V152R, phosphorous acid dehydr...

Embodiment 2

[0041] Example 2: Enzymatic preparation of reduced NAD analogs

[0042] The reaction system in Example 1 is scaled up and can be used to prepare reduced NAD analogs. Taking formate dehydrogenase FDH-G171Y to prepare NUDH from sodium formate as an example, the preparation process is illustrated. 20 mM NUD, 25 mM sodium formate and 5 mg of formate dehydrogenase FDH-G171Y were dissolved in 10 mL of sodium phosphate buffer solution with a concentration of 50 mM and a pH of 7.5, mixed well, and reacted at 30°C for 2 h. Freeze-dry directly after the reaction, concentrate to a total volume of about 4mL, separate with a formic acid-type anion-exchange resin column (201×4), track and collect the product at an ultraviolet wavelength of 340nm, and freeze-dry to obtain 11.6mg of white powder with a yield of about 90 %. 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 t...

Embodiment 3

[0044] Example 3: Chemical reduction of NAD analogues NCD, NTD and NUD to reduced NADH analogues NCDH, NTDH and NUDH.

[0045] The NAD analogs NCD, NTD and NUD, respectively, with Na 2 S 2 o 4 , NaBH 4 、NaBH(Et) 3 , NaBH 3 For CN reduction, the reaction was carried out as follows: 1 mM NAD analogue, 4 mM reducing agent (Na 2 S 2 o 4 , NaBH 4 、NaBH(Et) 3 , NaBH 3 CN) dissolved in 1 mL of H 2 Mix well in O, react at 30°C for 20 min, and take 20 μL for analysis.

[0046] The analysis found that all samples had characteristic absorption peaks at 340nm, indicating that Na 2 S 2 o 4 , NaBH 4 、NaBH(Et) 3 , NaBH 3 CN can reduce NAD analogs. The quantitative method is the same as in Example 1, and the results are shown in Table 2. Visible chemical reducing agent Na 2 S 2 o 4 , NaBH 4 、NaBH(Et) 3 , NaBH 3 CN can be used to reduce NAD analogs to prepare NADH analogs without substrate selectivity.

[0047] Table 2 Experimental results of reducing NAD analogues by...

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Abstract

The invention discloses an NADH analog dependent cytochrome P450 reductase and an application thereof. An NADH analogue is used as a cofactor and reducing power to catalyze electron transfer and reduce cytochrome P450 to complete catalytic circulation. The enzyme is subjected to fusion expression with different types of cytochrome P450 enzymes to construct the NADH analog-dependent, heterozygous and self-sufficient cytochrome P450 enzyme, the obtained NADH analog-dependent cytochrome P450 enzyme can be coupled with oxidoreductases of a regenerated NADH analogue, and the NADH analog is used tocatalyze corresponding substrates of different families of cytochrome P450 to be converted into products. The NADH analog-dependent cytochrome P450 reductase can be used for constructing a biologicalorthogonal metabolic pathway independent of natural cofactor NAD(P)H to realize uncoupling of P450 enzyme-catalyzed energy consumption and endogenous energy metabolism.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a coenzyme nicotinamide adenine dinucleotide (NADH) analogue-dependent cytochrome P450 reductase and its application, specifically, it is a genetically engineered BMR, with NAD(H) The analogue is a cofactor and a reducing power, which catalyzes the reduction of cytochrome P450 enzymes, and makes cytochrome P450 enzymes catalyze the conversion of substrates. The NADH analog-dependent hybrid self-sufficient cytochrome P450 enzyme constructed by fusion expression of NADH analog-dependent cytochrome P450 reductase and cytochrome P450 enzyme can be applied to the construction of bioorthogonal metabolic pathways to realize P450 enzyme catalysis Uncoupling of energy expenditure from endogenous energy metabolism. Background technique [0002] Cytochrome P450 enzymes are a class of thiolate-heme enzyme proteins with diverse functions, which can selectively activate C-H bonds, N-H bonds, S-H bo...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12P7/64
CPCC12N9/0038C12P7/6409
Inventor 赵宗保李青刘武军
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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