Method for preparing cofactor by coupling leucine dehydrogenase with photoelectrocatalysis

A technology of leucine dehydrogenase and photoelectric catalysis, which is applied in the field of biocatalytic coupling photocatalytic preparation of cofactors, can solve the problems of low reaction rate and yield, unstable existence of substrates and products, multi-biological catalysts, etc. To achieve the effect of reducing the cost of experiments

Pending Publication Date: 2021-10-08
HUAQIAO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The chemical method uses chemical reagents to directly realize the regeneration of NADH, but its reaction rate and yield are low; although the electrochemical method is simple, it often requires a high potential, and the substrate and product may not exist stably under high potential ; Although photochemical regeneration of NADH has great potential for development, it is still insufficient in terms of stability and large-scale production; although enzymatic regeneration of NADH has high specific activity and biocompatibility, it needs to consume more biological catalyst

Method used

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  • Method for preparing cofactor by coupling leucine dehydrogenase with photoelectrocatalysis
  • Method for preparing cofactor by coupling leucine dehydrogenase with photoelectrocatalysis
  • Method for preparing cofactor by coupling leucine dehydrogenase with photoelectrocatalysis

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Embodiment 1

[0020] A method for preparing a cofactor by coupling leucine dehydrogenase with photoelectric catalysis in this embodiment comprises the following steps:

[0021] (1) Induced expression of recombinant protein:

[0022] Add 10 μL of kanamycin to 10 mL of LB liquid medium, shake well, and insert 100 μL of preserved engineering bacteria containing leucine dehydrogenase (see Establishing a Mathematical Equations and Improving the Production of L-tert-Leucine by Uniform Design and Regression Analysis, Wei Jiang, et al, Applied Biochemistry and Biotechnology volume 181, pages 1454–1464 (2017)), 200rpm, 37°C shaker for 12h.

[0023] Add 100 μL of kanamycin to 100 mL of LB liquid medium, shake well, add 1000 μL of activated strains, and culture in a shaker at 200 rpm for 3-4 hours at 18°C. When OD 600 When it reaches 0.5-0.6, add 100 μL of IPTG and continue culturing for 20 h.

[0024] (2) Purification of crude enzyme liquid:

[0025] Divide the cultured bacterial solution into al...

Embodiment 2

[0047] The difference between embodiment 2 and embodiment 1 is:

[0048] In this example, the determination of system 2 is carried out under the condition of photoelectric catalysis. Add 400 μL leucine dehydrogenase enzyme solution obtained in step (2), 100 μL NAD + , 200 μL trimethylpyruvate, 1000 μL NH 4 Cl-NH 3 ·H 2 O buffer solution (pH 10) was used for the reaction, and samples were taken every 5 minutes within 30 minutes.

[0049] In this example, if figure 2 , OD of system 2 340 The value is floating between 0.2365-0.2875, indicating that there may be some NAD + Utilizes the e generated during the photocatalytic process - reduced to NADH, then leucine dehydrogenase consumes the generated NADH, making OD 340 The value increases first and then decreases.

Embodiment 3

[0051] The difference between embodiment 3 and embodiment 1 is:

[0052] In this example, the determination of system 3 is carried out under the condition of photoelectric catalysis. Add 400 μL leucine dehydrogenase enzyme solution obtained in step (2), 200 μL trimethylpyruvate, 1000 μL NH 4 Cl-NH 3 ·H 2 O buffer solution (pH 10) was used for the reaction, and samples were taken every 5 minutes within 30 minutes.

[0053] In this example, if image 3 , OD of system 3 340 Values ​​float between 0.2947-0.3154.

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Abstract

The invention discloses a method for preparing a cofactor by coupling leucine dehydrogenase with photoelectrocatalysis. The method comprises the following steps: inducible expression of recombinant protein, purification of crude enzyme liquid, determination of protein concentration and enzyme activity, detection of photoelectric properties of a catalyst, and preparation of the cofactor through coupling of biological catalysis and photoelectrocatalysis. Photoelectric properties and catalytic reactions of different systems are studied under a three-electrode system, results show that photoelectrocatalysis can provide cofactors for reactions, and feasibility of cofactor regeneration by coupling biocatalysis with photoelectrocatalysis is verified. The method is low in cost and environment-friendly, and has important significance on cofactor regeneration.

Description

technical field [0001] The invention belongs to the field of preparation of cofactors by coupling photoelectric catalysis with biocatalysis, and specifically relates to a method for preparing cofactors by coupling photoelectric catalysis with leucine dehydrogenase. Background technique [0002] Nicotinamide adenine dinucleotide (NAD) + / NADH) or its phosphorylated form (NADP + / NADPH) is a cofactor that initiates electron and proton transfer in biological redox reactions. Nearly 20% of known oxidoreductases require cofactors to provide stoichiometric hydrogen protons, and NAD(P)H is a required cofactor for nearly 700 known types of oxidoreductases. NAD(P)H is involved in a large number of biochemical processes, where it acts as a hydrogen or electron carrier. More than 1500 enzymatic reactions in microorganisms require the cofactors NAD(H) and NAD(P)H, so cofactor-mediated redox reactions play an important role in production. [0003] Cofactor regeneration methods includ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/36C12N9/06C25B3/05C25B3/07C25B3/09C25B3/21
CPCC12P19/36C12N9/0016C12Y104/01009C25B3/21C25B3/09C25B3/05C25B3/07
Inventor 江伟吴静芳肖静冉
Owner HUAQIAO UNIVERSITY
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