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Enzymatic preparation method of oxidized coenzyme II

An oxidized coenzyme, catalyzed preparation technology, applied in the direction of fermentation, etc., can solve the problems affecting the catalytic efficiency of immobilized NAD pyrophosphorylase and NAD kinase, the low yield of nicotinamide riboside phosphate synthesis, and the limitation of raw material sources. Easy to obtain in large quantities, strong stereospecificity, and high productivity

Active Publication Date: 2012-07-25
ENZYMEWORKS
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Problems solved by technology

However, this process is difficult to scale up because of the following reasons: First, the synthesis yield of nicotinamide riboside phosphate from raw materials is low, and it is not easy to scale up, and the source of raw materials is limited; second, the enzyme-catalyzed synthesis of NAD+ and NADP+ can only be obtained in a gram-level range. conversion rate, but the reaction time is as long as 16 days, and the production capacity is low; the third is due to the use of immobilized NAD pyrophosphorylase and NAD kinase, the interaction between nicotinamide riboside phosphate and NAD+ and immobilized NAD pyrophosphorylase and NAD kinase The mass transfer is hindered, which affects the catalytic efficiency of immobilized NAD pyrophosphorylase and NAD kinase, and the author pointed out that the method cannot be scaled up, so the process route is difficult to adapt to the scale-up requirements

Method used

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Experimental program
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Effect test

Embodiment 1

[0022] Add 10 mL of phosphate buffer solution (100 mM, pH 5.8) to a 20 mL three-necked flask, and then add nicotinamide riboside ( J. Med. Chem. 2007, 50, 6458-6461 ) 50 mg, adenosine triphosphate disodium salt 55 mg, nicotinamide riboside kinase ( PLoS Biology, 2007, 5(10), 2220-2230 ) 8 mg, inorganic pyrophosphatase ( 9024-82-2, EC 3.6.1.1 ) 10 mg, NAD kinase ( European Journal of Biochemistry, 2001, 268(15), 4359–4365 ) 5 mg and polyphosphokinase ( PNAS, 1997, 94(2), 439-442 ) 12 mg, magnesium chloride 20 mM, the reaction was stirred at 37°C, 200 rpm, and the conversion rate of the reaction was monitored by liquid chromatography-mass spectrometry. After 10 hours of reaction, it was detected that the adenosine triphosphate had been consumed, and the reaction was stopped. Through further filtration, the HZ-818 macroporous resin is adsorbed, lyophilized, and recrystallized with a mixed solvent of ethanol and water (15:1) to obtain the oxidized coenzyme II product with a yield...

Embodiment 2

[0024] Add 10 mL Tris-HCl buffer solution (200 mM, pH 7.5) to a 20 mL three-necked flask, and add nicotinamide riboside ( J. Med. Chem. 2007, 50, 6458-6461 ) 50 mg, adenosine triphosphate disodium salt 55 mg, nicotinamide riboside kinase ( PLoS Biology, 2007, 5(10), 2220-2230 ) 12 mg, inorganic pyrophosphatase ( 9024-82-2, EC 3.6.1.1 ) 15 mg, NAD kinase ( European Journal of Biochemistry, 2001, 268(15), 4359–4365 ) 12 mg and polyphosphokinase ( PNAS, 1997, 94(2), 439-442 ) 20 mg, manganese chloride 30 mM, magnesium chloride 20 mM, the reaction was stirred at 37°C, 200 rpm, and the conversion rate of the reaction was monitored by liquid chromatography-mass spectrometry. After 12 hours of reaction, it was detected that adenosine triphosphate had been consumed. Stop reaction. Through further filtration, D-101 type macroporous resin is adsorbed, freeze-dried, and recrystallized with a mixed solvent of ethanol and water (20:1) to obtain the oxidized coenzyme II product with a yie...

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Abstract

The invention relates to an enzymatic preparation method of an oxidized coenzyme II. Nicotinamide nucleotide (NR) and adenosine triphosphate disodium salt (ATP-Na2) are used as the raw material and subjected to one-pot reaction with nicotinamide nucleoside kinase (NRK), inorganic pyrophosphatase, NAD (Nicotinamide Adenine Dinucleotide) kinase and poly-pyrophosphokinase in a buffer solution with pH being 4.0-8.5 and at the temperature being 10-40 DEG C to obtain the oxidized coenzyme II. With the adoption of the enzymatic preparation method, the technical problems that in the existing enzymatic preparation method, the nicotinamide nucleotide is expensive and not easily obtained, the reaction time is long, the technical cost is relatively high and the technical conditions are not suitable for the industrialized scale-up production are solved; and the oxidized coenzyme II can be obtained with high efficiency and low lost in an industrialized scale-up production way.

Description

technical field [0001] The invention relates to an enzyme-catalyzed preparation method of oxidized coenzyme II. Background technique [0002] Oxidized coenzyme II (Nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide phosphate, referred to as: NADP+) is an extremely important nucleotide coenzyme, which is an oxidized coenzyme I (Nicotinamide adenine dinucleotide, nicotinamide adenine Purine dinucleotide, abbreviated as: NAD+) is a phosphorylated derivative of the 2'-position of the ribose ring system connected to adenine. It is an indispensable hydrogen transporter in the process of biological oxidation and participates in various anabolic reactions, such as lipid Synthesis of lipids, fatty acids and nucleotides. These reactions require NADPH as a reducing agent, hydrogen negative donor, and NADPH is the reduced form of NADP+. NADP+ is produced by phosphorylation of NAD+ catalyzed by NAD kinase. NAD+ and DPNA+ are coenzymes of various aerobic deh...

Claims

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Application Information

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IPC IPC(8): C12P19/36
Inventor 陶军华李斌谢磊庄季昌周永达张超刘根
Owner ENZYMEWORKS
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