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Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method

A technology for adenine phosphoribosyl and purine phosphoribosyl, which is applied in the field of enzymatic preparation of nicotinamide adenine dinucleotide phosphate, can solve the problems of high cost, high energy consumption, accidents and the like, and achieves low cost, safe and reliable preparation , the effect of reducing costs

Active Publication Date: 2020-01-03
杭州唯泰生物药业有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical enzymatic method uses nicotinamide riboside as the raw material, which also uses expensive reagents and explosive reagents, and accidents may occur due to high costs
Bio-fermentation technology is mature, but raw material consumption is huge, energy consumption is large, and output is limited

Method used

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  • Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method
  • Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method

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Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, preparation NADP + enzymes for production

[0031] According to the sequences of the four enzyme genes, four pairs of amplification primers were designed. Extract Escherichia coli (Escherichia coli) bacterial strain genomic DNA, use it as template, PCR amplifies APRT (AK enzyme gene) fragment, and it is connected to pET28a vector (purchased from Novagene Company); Extract Haemophilus ducreyi (Haemophilus ducreyi ) (purchased from Shanghai Jierui Bioengineering Co., Ltd.) genomic DNA, using it as a template, PCR amplified NmPRT gene fragment, and connecting it to the pET 28a vector (purchased from Novagene); extracting Methanococcus jannaschii (Methanococcus jannaschii ) strain genomic DNA, using it as a template, PCR amplified the NMNAT gene fragment, and connected it to the pET28a vector (purchased from Novagene); The ppnk gene fragment was amplified and connected to the pET28a vector (purchased from Novagene). After the 4 kinds of gene fragments were ...

Embodiment 2

[0040] Embodiment 2, preparation NADP +

[0041] Substrate adenylic acid 100g, sodium hexametaphosphate 300g, ATP 80g, MgCl 2 ·6H 2 O 30g was added to 5L pH 7.0 phosphate buffer solution, stirred evenly, and the pH was adjusted to 7.0. Add the enzyme composition that APRT, NmPRT, NMNAT and ppnk four kinds of enzymes are formed in reaction liquid, wherein the mass ratio of APRT in enzyme composition: NmPRT: NMNAT: ppnk is 1:2:1:1, controls pH during reaction and keeps At 7.0, the reaction temperature is 30-35 ° C, and after 12 hours of shaking reaction, HPLC detects NADP in the reaction supernatant + The yield is 38.5g / L, the purity is 80%, and the conversion rate of adenylic acid is 96%.

[0042] HPLC detection conditions: use octadecylsilane bonded silica gel as filler, mobile phase A is 25mM Tris-HAC, phase B is methanol, detection wavelength is 260nm, column temperature is 25°C, and the elution procedure is shown in Table 1.

[0043] Table 1 Elution program

[0044] ...

Embodiment 3

[0046] Embodiment 3, preparation NADP +

[0047] Substrate adenylic acid 100g, sodium hexametaphosphate 300g, ATP 80g, MgCl 2 ·6H 2 O 30g was added to 5L pH 7.0 phosphate buffer solution, stirred evenly, and the pH was adjusted to 7.0. Add the enzyme composition that APRT, NmPRT, NMNAT and ppnk four kinds of enzymes are formed in reaction solution, wherein the mass ratio of APRT:NmPRT:NMNAT:ppnk in the enzyme composition is 1:1:1:1, controls pH during reaction to maintain At 7.0, the reaction temperature is 30-35 ° C, and after 12 hours of shaking reaction, HPLC detects NADP in the reaction supernatant + The yield was 34g / L, the purity was 72%, and the conversion rate of adenylic acid was 90%. HPLC detection condition is the same as embodiment 2.

[0048] The supernatant collected by filtration passes through macroporous strong basic anion exchange resin ion exchange chromatography, concentrates, crystallizes, and dries to obtain the finished product NADP + 170g, purity ...

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Abstract

The invention belongs to the technical fields of biological pharmacy and biochemical engineering, and discloses an enzyme composition for production of nicotinamide adenine dinucleotide phosphate anda method for preparing the nicotinamide adenine dinucleotide phosphate by an enzyme method. The enzyme composition disclosed by the invention consists of adenine phosphoribosyl transferase, nicotinamide phosphoribosyl transferase, nicotinamide phosphate ribose transferase and polyphosphate dependent form NAD kinase. The four kinds of enzymes are in reasonable combination, so that the nicotinamideadenine dinucleotide phosphate can be efficiently catalyzed and prepared. The enzyme composition disclosed by the invention can be in cyclic utilization, and is low in cost, energy-saving and environmentally-friendly. According to the method for preparing the nicotinamide adenine dinucleotide phosphate by an enzyme method disclosed by the invention, adenylic acid is used as a substrate, and the enzyme composition is added, so that the nicotinamide adenine dinucleotide phosphate can be prepared safely and reliably at a low cost, the cost by a conventional route is reduced, the method is adaptedto large-scale production, and guarantee is provided for usage of the nicotinamide adenine dinucleotide phosphate in the fields of biocatalysis and medicines.

Description

technical field [0001] The invention belongs to the technical fields of biopharmaceutical and biochemical industry, and in particular relates to a method for enzymatically preparing nicotinamide adenine dinucleotide phosphate. Background technique [0002] Nicotinamide adenine dinucleotide phosphate, also known as oxidized coenzyme II, (NADP + ) is an important coenzyme, is nicotinamide adenine dinucleotide (NAD + ) is a phosphorylated derivative at the 2'-position of the ribose ring linked to adenine. Oxidized coenzyme II transfers protons, electrons and energy in the form of oxidation-reduction reactions, and participates in many metabolic reactions of cells, such as the synthesis of lipids, fatty acids and nucleotides, can activate multi-enzyme systems, and promote the synthesis of nucleic acids, proteins and polysaccharides. Synthesis and metabolism, improve material transport and regulation control, improve metabolic function. Studies have shown that oxidized coenzym...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N9/12C12P19/36
CPCC12N9/1077C12N9/1205C12N9/1241C12P19/36C12Y204/02007C12Y204/02012C12Y207/01023C12Y207/07001
Inventor 周浩
Owner 杭州唯泰生物药业有限公司
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