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Method for preparing nicotinamide adenine dinucleotide by virtue of enzyme method

A technology of nicotinamide adenine and adenine phosphoribose, applied in the field of enzymatic preparation of nicotinamide adenine dinucleotide, can solve the problems of high cost, long reaction route, environmental pollution, etc., and achieve low cost, safe and reliable preparation , cost reduction effect

Inactive Publication Date: 2020-05-15
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 method uses nicotinamide as raw material to synthesize NAD through multi-steps. The reaction route is long, and expensive reagents and a large amount of organic solvents are used to cause environmental pollution.
The chemical enzymatic method uses nicotinamide riboside as the raw material, which also uses expensive and explosive reagents, which is costly and prone to dangerous accidents
The bio-fermentation process technology is mature, but the consumption of raw materials is huge, the energy consumption is large, and the output is limited

Method used

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  • Method for preparing nicotinamide adenine dinucleotide by virtue of enzyme method
  • Method for preparing nicotinamide adenine dinucleotide by virtue of enzyme method
  • Method for preparing nicotinamide adenine dinucleotide by virtue of enzyme method

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Embodiment 1

[0026] Embodiment 1, preparation NADP + enzymes for production

[0027] 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 amplify AK fragment, and connect it to pET28a vector (purchased from Novagene Company); Extract Escherichia coli (Escherichia coli) bacterial strain genomic DNA, use it as Template, PCR amplifies the APRT fragment, and connects it to the pET28a vector (purchased from Novagene); extracts Haemophilusducreyi (purchased from Shanghai Jierui Biological Engineering Co., Ltd.) genomic DNA, using it as a template, PCR amplified the NmPRT gene fragment, and connected it to the pET 28a vector (purchased from Novagene); extracted Genomic DNA of Methanococcus jannaschii (Methanococcus jannaschii) strain, using it as a template, PCR amplified the NMNAT gene fragment, and connected it to the pET28a vector (purchased fro...

Embodiment 2

[0036] Embodiment 2, preparation NAD

[0037] Substrate adenosine 100g, nicotinamide 68.5g, sodium hexametaphosphate 330g, ATP 300g, MgCl 2 ·6H 2 O110g 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 AK, APRT, NmPRT and NMNAT four kinds of enzymes are formed in reaction liquid, wherein the mass ratio of AK:APRT:NmPRT:NMNAT in the enzyme composition is 1:1:1:1, controls pH during the reaction to maintain At 7.0, the reaction temperature was 30-35° C., and after 13 hours of shaking reaction, the amount of NAD produced in the reaction supernatant was detected by HPLC to be 35 g / L, the purity was 70%, and the conversion rate of adenosine was 94%.

[0038] 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.

[0039] T...

Embodiment 3

[0043] Embodiment 3, preparation NAD

[0044] Add 100g of substrate adenosine, 68.5g of nicotinamide, 330g of sodium hexametaphosphate, 300g of ATP, and 110g of MgCl2·6H2O into 5L of pH 7.0 phosphate buffer solution, stir well, and adjust the pH to 7.0. Add the enzyme composition that AK, APRT, NmPRT and NMNAT four kinds of enzymes are formed in reaction solution, wherein the mass ratio of AK:APRT:NmPRT:NMNAT in the enzyme composition is 1:2:1:1, controls pH during the reaction to maintain At 7.0, the reaction temperature was 30-35° C., and after 12 hours of shaking reaction, the amount of NAD produced in the reaction supernatant was detected by HPLC to be 37.7 g / L, the purity was 75%, and the conversion rate of adenosine was 95%. HPLC detection condition is the same as embodiment 2.

[0045] The supernatant collected by filtration was subjected to ion-exchange chromatography on a macroporous strongly basic anion-exchange resin, concentrated, crystallized, and dried to obtain...

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Abstract

The invention belongs to the technical fields of biological pharmacy and biochemical engineering and discloses an enzyme composition for preparing nicotinamide adenine dinucleotide and a method for preparing the nicotinamide adenine dinucleotide by virtue of an enzyme method. The enzyme composition consists of adenosine kinase, adenine phosphoribosyltransferase, nicotinamide phosphoribose transferase and nicotinamide mononucleotide adenosine transferase. By virtue of reasonable combination of four enzymes, the nicotinamide adenine dinucleotide can be prepared through efficient catalysis. The enzyme composition can be circularly utilized and is low in cost, energy-saving and environment-friendly. According to the method for preparing the nicotinamide adenine dinucleotide by virtue of the enzyme method, adenosine is taken as a substrate, and the enzyme composition is added, so that the nicotinamide adenine dinucleotide can be safely and reliably prepared in a low-cost way, the cost of anexisting route is lowered, the existing route is applicable to large-scale production, and the use of the nicotinamide adenine dinucleotide in the fields of biological catalysis and drugs is guaranteed.

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. Background technique [0002] Nicotinamide adenine dinucleotide, also known as pyridine nucleotide diphosphate (abbreviated as DPN), or co-dehydrogenase I or coenzyme I. In mammals, there are two states of oxidized (NAD+) and reduced (NADH), the oxidized (NAD + ) has a maximum ultraviolet absorption spectrum at 260nm, through various deaminases, accepts a hydrogen atom and an electron from the substrate, and becomes a reduced form (NADH), and has a maximum absorption at 340nm. Studies have shown that nicotinamide adenine dinucleotide participates in various physiological activities such as cell substance metabolism, energy synthesis, and cell DNA repair, and can promote substance metabolism, energy metabolism, resist cell aging and antioxidant effects (ying, W....

Claims

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Application Information

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IPC IPC(8): C12P19/36C12P19/18
CPCC12P19/18C12P19/36
Inventor 周浩
Owner 杭州唯泰生物药业有限公司
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