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Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by enzymatic process

An enzyme composition and single nucleotide technology, applied in biochemical equipment and methods, enzymes, transferases, etc., to achieve cost-saving, safe and reliable preparation, and low-cost effects

Inactive Publication Date: 2020-05-22
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

At present, there is no report of using adenosine as a substrate to produce NMN in the prior art

Method used

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  • Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by enzymatic process
  • Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by enzymatic process

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Experimental program
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Effect test

Embodiment 1

[0049] Embodiment 1, preparation NMN production enzyme

[0050] According to the sequences of the three enzyme genes, three 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, The NmPRT gene fragment was amplified by PCR and connected to the pET 28a vector (purchased from Novagene); after the three gene fragments were successfully connected and sequenced correctly, they were respectively transferred into E.coli BL21 (DE3) strain (Shanghai Weidi Biotechnology Co., Ltd. Ltd.).

[0051...

Embodiment 2

[0058] Embodiment 2, preparation NMN

[0059] Substrate adenosine 100g, nicotinamide 68.5g, ATP 300g, MgCl 2 ·6H 2 O 110g was added to 5L pH7.0 phosphate buffer solution, stirred evenly, and the pH was adjusted to 7.0. Add the enzyme composition that AK, APRT and NmPRT three kinds of enzymes are formed in reaction solution, wherein the mass ratio of AK:APRT:NmPRT in the enzyme composition is 1:1:1, control pH during reaction and keep at 7.0, reaction temperature 33 ℃, after 8 hours of shaking reaction, the amount of NMN produced in the reaction supernatant was detected by HPLC as 19.67g / L, the purity was 55%, and the conversion rate of adenosine was 96%.

[0060] HPLC detection conditions: use octadecylsilane bonded silica gel as filler, mobile phase A is 0.1% TFA, B phase is methanol, detection wavelength is 260nm, column temperature is 25°C, and the elution procedure is shown in Table 1.

[0061] Table 1 Elution program

[0062] time (min) Mobile phase A(%) ...

Embodiment 3

[0064] Embodiment 3, preparation NMN

[0065] Substrate adenosine 100g, nicotinamide 68.5g, ATP 300g, MgCl 2 ·6H 2 O 110g was added to 5L pH7.0 phosphate buffer solution, stirred evenly, and the pH was adjusted to 7.0. Add the enzyme composition that AK, APRT and NmPRT three kinds of enzymes are formed in reaction solution, wherein the mass ratio of AK:APRT:NmPRT in the enzyme composition is 1:2:1, control pH during reaction and keep on 7.0, reaction temperature 30 After 8 hours of shaking reaction at -35°C, the amount of NMN produced in the reaction supernatant was detected by HPLC to be 17.4g / L, the purity was 50%, and the conversion rate of adenosine was 92%. HPLC detection condition is the same as embodiment 2.

[0066] 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 87.4 g of finished product NMN with a purity of 98.8% and a total y...

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Abstract

The invention relates to the technical fields of biopharmacy and biochemical industry, and in particular relates to an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing the nicotinamide mononucleotide by an enzymatic process. The enzyme composition is composed of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase.The three enzymes provided by the invention can be reasonably combined for efficient catalytic preparation of the nicotinamide mononucleotide. The enzyme composition provided by the invention can be recycled, has low costs, saves energy and protects the environment; the method for preparing the nicotinamide mononucleotide by the enzymatic process provided by the invention uses adenosine as a substrate, the enzyme composition is added, so that the nicotinamide mononucleotide can be prepared at low cost, safely and reliably, the cost of a current route can be reduced, and the method can be suitable for large-scale production and provides guarantee for the use of the nicotinamide mononucleotide in the fields of biocatalysis and drugs.

Description

technical field [0001] The invention relates to the technical fields of biopharmaceuticals and biochemical engineering, in particular to an enzyme composition for preparing nicotinamide mononucleotide and a method for enzymatically preparing nicotinamide mononucleotide. Background technique [0002] Nicotinamide mononucleotide (NMN) is the synthetic substrate of coenzyme I, and its structural formula is as follows: [0003] [0004] It becomes coenzyme I (NAD) upon adenylation by nicotinamide nucleotide adenylyltransferase. In vivo, the level of NMN and the activity of nicotinamide nucleotide adenylyltransferase (NAMPT) directly affect the concentration of NAD. At the same time, NMN directly participates in the transfer of adenosine in the body, and is an important synthetic substrate and functional regulator in the body. . In terms of therapeutic applications, NMN can be used for anti-aging, treatment of chronic diseases, etc. At the same time, studies have shown that ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N9/12C12P19/18C12P19/30
CPCC12N9/1077C12N9/1205C12P19/18C12P19/30C12Y204/02007C12Y204/02012C12Y207/0102
Inventor 周浩
Owner 杭州唯泰生物药业有限公司
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