Method for synthesizing nicotinamide mononucleotide based on enzyme method

A single nucleotide and enzymatic synthesis technology, applied in the direction of fermentation, etc., can solve the problems of limited production and application, high product impurities, environmental pollution, etc., and achieve the effect of low cost, high product purity and cost reduction

Active Publication Date: 2021-06-01
HUNAN FLAG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

People try to produce NMN by chemical method, which is usually obtained by phosphorylating nicotinamide ribose (NR) with phosphorus oxychloride etc. (Chem.Commun.1999,729-730, CN 107613990 A), but The obtained product has many impurities, low yield, high cost, and involves the use of a large amount of toxic and harmful reagents, causing serious environmental pollution, which limits its production and application in food-grade NMN

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  • Method for synthesizing nicotinamide mononucleotide based on enzyme method
  • Method for synthesizing nicotinamide mononucleotide based on enzyme method
  • Method for synthesizing nicotinamide mononucleotide based on enzyme method

Examples

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Embodiment 1

[0044] The construction of embodiment 1 enzyme gene expression engineering strain

[0045] The ribokinase, phosphoribosyl mutase and nicotinamide ribokinase amino acid sequences involved in the present invention were sent to Gene Synthesis Company for codon optimization of Escherichia coli and artificially synthesized coding genes, respectively cloned into NdeI of the prokaryotic expression vector pET30a(+) Between the XhoI restriction site and the C-terminus of the above genes, 6 histidine coding sequences are added to facilitate subsequent protein purification. The recombinant expression vectors containing the above three enzyme genes were introduced into Escherichia coli BL21(DE3) by electrotransformation to obtain corresponding engineering strains for enzyme gene expression. The fermentation of engineering strains adopts conventional fermentation medium TB, and first cultivates OD at 37°C and 200rpm. 600 reach 0.6-0.8, and then use the final concentration of 0.5mM IPTG or...

Embodiment 2

[0046] The preparation of embodiment 2 immobilized enzyme

[0047] Use a low-temperature high-speed centrifuge (10000rpm, 4°C, 10min) to collect ribokinase, phosphoribosyl mutase and nicotinamide ribokinase fermentation cells after induction for 10 hours, and wash repeatedly with pH 8.0, 0.1mol / L phosphate buffer Bacteria were resuspended 20 times concentrated in 50ml of phosphate buffer, and ultrasonically disrupted in an ice bath (ultrasonic conditions: working 2s, interval 5s) until clarified. Centrifuge the above broken solution in a low-temperature high-speed centrifuge (10000rpm, 4°C, 20min), collect the supernatant to obtain the crude enzyme solutions of ribokinase, phosphoribosylmutase and nicotinamide ribokinase, and then use vacuum freeze-drying to prepare into crude enzyme protein powder. Further inject the above crude enzyme protein solution onto the IDA resin that has been activated and bound to Ni+, first use 1-2 column volumes of 10 and 30mM low-concentration i...

Embodiment 3

[0048] Example 3 Using D-ribose as a substrate, using crude enzyme one-pot method to produce NMN

[0049] Add D-ribose with a final concentration of 50mM, 50mM nicotinamide, 60mM ATP, 5mM manganese chloride, and 20mM magnesium chloride in sequence to the reaction system, and use 1L of pH 6.0 and 50mM phosphate buffer solution to fully shake and dissolve, then adjust the pH to 5.00 . Add 300 mg of ribokinase crude enzyme freeze-dried powder prepared in Example 2, 300 mg of nicotinamide ribokinase crude enzyme freeze-dried powder, and 600 mg of phosphoribosylmutase crude enzyme freeze-dried powder, mix well, and control the reaction temperature to 25 ° C. 300rpm stirred reaction, adopts automatic titrator, controls pH 5.0 with 1mol / l sodium hydroxide in the whole process, detects the generation concentration of NMN with liquid chromatography HPLC in the reaction process, reacts and finishes in 4 hours, and reaction obtains NMN 14.8g, and reaction yield The yield was 88.62%. The...

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Abstract

The invention provides a method for synthesizing nicotinamide mononucleotide (NMN) based on an enzymic method, which comprises the following steps: taking D-ribose, nicotinamide and ATP (adenosine triphosphate) as substrates, and synthesizing the nicotinamide mononucleotide by a one-pot method under the coupling catalytic action of ribose kinase, phosphoribose mutase and nicotinamide ribose kinase. The method for preparing the NMN opens up a new way for synthesizing the NMN by an enzyme method. The three enzymes can be recycled, and the method is low in cost, energy-saving, environment-friendly and suitable for large-scale industrial production.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals and biocatalysis, and relates to a novel method for synthesizing nicotinamide mononucleotide based on an enzymatic method. Background technique [0002] Nicotinamide mononucleotide (β-Nicotinamide mononucleotide, referred to as β-NMN or NMN) is the product of nicotinamide phosphoribosyltransferase reaction, which is the key precursor of NAD+. After glycosylation, it becomes NAD+. Human extracellular NMN needs to be dephosphorylated and converted to nicotinamide riboside (NR) to enter the interior of liver cells, and then NR generates NMN under the action of nicotinamide riboside kinase 1. NMN exerts its physiological functions by converting into NAD+ in the human body, such as activating NAD+ substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulating cell survival and death, maintaining redox state, etc. Recent studies have found that NMN supplementation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30
CPCC12P19/30
Inventor 赵强赵士敏周晶辉曾红宇许岗
Owner HUNAN FLAG BIOTECHNOLOGY CO LTD
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