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Method for preparing beta-nicotinamide mononucleotide by using enzymic method

A single nucleotide and enzymatic preparation technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the problems of environmental damage, low product purity, and low yield, and achieve easy purification and environmental protection. The effect of friendliness and expanding the scope of application

Active Publication Date: 2017-05-31
ENZYMEWORKS
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AI Technical Summary

Problems solved by technology

[0003] The traditional production method of β-nicotinamide mononucleotide is obtained by phosphorylating nicotinamide riboside with phosphorus oxychloride (see Chem. Commun. 1999, 729–730), with low yield and low product purity. Moreover, a large amount of organic solvents are used, which seriously damages the environment, among which phosphorus oxychloride is a relatively dangerous reagent

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation of recombinant Escherichia coli cells containing nicotinamide ribokinase

[0029] According to Sequence 1 and Sequence 2 in the sequence list, the nicotinamide ribokinase gene fragment was gene-synthesized, and NdeI and BamHI restriction sites were added to both ends, respectively, and connected into pET30a vector (produced by Suzhou Jinweizhi Biotechnology Co., Ltd.) to transform BL21 (DE3) strain.

[0030] Inoculate 4 ml of liquid LB medium with 1% nicotinamide ribokinase strain, shake culture at 37°C (200rpm) overnight, transfer the overnight culture to 50 ml of liquid LB medium with 1% inoculum, and incubate at 37°C Shake culture (200 rpm) until the OD600 value reaches 0.6-0.8, add a final concentration of 0.4 mM IPTG and shake culture at 20°C overnight. After the induction, the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended with 5 ml of 20 mmol / L triethanolamine buffer (pH 8.0) to obtain recombina...

Embodiment 2

[0031] Embodiment 2: Preparation of nicotinamide ribokinase freeze-dried powder

[0032] The recombinant cells of nicotinamide ribokinase prepared in Example 1 were ultrasonically disrupted in an ice bath, the broken liquid was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and freeze-dried to obtain the lyophilized nicotinamide ribokinase pink.

Embodiment 3

[0033] Example 3: Enzymatic synthesis of β-nicotinamide mononucleotide using nicotinamide ribose as a substrate

[0034] In this example, the lyophilized powder of nicotinamide ribokinase prepared according to the method in Example 2 was used to catalyze the synthesis of β-nicotinamide mononucleotide.

[0035] Add 1 L of 20 mmol / L triethanolamine buffer solution (pH8.0), final concentration of 18 mM nicotinamide riboside, final concentration of 20 mM ATP, final concentration of 20 mM MgCl2, and 5 g of nicotinamide ribokinase lyophilized powder to the reaction system in sequence After mixing evenly, place in a water bath at 33 °C and stir at 300 rpm for 24 h. After the reaction, the conversion rate of nicotinamide ribose was detected by high performance liquid chromatography to be over 90%. 4.9 g of β-nicotinamide mononucleotide was obtained after ion exchange resin separation, lyophilization and other post-treatment purification, with a purity of more than 95%.

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PUM

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Abstract

The invention relates to a method for preparing beta-nicotinamide mononucleotide by using an enzymic method. According to the method for preparing beta-nicotinamide mononucleotide by using the enzymic method, with nicotinamide riboside as a substrate and in the presence of a phosphoryl donor, the beta-nicotinamide mononucleotide is prepared by enabling the nicotinamide riboside to react with the phosphoryl donor under catalysis of nicotinamide riboside kinase and / or a reconstituted cell containing the nicotinamide riboside kinase. The method for preparing beta-nicotinamide mononucleotide by using the enzymic method has important application values. Compared with the conventional technology for chemically synthesizing the beta-nicotinamide mononucleotide, the method for preparing beta-nicotinamide mononucleotide by using the enzymic method is more environment-friendly, is lower in cost and can provide a product with higher purity; therefore, the method for preparing beta-nicotinamide mononucleotide by using the enzymic method can be more economically used for the field of health care products and biological medicines.

Description

technical field [0001] The invention relates to a preparation method of β-nicotinamide mononucleotide, in particular to a biological preparation method of β-nicotinamide mononucleotide. Background technique [0002] β-nicotinamide mononucleotide is a health product that has received extensive research and attention in recent years, and it is also a key intermediate for the synthesis of nicotinamide adenine dinucleotide (coenzyme I). It is reported that β-nicotinamide mononucleotide is anti-aging (see JNutr Sci Vitaminol (Tokyo). 2016;62(4):272-276), treating diabetes (Cell Metab. 2011Oct 5; 14(4): 528– 536) and other aspects have great potential, and related health products are on the market in Japan and the United States. [0003] The traditional production method of β-nicotinamide mononucleotide is obtained by phosphorylating nicotinamide riboside with phosphorus oxychloride (see Chem. Commun. 1999, 729–730), with low yield and low product purity. And use a large amount ...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12R1/19C12R1/84C12R1/865
CPCC12P19/305
Inventor 陶军华付敏杰梁晓亮
Owner ENZYMEWORKS
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