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A strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application

A single nucleotide, nicotinamide technology, applied in bacteria, microorganism-based methods, resistance to vector-borne diseases, etc., can solve the problems of long time catalysis, low yield, low enzyme activity, etc.

Active Publication Date: 2021-11-05
HOBOOMLIFE BIO TECH SHENZHEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, enzymatic reaction is mostly used to realize the large-scale synthesis of nicotinamide mononucleotide, but the cost of enzymatic reaction is high, the reaction conditions are harsh, the production process is unstable, the product index of each batch varies greatly, and the reaction capacity is low; In recent years, there is also a method of producing NMN by fermentation by biological means, but the natural nicotinamide phosphoribosyltransferase (NiaciNamidephosphoribosyltransferase, Nampt) has the problem of low enzymatic activity, and the catalysis takes a long time, high cost, and low yield, making it difficult to realize industrialization. Large-scale production limits the large-scale application of NMN
For this reason, Chinese patent CN201811606780.9 discloses a method of using recombinant Escherichia coli to ferment NMN, but the cost of transgenic recombinant strains is high, and the biological safety is doubtful, especially when it is applied to cosmetics, food or medicine

Method used

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  • A strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application
  • A strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application
  • A strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 strain screening and identification

[0049] 1. Soil collection and pretreatment

[0050] The soil near the sewer pipe of Bangtai Bioengineering (Shenzhen) Co., Ltd. factory was collected, and the sampling depth was 5cm-10cm. Weigh 1g of soil sample into a 50ml Erlenmeyer flask, add 20ml of normal saline and place it in a shaker, shake it well, take it out and let it stand for later use.

[0051] 2. Enrichment culture: Inoculate 1ml of the supernatant of the pretreated soil suspension into the enrichment medium (10ml / 50ml Erlenmeyer flask), and cultivate in a constant temperature incubator at 37°C and 150r / min for 12h. The composition of the enrichment medium includes: 2g / L nicotinamide, 5g / L glucose, 5g / L yeast extract, 5g / L peptone, 14g / LK 2 HPO 4 ·3H 2 O, 5.2g / LKH 2 PO 4 and 2g / LMgSO 4 ·7H 2 O, the pH of the enrichment medium is 7.0.

[0052] 3. Screening

[0053] Preliminary screening: the enrichment solution was diluted step by step with normal ...

Embodiment 2

[0064] Example 2 Effect of optimization of fermentation and enrichment conditions of Enterobacter chengdu 2021T4.7 on NMN yield

[0065] When Chengdu Enterobacter 2021T4.7 was fermented and enriched in the fermentation medium, the concentration of each component of the fermentation medium (including glucose concentration, tryptone concentration, KH 2 PO 4 Concentration, MgSO 4 ·7H 2 (2 concentration), the initial pH value of the fermentation medium, the concentration of the inducer Nam, the fermentation enrichment temperature and time, while other conditions were constant, and cultured in a 200rpm constant temperature shaking table; then under the conditions of Example 1 "enzyme conversion reaction experiment" Carry out fermentation and measure OD after the end 600 and enzyme activity.

[0066] 1. Add different concentrations of glucose (0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, mass fraction) respectively in the fermentation medium, while other components are unchanged. The re...

Embodiment 3

[0072] The influence of embodiment 3 substrate Nam concentration on NMN output

[0073] When the optimum conditions for fermentation and enrichment of Enterobacter Chengdu 2021T4.7 in Example 2 (that is, the fermentation medium was: 1% glucose, 1.25% tryptone, 0.75% KH 2 PO 4 , 0.025% MgSO 4 ·7H 2 O, inducer nicotinamide 1%, pH value 7.0, fermentation temperature 37°C, fermentation time 20h), only change the substrate Nam concentration (100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600μM, 700μM, 800μM, 900μM, 1000μM) for fermentation, other conditions remain unchanged, and the OD is measured after the end 600 and enzyme activity.

[0074] The result is as Figure 12 As shown, when the substrate Nam concentration was 400 μM, the NMN production was the highest, which was 66.47 μM.

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Abstract

The invention provides a strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application, and relates to the technical field of strain screening and application. The Chengdu Enterobacteriaceae described in the present invention collect soil from the vicinity of the sewer of a factory that produces NMN-related products, and through primary screening and re-screening, strains with strong ability to convert nicotinamide into nicotinamide mononucleotide in the soil are selected, and then tested After identification and preservation, the Enterobacter is determined to be a Gram-negative bacterium belonging to the genus Enterobacter Chengdu. When using nicotinamide as a substrate to ferment and produce NMN with Enterobacter chengdu, the NMN production can reach 67.66 μM, or 22.6 mg / L, in 15 minutes, indicating that the wild strain has a strong activity of synthesizing NMN and reduces the activity of synthesizing NMN. When it is dependent on Nampt, it has a high prospect of large-scale application.

Description

technical field [0001] The invention belongs to the technical field of strain screening and application, and in particular relates to a strain of Enterobacter chengdu producing nicotinamide mononucleotide and its application. Background technique [0002] Nicotinamide Mononucleotide (NMN) is an organic molecule and a nucleotide that can reverse aging and prolong life. [0003] At present, enzymatic reaction is mostly used to realize the large-scale synthesis of nicotinamide mononucleotide, but the cost of enzymatic reaction is high, the reaction conditions are harsh, the production process is unstable, the product index of each batch varies greatly, and the reaction capacity is low; In recent years, there is also a method of producing NMN by fermentation by biological means, but the natural nicotinamide phosphoribosyltransferase (NiaciNamidephosphoribosyltransferase, Nampt) has the problem of low enzymatic activity, and the catalysis takes a long time, high cost, and low yie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P19/30C12R1/01
CPCC12N1/20C12P19/305C12N1/205C12R2001/01C12P19/30C12M33/10
Inventor 赵丽青陈建生段志刚张海潮
Owner HOBOOMLIFE BIO TECH SHENZHEN CO LTD
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